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Sample records for protein specific cd4

  1. CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.

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    Andreas Schweizer

    2008-07-01

    Full Text Available Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.

  2. Heat Shock Proteins 60 and 70 Specific Proinflammatory and Cytotoxic Response of CD4+CD28null Cells in Chronic Kidney Disease

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    Ashok K. Yadav

    2013-01-01

    Full Text Available Background. CD4+CD28null T cells are expanded in peripheral blood of patients with chronic kidney disease and associated with subclinical atherosclerosis. However, triggers for the oligoclonal expansion and activation of these cells are not clear. Methods. We investigated twenty-five stage V-IV chronic kidney disease (CKD patients and eight healthy subjects (HC. Peripheral mononuclear cells were isolated and incubated with heat shock protein- (HSP 60 and 70. CD4+CD28null and CD4+CD28+ cells were sorted by flowcytometry and antigen specific response was assessed by the mRNA and protein expression of interferon (IFN-γ, perforin, and granzyme B using qRT-PCR and Elispot. Results. The basal mRNA expression of IFN-γ, perforin, and granzyme B in CD4+CD28null cells was higher in subjects with CKD compared to that in HC (P<0.0001. Subjects with CKD also showed expression of IFN-γ, perforin, and granzyme B in the CD4+CD28+ subset, but this was much weaker than that seen in the CD4+CD28null population (P<0.0001. We did not note the expression of these molecules at mRNA or protein level in either subset of CD4 cells in HC. After incubation with HSP60 and HSP70, CD4+CD28null cells showed increased expression at mRNA (P<0.001 and protein level (P<0.001. CD4+CD28+ cells also showed a weak increase in expression. No antigen-specific response was noted in HC. Conclusion. These data show that CD4+CD28null cells in subjects with CKD react with HSP60 and HSP70 by upregulating the expression of IFN-γ, perforin and granzyme B. Increased circulating level of HSP60 and HSP70 might play a role in initiation and/or progression of atherosclerosis in CKD subjects through perturbation of CD4+CD28null cells.

  3. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    by a defect in the cytotoxic capacity of the individual CD4+ lymphocyte after antigen stimulation, and it could not be explained by a reduction in the total number of CD4+ cells before antigen stimulation. The antigen-specific cytotoxic activity was, however, closely related to the ability of the CD4+ T cells...

  4. Ex vivo detection of adenovirus specific CD4+ T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of THELPER cells following stem cell transplantation

    International Nuclear Information System (INIS)

    Serangeli, Celine; Bicanic, Oliver; Scheible, Michael H.; Wernet, Dorothee; Lang, Peter; Rammensee, Hans-Georg; Stevanovic, Stefan; Handgretinger, Rupert; Feuchtinger, Tobias

    2010-01-01

    Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4 + T-cell responses against the Hexon-protein, but the frequency of specific T HELPER cells is extremely low or not detectable ex vivo and preference for different CD4 + T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4 + -responses ex vivo, to identify epitopes which would be broadly applicable. We selected 19 candidate epitopes with predicted restriction to HLA-DR1/DR3/DR4/DR7; 16 were located within the highly conserved regions, indicating cross-reactivity of T cells among HAdV-subspecies. Ten epitopes induced CD4 + -proliferation in >50% of individuals, confirmed by intracellular IFN-γ detection. Three SCT recipients who recovered from an infection with HAdV displayed reactivity towards only a single hexon epitope, whereas healthy individuals were responsive to two to eight epitopes (median 3). The ex vivo detection of Hexon-specific CD4 + T-cells, without any long-term culture in vitro, enables the detection and generation of HAdV-specific CD4 + T cells for adoptive T-cell transfer against HAdV-infection post SCT.

  5. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

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    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  6. Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.

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    Abana, Chike O; Pilkinton, Mark A; Gaudieri, Silvana; Chopra, Abha; McDonnell, Wyatt J; Wanjalla, Celestine; Barnett, Louise; Gangula, Rama; Hager, Cindy; Jung, Dae K; Engelhardt, Brian G; Jagasia, Madan H; Klenerman, Paul; Phillips, Elizabeth J; Koelle, David M; Kalams, Spyros A; Mallal, Simon A

    2017-11-01

    Select CMV epitopes drive life-long CD8 + T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4 + T cells specific for human CMV (HCMV) are elevated in HIV + HCMV + subjects. To determine whether HCMV epitope-specific CD4 + T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4 + T cells in coinfected HLA-DR7 + long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4 + T cells were inflated among these HIV + subjects compared with those from an HIV - HCMV + HLA-DR7 + cohort or with HLA-DR7-restricted CD4 + T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4 + T cells consisted of effector memory or effector memory-RA + subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX 3 CR1, CD38, or HLA-DR but less often coexpressed CD38 + and HLA-DR + The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4 + T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection.

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    Michaela Lucas

    2007-07-01

    Full Text Available CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays.Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C.During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

  8. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    mycobacteria. Our objective was to investigate the M.tuberculosis-and M. avium-specific cytotoxic capacity of T cells from healthy, bacille Calmette-Guérin-vaccinated, HIV-seropositive individuals. Blood mononuclear cells were obtained from 10 healthy HIV-seropositive and 10 healthy seronegative persons...... with no history of previous or active mycobacterial infection. Antigen-specific killing of macrophages presenting mycobacterial antigens (purified protein derivative or M. avium culture filtrate) was conducted. The phenotype of the killer cells was determined by a fluorescence-activated cell sorter after antigen...

  9. High Frequency of CD4+ T Cells Specific for the TB10.4 Protein Correlates with Protection against Mycobacterium tuberculosis Infection

    Czech Academy of Sciences Publication Activity Database

    Hervas-Stubbs, S.; Majlessi, L.; Šimšová, Marcela; Morová, Jana; Rojas, M. J.; Nouzé, C.; Brodin, P.; Šebo, Peter; Leclerc, C.

    2006-01-01

    Roč. 74, č. 6 (2006), s. 3396-3407 ISSN 0019-9567 R&D Projects: GA AV ČR IBS5020311 Institutional research plan: CEZ:AV0Z50200510 Keywords : t cell s * mycobacterium tuberculosis * protein Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2006

  10. CD27 instructs CD4+ T cells to provide help for the memory CD8+ T cell response after protein immunization

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    Xiao, Yanling; Peperzak, Victor; Keller, Anna M.; Borst, Jannie

    2008-01-01

    For optimal quality, memory CD8(+) T cells require CD4(+) T cell help. We have examined whether CD4(+) T cells require CD27 to deliver this help, in a model of intranasal OVA protein immunization. CD27 deficiency reduced the capacity of CD4(+) T cells to support Ag-specific CD8(+) T cell

  11. Regulatory function of cytomegalovirus-specific CD4+CD27-CD28- T cells

    International Nuclear Information System (INIS)

    Tovar-Salazar, Adriana; Patterson-Bartlett, Julie; Jesser, Renee; Weinberg, Adriana

    2010-01-01

    CMV infection is characterized by high of frequencies of CD27 - CD28 - T cells. Here we demonstrate that CMV-specific CD4 + CD27 - CD28 - cells are regulatory T cells (T R ). CD4 + CD27 - CD28 - cells sorted from CMV-stimulated PBMC of CMV-seropositive donors inhibited de novo CMV-specific proliferation of autologous PBMC in a dose-dependent fashion. Compared with the entire CMV-stimulated CD4 + T-cell population, higher proportions of CD4 + CD27 - CD28 - T R expressed FoxP3, TGFβ, granzyme B, perforin, GITR and PD-1, lower proportions expressed CD127 and PD1-L and similar proportions expressed CD25, CTLA4, Fas-L and GITR-L. CMV-CD4 + CD27 - CD28 - T R expanded in response to IL-2, but not to CMV antigenic restimulation. The anti-proliferative effect of CMV-CD4 + CD27 - CD28 - T R significantly decreased after granzyme B or TGFβ inhibition. The CMV-CD4 + CD27 - CD28 - T R of HIV-infected and uninfected donors had similar phenotypes and anti-proliferative potency, but HIV-infected individuals had higher proportions of CMV-CD4 + CD27 - CD28 - T R . The CMV-CD4 + CD27 - CD28 - T R may contribute to the downregulation of CMV-specific and nonspecific immune responses of CMV-infected individuals.

  12. Cytokines affecting CD4+T regulatory cells in transplant tolerance. II. Interferon gamma (IFN-γ) promotes survival of alloantigen-specific CD4+T regulatory cells.

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    Nomura, Masaru; Hodgkinson, Suzanne J; Tran, Giang T; Verma, Nirupama D; Robinson, Catherine; Plain, Karren M; Boyd, Rochelle; Hall, Bruce M

    2017-06-01

    CD4 + T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4 + T cells. Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for >100days with no further immunosuppression. In previous studies, we found the CD4 + T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4 + T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4 + T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4 + T cells. Tolerant CD4 + CD25 + T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4 + CD25 + T cells to third-party Lewis. Tolerant CD4 + CD25 + T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4 + CD25 + T cells. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  13. Cytokines affecting CD4+T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells.

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    Hall, Bruce M; Plain, Karren M; Tran, Giang T; Verma, Nirupama D; Robinson, Catherine M; Nomura, Masaru; Boyd, Rochelle; Hodgkinson, Suzanne J

    2017-08-01

    CD4 + T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4 + CD25 + FOXP3 + Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg. This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4 + , especially CD4 + CD25 + T cells. CD4 + T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4 + T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4 + CD25 + T cells' response to rIL-5 and expression of IL-5Rα was also assessed. rIL-5 was sufficient to promote transplant tolerance mediating CD4 + T cells' survival in culture with specific-donor alloantigen. Tolerant CD4 + T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4 + T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4 + CD25 + T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4 + CD25 + T cells expressed IL-5Rα. This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4 + CD25 + T cells that mediate transplant tolerance. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. In-Depth Analysis of Citrulline Specific CD4 T-Cells in Rheumatoid Arthritis

    Science.gov (United States)

    2017-01-01

    AWARD NUMBER: W81XWH-15-1-0004 TITLE: In-Depth Analysis of Citrulline-Specific CD4 T - Cells in Rheumatoid Arthritis PRINCIPAL INVESTIGATOR...2016 4. TITLE AND SUBTITLE In-Depth Analysis of Citrulline-Specific CD4 T Cells in Rheumatoid Arthritis 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH...NOTES 14. ABSTRACT The goal of this project is to test the hypothesis that cit-specific CD4 T cells present in rheumatoid arthritis (RA) patients

  15. Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

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    Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

    2018-03-01

    During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Granzyme B mediated function of Parvovirus B19-specific CD4+ T cells

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    Kumar, Arun; Perdomo, Maria F; Kantele, Anu; Hedman, Lea; Hedman, Klaus; Franssila, Rauli

    2015-01-01

    A novel conception of CD4+ T cells with cytolytic potential (CD4+ CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4+ CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4+ T cells. CD4+ T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4+ T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4+ CTLs co-expressing CD56 antigen. Our results suggest a role for CD4+ CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. PMID:26246896

  17. In-Depth Analysis of Citrulline-Specific CD4 T-Cells in Rheumatoid Arthritis

    Science.gov (United States)

    2016-01-01

    be used to identify and characterize CD4+T cells specific for influenza A, West Nile, Yellow fever , Dengue and Japanese Encephalitis viruses. The...four difference species of Flavivirus using the TGEM approach. These will include: Yellow Fever Virus, West Nile Virus, Dengue Virus and Japanese...1 AWARD NUMBER: W81XWH-15-1-0004 TITLE: In-Depth Analysis of Citrulline-Specific CD4 T-Cells in Rheumatoid Arthritis PRINCIPAL INVESTIGATOR

  18. Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells.

    Science.gov (United States)

    Bronke, Corine; Palmer, Nanette M; Westerlaken, Geertje H A; Toebes, Mireille; van Schijndel, Gijs M W; Purwaha, Veenu; van Meijgaarden, Krista E; Schumacher, Ton N M; van Baarle, Debbie; Tesselaar, Kiki; Geluk, Annemieke

    2005-09-01

    In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4+ T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated, or CMV-seropositive individuals, we were able to directly detect with both tetramers epitope-specific T cells up to 0.62% and 0.45% of the CD4+ T-cell population reactive to M. tuberculosis and CMV, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer+ CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4+ T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4+ T cells in clinical disease or after vaccination.

  19. Characterisation of CD4 T cells in healthy and diseased koalas (Phascolarctos cinereus) using cell-type-specific monoclonal antibodies.

    Science.gov (United States)

    Mangar, Chandan; Armitage, Charles W; Timms, Peter; Corcoran, Lynn M; Beagley, Kenneth W

    2016-07-01

    The koala (Phascolarctos cinereus) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4(+) lymphocytes collected from koala spleens (41.1%, range 20-45.1%) lymph nodes (36.3%, range 19-55.9%) and peripheral blood (23.8%, range 17.3-35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4(+) cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4(+) T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4(+) T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells.

    Science.gov (United States)

    Baldauf, Hanna-Mari; Stegmann, Lena; Schwarz, Sarah-Marie; Ambiel, Ina; Trotard, Maud; Martin, Margarethe; Burggraf, Manja; Lenzi, Gina M; Lejk, Helena; Pan, Xiaoyu; Fregoso, Oliver I; Lim, Efrem S; Abraham, Libin; Nguyen, Laura A; Rutsch, Frank; König, Renate; Kim, Baek; Emerman, Michael; Fackler, Oliver T; Keppler, Oliver T

    2017-03-07

    Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

  1. Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones

    International Nuclear Information System (INIS)

    Honda, S.; Okuno, K.; Yasutomi, M.; Takasaki, T.; Kurane, I.

    1998-01-01

    We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells were cultured with EBV; and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+ , CD4+ , and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-super-infected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+ , CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-super-infected and non-super-infected LCLs. Proliferative and cytotoxic responses to allogeneic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections. (authors)

  2. A dominant EV71-specific CD4+ T cell epitope is highly conserved among human enteroviruses.

    Directory of Open Access Journals (Sweden)

    Ruicheng Wei

    Full Text Available CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD, has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine.

  3. CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

    Science.gov (United States)

    Aguado, Enrique; Garcia-Cozar, Francisco

    2014-01-01

    Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127lowPD-1highTIM-3high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein. PMID:24465502

  4. CD4+ primary T cells expressing HCV-core protein upregulate Foxp3 and IL-10, suppressing CD4 and CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Cecilia Fernandez-Ponce

    Full Text Available Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127(lowPD-1(highTIM-3(high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.

  5. ImmunoPET Imaging of Murine CD4+ T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging.

    Science.gov (United States)

    Freise, Amanda C; Zettlitz, Kirstin A; Salazar, Felix B; Lu, Xiang; Tavaré, Richard; Wu, Anna M

    2017-08-01

    Molecular imaging of CD4 + T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4 + T cell viability, proliferation, CD4 expression, and function. The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of 89 Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed. For immunoPET imaging, the lowest protein dose of 2 μg of 89 Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-μg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 μg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 μg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 μg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4 + T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4 + T cells. Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4 + T cells in the context of

  6. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

    1990-01-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  7. Functional, Antigen-Specific Stem Cell Memory (TSCM CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Cheleka A. M. Mpande

    2018-03-01

    Full Text Available BackgroundMaintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM, which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM or effector (TEFF T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb, the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology.MethodsWe studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette–Guerin (BCG vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+ were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.ResultsM. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also

  8. Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection

    NARCIS (Netherlands)

    Geldmacher, Christof; Ngwenyama, Njabulo; Schuetz, Alexandra; Petrovas, Constantinos; Reither, Klaus; Heeregrave, Edwin J.; Casazza, Joseph P.; Ambrozak, David R.; Louder, Mark; Ampofo, William; Pollakis, Georgios; Hill, Brenna; Sanga, Erica; Saathoff, Elmar; Maboko, Leonard; Roederer, Mario; Paxton, William A.; Hoelscher, Michael; Koup, Richard A.

    2010-01-01

    HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common

  9. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor

    International Nuclear Information System (INIS)

    Schols, D.; Baba, M.; Pauwels, R.; Desmyter, J.; De Clercq, E.

    1989-01-01

    The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a monoclonal antibody (mAb) and, to a lesser extent, OKT4 mAb to the CD4 cell receptor for human immunodeficiency virus type 1 (HIV-1). The effect was seen within 1 min at an ATA concentration of 10 μM in various T4 + cells (MT-4, U-937, peripheral blood lymphocytes, and monocytes). It was dose-dependent and reversible. ATA prevented the attachment of radiolabeled HIV-1 particles to MT-4 cells, which could be expected as the result of its specific binding to the HIV/CD4 receptor. Other HIV inhibitors such as suramin, fuchsin acid, azidothymidine, dextran sulfate, heparin, and pentosan polysulfate did not affect OKT4A/Leu-3a mAb binding to the CD4 receptor, although the sulfated polysaccharides suppressed HIV-1 adsorption to the cells at concentrations required for complete protection against HIV-1 cytopathogenicity. Thus, ATA is a selective marker molecule for the CD4 receptor. ATA also interfered with the staining of membrane-associated HIV-1 glycoprotein gp120 by a mAb against it. These unusual properties of a small molecule of nonimmunological origin may have important implications for the study of CD4/HIV/AIDS pathogenesis and possibly treatment

  10. Scaffold protein JLP mediates TCR-initiated CD4+T cell activation and CD154 expression.

    Science.gov (United States)

    Yan, Qi; Yang, Cheng; Fu, Qiang; Chen, Zhaowei; Liu, Shan; Fu, Dou; Rahman, Rahmat N; Nakazato, Ryota; Yoshioka, Katsuji; Kung, Sam K P; Ding, Guohua; Wang, Huiming

    2017-07-01

    CD4 + T-cell activation and its subsequent induction of CD154 (CD40 ligand, CD40L) expression are pivotal in shaping both the humoral and cellular immune responses. Scaffold protein JLP regulates signal transduction pathways and molecular trafficking inside cells, thus represents a critical component in maintaining cellular functions. Its role in regulating CD4 + T-cell activation and CD154 expression, however, is unclear. Here, we demonstrated expression of JLP in mouse tissues of lymph nodes, thymus, spleen, and also CD4 + T cells. Using CD4+ T cells from jlp-deficient and jlp-wild-type mice, we demonstrated that JLP-deficiency impaired T-cell proliferation, IL-2 production, and CD154 induction upon TCR stimulations, but had no impacts on the expression of other surface molecules such as CD25, CD69, and TCR. These observed impaired T-cell functions in the jlp-/- CD4 + T cells were associated with defective NF-AT activation and Ca 2 + influx, but not the MAPK, NF-κB, as well as AP-1 signaling pathways. Our findings indicated that, for the first time, JLP plays a critical role in regulating CD4 + T cells response to TCR stimulation partly by mediating the activation of TCR-initiated Ca 2+ /NF-AT. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination

    OpenAIRE

    Li, Xi; Miao, Hongyu; Henn, Alicia; Topham, David J.; Wu, Hulin; Zand, Martin S.; Mosmann, Tim R.

    2012-01-01

    Although previous studies have found minimal changes in CD4 T cell responses after vaccination of adults with trivalent inactivated influenza vaccine, daily sampling and monitoring of the proliferation marker Ki-67 have now been used to reveal that a substantial fraction of influenza-specific CD4 T cells respond to vaccination. At 4–6 days after vaccination, there is a sharp rise in the numbers of Ki-67-expressing PBMC that produce IFNγ, IL-2 and/or TNFα in vitro in response to influenza vacc...

  12. The Ixodes scapularis Salivary Protein, Salp15, Prevents the Association of HIV-1 gp120 and CD4

    OpenAIRE

    Juncadella, Ignacio J.; Garg, Renu; Bates, Tonya C.; Olivera, Elias R.; Anguita, Juan

    2007-01-01

    Ixodes scapularis salivary protein, Salp15, inhibits CD4+ T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interactio...

  13. Global Assessment of Dengue Virus-Specific CD4+ T Cell Responses in Dengue-Endemic Areas

    Directory of Open Access Journals (Sweden)

    Alba Grifoni

    2017-10-01

    Full Text Available BackgroundDengue is a major public health problem worldwide. Assessment of adaptive immunity is important to understanding immunopathology and to define correlates of protection against dengue virus (DENV. To enable global assessment of CD4+ T cell responses, we mapped HLA-DRB1-restricted DENV-specific CD4+ T cell epitopes in individuals previously exposed to DENV in the general population of the dengue-endemic region of Managua, Nicaragua.MethodsHLA class II epitopes in the population of Managua were identified by an in vitro IFNγ ELISPOT assay. CD4+ T cells purified by magnetic bead negative selection were stimulated with HLA-matched epitope pools in the presence of autologous antigen-presenting cells, followed by pool deconvolution to identify specific epitopes. The epitopes identified in this study were combined with those previously identified in the DENV endemic region of Sri Lanka, to generate a “megapool” (MP consisting of 180 peptides specifically designed to achieve balanced HLA and DENV serotype coverage. The DENV CD4MP180 was validated by intracellular cytokine staining assays.ResultsWe detected responses directed against a total of 431 epitopes, representing all 4 DENV serotypes, restricted by 15 different HLA-DRB1 alleles. The responses were associated with a similar pattern of protein immunodominance, overall higher magnitude of responses, as compared to what was observed previously in the Sri Lanka region. Based on these epitope mapping studies, we designed a DENV CD4 MP180 with higher and more consistent coverage, which allowed the detection of CD4+ T cell DENV responses ex vivo in various cohorts of DENV exposed donors worldwide, including donors from Nicaragua, Brazil, Singapore, Sri Lanka, and U.S. domestic flavivirus-naïve subjects immunized with Tetravalent Dengue Live-Attenuated Vaccine (TV005. This broad reactivity reflects that the 21 HLA-DRB1 alleles analyzed in this and previous studies account for more than 80

  14. Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

    Science.gov (United States)

    Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

    2015-10-23

    The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Characterization of HIV-Specific CD4+T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

    DEFF Research Database (Denmark)

    Buggert, Marcus; Norstrom, Melissa M.; Czarnecki, Chris

    2012-01-01

    for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve...... this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73...... II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines....

  16. Antigen-specific and non-specific CD4+ T cell recruitment and proliferation during influenza infection

    International Nuclear Information System (INIS)

    Chapman, Timothy J.; Castrucci, Maria R.; Padrick, Ryan C.; Bradley, Linda M.; Topham, David J.

    2005-01-01

    To track epitope-specific CD4 + T cells at a single-cell level during influenza infection, the MHC class II-restricted OVA 323-339 epitope was engineered into the neuraminidase stalk of influenza/A/WSN, creating a surrogate viral antigen. The recombinant virus, influenza A/WSN/OVA II , replicated well, was cleared normally, and stimulated both wild-type and DO11.10 or OT-II TCR transgenic OVA-specific CD4 + T cells. OVA-specific CD4 T cells proliferated during infection only when the OVA epitope was present. However, previously primed (but not naive) transgenic CD4 + T cells were recruited to the infected lung both in the presence and absence of the OVA 323-339 epitope. These data show that, when primed, CD4 + T cells may traffic to the lung in the absence of antigen, but do not proliferate. These results also document a useful tool for the study of CD4 T cells in influenza infection

  17. Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection.

    Science.gov (United States)

    Richards, Katherine A; DiPiazza, Anthony T; Rattan, Ajitanuj; Knowlden, Zackery A G; Yang, Hongmei; Sant, Andrea J

    2018-01-01

    One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells.

  18. 5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4+ T Cells

    Directory of Open Access Journals (Sweden)

    Colm E. Nestor

    2016-07-01

    Full Text Available 5-methylcytosine (5mC is converted to 5-hydroxymethylcytosine (5hmC by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4+ T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4+ memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4+ T cell biology in humans, with important implications for gene regulation and lineage commitment.

  19. Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination.

    Science.gov (United States)

    Li, Xi; Miao, Hongyu; Henn, Alicia; Topham, David J; Wu, Hulin; Zand, Martin S; Mosmann, Tim R

    2012-06-29

    Although previous studies have found minimal changes in CD4 T cell responses after vaccination of adults with trivalent inactivated influenza vaccine, daily sampling and monitoring of the proliferation marker Ki-67 have now been used to reveal that a substantial fraction of influenza-specific CD4 T cells respond to vaccination. At 4-6 days after vaccination, there is a sharp rise in the numbers of Ki-67-expressing PBMC that produce IFNγ, IL-2 and/or TNFα in vitro in response to influenza vaccine or peptide. Ki-67(+) cell numbers then decline rapidly, and 10 days after vaccination, both Ki-67(+) and overall influenza-specific cell numbers are similar to pre-vaccination levels. These results provide a tool for assessing the quality and quantity of CD4 T cell responses to different influenza vaccines, and raise the possibility that the anti-influenza T cell memory response may be qualitatively altered by vaccination, even if the overall memory cell numbers do not change significantly. Copyright © 2012. Published by Elsevier Ltd.

  20. Autocrine production of beta-chemokines protects CMV-Specific CD4 T cells from HIV infection.

    Directory of Open Access Journals (Sweden)

    Joseph P Casazza

    2009-10-01

    Full Text Available Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1alpha and MIP-1beta mRNA, resulting in a rapid increase in production of MIP-1alpha and MIP-1beta after cognate antigen stimulation. Production of beta-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of beta-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1beta contained 10 times less Gag DNA than did those which failed to produce MIP-1beta. These data suggest that CD4+ T cells which produce MIP-1alpha and MIP-1beta bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.

  1. Diminished primary and secondary influenza virus-specific CD8(+) T-cell responses in CD4-depleted Ig(-/-) mice

    DEFF Research Database (Denmark)

    Riberdy, J M; Christensen, Jan Pravsgaard; Branum, K

    2000-01-01

    Optimal expansion of influenza virus nucleoprotein (D(b)NP(366))-specific CD8(+) T cells following respiratory challenge of naive Ig(-/-) microMT mice was found to require CD4(+) T-cell help, and this effect was also observed in primed animals. Absence of the CD4(+) population was consistently...

  2. Applying machine learning to predict patient-specific current CD 4 ...

    African Journals Online (AJOL)

    This work shows the application of machine learning to predict current CD4 cell count of an HIV-positive patient using genome sequences, viral load and time. A regression model predicting actual CD4 cell counts and a classification model predicting if a patient's CD4 cell count is less than 200 was built using a support ...

  3. Heat shock protein 90-mediated peptide-selective presentation of cytosolic tumor antigen for direct recognition of tumors by CD4(+) T cells.

    Science.gov (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Caballero, Otavia L; Jungbluth, Achim A; Ritter, Gerd; Odunsi, Kunle; Old, Lloyd J; Gnjatic, Sacha

    2012-04-15

    Tumor Ag-specific CD4(+) T cells play important functions in tumor immunosurveillance, and in certain cases they can directly recognize HLA class II-expressing tumor cells. However, the underlying mechanism of intracellular Ag presentation to CD4(+) T cells by tumor cells has not yet been well characterized. We analyzed two naturally occurring human CD4(+) T cell lines specific for different peptides from cytosolic tumor Ag NY-ESO-1. Whereas both lines had the same HLA restriction and a similar ability to recognize exogenous NY-ESO-1 protein, only one CD4(+) T cell line recognized NY-ESO-1(+) HLA class II-expressing melanoma cells. Modulation of Ag processing in melanoma cells using specific molecular inhibitors and small interfering RNA revealed a previously undescribed peptide-selective Ag-presentation pathway by HLA class II(+) melanoma cells. The presentation required both proteasome and endosomal protease-dependent processing mechanisms, as well as cytosolic heat shock protein 90-mediated chaperoning. Such tumor-specific pathway of endogenous HLA class II Ag presentation is expected to play an important role in immunosurveillance or immunosuppression mediated by various subsets of CD4(+) T cells at the tumor local site. Furthermore, targeted activation of tumor-recognizing CD4(+) T cells by vaccination or adoptive transfer could be a suitable strategy for enhancing the efficacy of tumor immunotherapy.

  4. Sequential Dysfunction and Progressive Depletion of Candida albicans-Specific CD4 T Cell Response in HIV-1 Infection

    Science.gov (United States)

    Liu, Fengliang; Fan, Xiuzhen; Auclair, Sarah; Ferguson, Monique; Sun, Jiaren; Soong, Lynn; Hou, Wei; Redfield, Robert R.; Birx, Deborah L.; Ratto-Kim, Silvia; Robb, Merlin L.; Kim, Jerome H.; Michael, Nelson L.; Hu, Haitao

    2016-01-01

    Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV) disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV)-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART) naïve HIV infection cohort (RV21), we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1β) functional subsets, the Th17 functional subsets (IL-17, IL-22) of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis. PMID:27280548

  5. Cooperativity of HIV-Specific Cytolytic CD4 T Cells and CD8 T Cells in Control of HIV Viremia

    Science.gov (United States)

    Johnson, Susan; Eller, Michael; Teigler, Jeffrey E.; Maloveste, Sebastien M.; Schultz, Bruce T.; Soghoian, Damien Z.; Lu, Richard; Oster, Alexander F.; Chenine, Agnès-Laurence; Alter, Galit; Dittmer, Ulf; Marovich, Mary; Robb, Merlin L.; Michael, Nelson L.; Bolton, Diane

    2015-01-01

    ABSTRACT CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, nontraditional CD4+ T cell functions beyond helper effects have been described, and a role for cytolytic CD4+ T cells in the control of HIV infection has been suggested. We define here the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of CD4 T cell susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection. IMPORTANCE The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. We show here that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypic differences compared to HIV-specific Th1 cells but with similarities to CD8+ T cells. These findings are

  6. Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

    Directory of Open Access Journals (Sweden)

    Dae-Hee Sohn

    Full Text Available An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs, recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs as stimulators of CD8(+ and CD4(+ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ enzyme-linked immunospot (ELISPOT assay by using isolated CD8(+ and CD4(+ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1-specific IFN-γ producing CD4(+ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+ T cells was significantly correlated with that of CD8(+ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001. To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+ T cell responses showed more significant changes than CD8(+ T cell responses. CD8(+ and CD4(+ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4 and Th17 (IL-17a cytokines were not detected. CD4(+ T cells secreted significantly higher cytokine levels than did CD8(+ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.

  7. Tracking of peptide-specific CD4+ T-cell responses after an acute resolving viral infection: a study of parvovirus B19

    DEFF Research Database (Denmark)

    Kasprowicz, Victoria; Isa, Adiba; Tolfvenstam, Thomas

    2006-01-01

    The evolution of peptide-specific CD4(+) T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4(+) T......-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared...... to those for a cohort of B19-specific immunoglobulin M-negative (IgM(-)), IgG(+) remotely infected individuals. Both cohorts of individuals were found to make broad CD4(+) responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals...

  8. HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

    Science.gov (United States)

    Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D; Ndung'u, Thumbi; Ndhlovu, Zaza M

    2017-04-01

    Immune control of viral infections is heavily dependent on helper CD4 + T cell function. However, the understanding of the contribution of HIV-specific CD4 + T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4 + T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4 + T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4 + T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4 + T cells in HIV controllers than progressors ( P = 0.0001), and these expanded Gag-specific CD4 + T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control ( r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4 + T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4 + T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4 + T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

  9. CXCR3 Directs Antigen-Specific Effector CD4+ T Cell Migration to the Lung During Parainfluenza Virus Infection

    DEFF Research Database (Denmark)

    Kohlmeier, Jacob E; Cookenham, Tres; Miller, Shannon C

    2009-01-01

    effector CD4(+) T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4......(+) T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4(+) T cell proliferation, phenotype......, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4(+) T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections....

  10. Transcriptome Analysis of Mycobacteria-Specific CD4+ T Cells Identified by Activation-Induced Expression of CD154.

    Science.gov (United States)

    Kunnath-Velayudhan, Shajo; Goldberg, Michael F; Saini, Neeraj K; Johndrow, Christopher T; Ng, Tony W; Johnson, Alison J; Xu, Jiayong; Chan, John; Jacobs, William R; Porcelli, Steven A

    2017-10-01

    Analysis of Ag-specific CD4 + T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4 + T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4 + T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4 + T cells from bacillus Calmette-Guérin-vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154 + cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4 + CD154 + cells was distinct from that of CD154 - cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4 + T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4 + T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts. Copyright © 2017 by The American Association of Immunologists, Inc.

  11. Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

    Science.gov (United States)

    Gardyan, Adriane; Osen, Wolfram; Zörnig, Inka; Podola, Lilli; Agarwal, Maria; Aulmann, Sebastian; Ruggiero, Eliana; Schmidt, Manfred; Halama, Niels; Leuchs, Barbara; von Kalle, Christof; Beckhove, Philipp; Schneeweiss, Andreas; Jäger, Dirk; Eichmüller, Stefan B

    2015-06-01

    Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer-related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4(+) effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4(+) T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4(+) T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4(+) T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4(+) T cells specific for all four CD4(+) T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4(+) T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific HLA-DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer. © 2014 UICC.

  12. CD4+ T-cell Responses Among Adults and Young Children In Response to Streptococcus pneumoniae and Haemophilus influenzae Vaccine Candidate Protein Antigens

    OpenAIRE

    Sharma, Sharad K.; Roumanes, David; Almudevar, Anthony; Mosmann, Tim R.; Pichichero, Michael E.

    2013-01-01

    We characterized cytokine profiles of CD4+ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and Protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 - and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4+ T cells producing...

  13. Specific interaction of CXCR4 with CD4 and CD8α: Functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion

    International Nuclear Information System (INIS)

    Basmaciogullari, Stephane; Pacheco, Beatriz; Bour, Stephan; Sodroski, Joseph

    2006-01-01

    We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8α in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8α/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8α molecules

  14. Applying machine learning to predict patient-specific current CD4 ...

    African Journals Online (AJOL)

    Apple apple

    This work shows the application of machine learning to predict current CD4 cell count of an HIV- .... Pre-processing ... remaining data elements of the PR and RT datasets. ... technique based on the structure of the human brain's neuron.

  15. Specific immunotherapy modifies allergen-specific CD4+ T cell responses in an epitope-dependent manner

    Science.gov (United States)

    Wambre, Erik; DeLong, Jonathan H.; James, Eddie A.; Torres-Chinn, Nadia; Pfützner, Wolfgang; Möbs, Christian; Durham, Stephen R.; Till, Stephen J.; Robinson, David; Kwok, William W.

    2014-01-01

    Background Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T cell epitope level is critical for the design of new allergy vaccine strategies. Objective To characterize allergen-specific T cell responses linked with allergy or peripheral tolerance and to determine how CD4+ T cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy (ASIT). Methods Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy and phenotype of the DR04:01-restricted TGP-specific T cell responses in ten grass pollen allergic, five non-atopic and six allergy vaccine-treated individuals was determined using an ex vivo pMHCII-tetramer approach. Results CD4+ T cells in allergic individuals are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. ASIT was associated with preferential deletion of allergen-specific TH2 cells and without significant change in frequency of TH1/TR1 cells. Conclusions Preferential allergen-specific TH2-cells deletion after repeated high doses antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy. PMID:24373351

  16. IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity.

    Science.gov (United States)

    Tran, Giang T; Hodgkinson, Suzanne J; Carter, Nicole M; Verma, Nirupama D; Plain, Karren M; Boyd, Rochelle; Robinson, Catherine M; Nomura, Masaru; Killingsworth, Murray; Hall, Bruce M

    2012-05-10

    Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.

  17. The immune checkpoint regulator PD-L1 is a specific target for naturally occurring CD4(+) T cells

    DEFF Research Database (Denmark)

    Munir, Shamaila; Andersen, Gitte Holmen; Svane, Inge Marie

    2013-01-01

    Programmed cell death 1 ligand 1 (PD-L1) is an important regulator of T-cell responses and may consequently limit anticancer immunity. We have recently identified PD-L1-specific, cytotoxic CD8(+) T cells. In the present study, we develop these findings and report that CD4(+) helper T cells...... spontaneously recognize PD-L1. We examined the locality of a previously identified HLA-A*0201-restricted PD-L1-epitope for the presence of possible CD4(+) T-cell epitopes. Thus, we identified naturally occurring PD-L1-specific CD4(+) T cells among the peripheral blood lymphocytes of cancer patients...... and - to lesser extents - healthy donors, by means of ELISPOT assays. PD-L1-specific CD4(+) T cells appeared to be TH17 cells exhibiting an effector T-cell cytokine profile. Hence, PD-L1-specific CD4(+) T cells released interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-17 (IL-17) in response...

  18. Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

    International Nuclear Information System (INIS)

    Rojas, Olga Lucia; Gonzalez, Ana Maria; Gonzalez, Rosabel; Perez-Schael, Irene; Greenberg, Harry B.; Franco, Manuel A.; Angel, Juana

    2003-01-01

    Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4 + and CD8 + T cells secreting INFγ, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFγ-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4 + and CD8 + T cell subsets, IFNγ-secreting RV-specific CD8 + but not CD4 + T cells were detected in recently infected children. Using the same approach, both CD4 + and CD8 + RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFγ-secreting CD4 + T cells from adult volunteers preferentially express the intestinal homing receptor α4β7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFγ-secreting CD4 + T cells preferentially express L-selectin but not α4β7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFγ is independent of the expression of these homing receptors

  19. Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

    Directory of Open Access Journals (Sweden)

    Katarzyna Wachowicz

    Full Text Available Protein kinase C θ (PKCθ is involved in signaling downstream of the T cell antigen receptor (TCR and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures and in vivo (experimental autoimmune encephalomyelitis model, EAE techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt, accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ-/- CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.

  20. A novel SIV gag-specific CD4(+)T-cell clone suppresses SIVmac239 replication in CD4(+)T cells revealing the interplay between antiviral effector cells and their infected targets.

    Science.gov (United States)

    Ayala, Victor I; Trivett, Matthew T; Coren, Lori V; Jain, Sumiti; Bohn, Patrick S; Wiseman, Roger W; O'Connor, David H; Ohlen, Claes; Ott, David E

    2016-06-01

    To study CD4(+)T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4(+)T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8(+)T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4(+)T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4(+)effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4(+)T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4(+)T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4(+)T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Detailed analysis of Epstein–Barr virus-specific CD4+ and CD8+ T cell responses during infectious mononucleosis

    Science.gov (United States)

    Scherrenburg, J; Piriou, E R W A N; Nanlohy, N M; van Baarle, D

    2008-01-01

    We studied simultaneously Epstein–Barr virus (EBV)-specific CD4+ and CD8+ T cell responses during and after infectious mononucleosis (IM), using a previously described 12-day stimulation protocol with EBNA1 or BZLF1 peptide pools. Effector function of EBV-specific T cells was determined after restimulation by measuring intracellular interferon-γ production. During IM, BZLF1-specifc CD4+ T cell responses were dominant compared with CD8+ T cell responses. EBNA1-specific CD4+ and CD8+ T cell responses were low and remained similar for 6 months. However, 6 months after IM, BZLF1-specific CD4+ T cell responses had declined, but CD8+ T cell responses had increased. At diagnosis, EBV-specific CD8+ T cells as studied by human leucocyte antigen class I tetramer staining comprised a tetramerbrightCD8bright population consisting mainly of CD27+ memory T cells and a tetramerdimCD8dim population consisting primarily of CD27- effector T cells. The remaining EBV-specific CD8+ T cell population 6 months after the diagnosis of IM consisted mainly of tetramerbrightCD8bright CD27+ T cells, suggesting preferential preservation of memory T cells after contraction of the EBV-specific T cell pool. PMID:18549439

  2. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

    OpenAIRE

    Day, Cheryl L.; Seth, Nilufer P.; Lucas, Michaela; Appel, Heiner; Gauthier, Laurent; Lauer, Georg M.; Robbins, Gregory K.; Szczepiorkowski, Zbigniew M.; Casson, Deborah R.; Chung, Raymond T.; Bell, Shannon; Harcourt, Gillian; Walker, Bruce D.; Klenerman, Paul; Wucherpfennig, Kai W.

    2003-01-01

    Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a c...

  3. Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro.

    Science.gov (United States)

    Jackson, Sarah E; Sedikides, George X; Mason, Gavin M; Okecha, Georgina; Wills, Mark R

    2017-03-15

    Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8 + T cell response to HCMV has been extensively studied, the HCMV-specific CD4 + T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-γ) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4 + T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-γ response was still dominant. CD4 + T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1β secretion) effector responses. Importantly, when we measured the CD4 + T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro , we observed that the CD4 + T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4 + T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4 + T cell responses, even those from elderly individuals, are highly functional and are directly antiviral. IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated

  4. Phenotypic analysis of perennial airborne allergen-specific CD4+ T cells in atopic and non-atopic individuals.

    Science.gov (United States)

    Crack, L R; Chan, H W; McPherson, T; Ogg, G S

    2011-11-01

    Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. © 2011 Blackwell Publishing Ltd.

  5. Human cerebrospinal fluid contains CD4+ memory T cells expressing gut- or skin-specific trafficking determinants: relevance for immunotherapy

    Directory of Open Access Journals (Sweden)

    Campbell James J

    2006-07-01

    Full Text Available Abstract Background Circulating memory T cells can be divided into tissue-specific subsets, which traffic through distinct tissue compartments during physiologic immune surveillance, based on their expression of adhesion molecules and chemokine receptors. We reasoned that a bias (either enrichment or depletion of CSF T cell expression of known organ-specific trafficking determinants might suggest that homing of T cells to the subarachnoid space could be governed by a CNS-specific adhesion molecule or chemokine receptor. Results The expression of cutaneous leukocyte antigen (CLA and CC-chemokine receptor 4 (CCR4; associated with skin-homing as well as the expression of integrin α4β7 and CCR9 (associated with gut-homing was analyzed on CD4+ memory T cells in CSF from individuals with non-inflammatory neurological diseases using flow cytometry. CSF contained similar proportions of CD4+ memory T cells expressing CLA, CCR4, integrin α4β7 and CCR9 as paired blood samples. Conclusion The results extend our previous findings that antigen-experienced CD4+ memory T cells traffic through the CSF in proportion to their abundance in the peripheral circulation. Furthermore, the ready access of skin- and gut-homing CD4+ memory T cells to the CNS compartment via CSF has implications for the mechanisms of action of immunotherapeutic strategies, such as oral tolerance or therapeutic immunization, where immunogens are administered using an oral or subcutaneous route.

  6. Profiling MHC II immunopeptidome of blood-stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells.

    Science.gov (United States)

    Draheim, Marion; Wlodarczyk, Myriam F; Crozat, Karine; Saliou, Jean-Michel; Alayi, Tchilabalo Dilezitoko; Tomavo, Stanislas; Hassan, Ali; Salvioni, Anna; Demarta-Gatsi, Claudia; Sidney, John; Sette, Alessandro; Dalod, Marc; Berry, Antoine; Silvie, Olivier; Blanchard, Nicolas

    2017-11-01

    In malaria, CD4 Th1 and T follicular helper (T FH ) cells are important for controlling parasite growth, but Th1 cells also contribute to immunopathology. Moreover, various regulatory CD4 T-cell subsets are critical to hamper pathology. Yet the antigen-presenting cells controlling Th functionality, as well as the antigens recognized by CD4 T cells, are largely unknown. Here, we characterize the MHC II immunopeptidome presented by DC during blood-stage malaria in mice. We establish the immunodominance hierarchy of 14 MHC II ligands derived from conserved parasite proteins. Immunodominance is shaped differently whether blood stage is preceded or not by liver stage, but the same ETRAMP-specific dominant response develops in both contexts. In naïve mice and at the onset of cerebral malaria, CD8α + dendritic cells (cDC1) are superior to other DC subsets for MHC II presentation of the ETRAMP epitope. Using in vivo depletion of cDC1, we show that cDC1 promote parasite-specific Th1 cells and inhibit the development of IL-10 + CD4 T cells. This work profiles the P. berghei blood-stage MHC II immunopeptidome, highlights the potency of cDC1 to present malaria antigens on MHC II, and reveals a major role for cDC1 in regulating malaria-specific CD4 T-cell responses. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  7. Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 onto plates coated with anti-CD4bs antibodies b12, b6 or the CD4 receptor mimetic, CD4-IgG2...

  8. A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Therapy

    NARCIS (Netherlands)

    DeMaster, Laura K.; Liu, Xiaohe; VanBelzen, D. Jake; Trinité, Benjamin; Zheng, Lingjie; Agosto, Luis M.; Migueles, Stephen A.; Connors, Mark; Sambucetti, Lidia; Levy, David N.; Pasternak, Alexander O.; O'Doherty, Una

    2016-01-01

    A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4(+) T cells that express no viral proteins.

  9. Prion protein-deficient mice exhibit decreased CD4 T and LTi cell numbers and impaired spleen structure.

    Science.gov (United States)

    Kim, Soochan; Han, Sinsuk; Lee, Ye Eun; Jung, Woong-Jae; Lee, Hyung Soo; Kim, Yong-Sun; Choi, Eun-Kyoung; Kim, Mi-Yeon

    2016-01-01

    The cellular prion protein is expressed in almost all tissues, including the central nervous system and lymphoid tissues. To investigate the effects of the prion protein in lymphoid cells and spleen structure formation, we used prion protein-deficient (Prnp(0/0)) Zürich I mice generated by inactivation of the Prnp gene. Prnp(0/0) mice had decreased lymphocytes, in particular, CD4 T cells and lymphoid tissue inducer (LTi) cells. Decreased CD4 T cells resulted from impaired expression of CCL19 and CCL21 in the spleen rather than altered chemokine receptor CCR7 expression. Importantly, some of the white pulp regions in spleens from Prnp(0/0) mice displayed impaired T zone structure as a result of decreased LTi cell numbers and altered expression of the lymphoid tissue-organizing genes lymphotoxin-α and CXCR5, although expression of the lymphatic marker podoplanin and CXCL13 by stromal cells was not affected. In addition, CD3(-)CD4(+)IL-7Rα(+) LTi cells were rarely detected in impaired white pulp in spleens of these mice. These data suggest that the prion protein is required to form the splenic white pulp structure and for development of normal levels of CD4 T and LTi cells. Copyright © 2015. Published by Elsevier GmbH.

  10. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells

    Science.gov (United States)

    Hepworth, Matthew R.; Fung, Thomas C.; Masur, Samuel H.; Kelsen, Judith R.; McConnell, Fiona M.; Dubrot, Juan; Withers, David R.; Hugues, Stephanie; Farrar, Michael A.; Reith, Walter; Eberl, Gerard; Baldassano, Robert N.; Laufer, Terri M.; Elson, Charles O.; Sonnenberg, Gregory F.

    2015-01-01

    Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. While selection of self-specific T cells in the thymus limits responses to tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells, and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on human colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4+ T cells in the intestine, and suggest that this process is dysregulated in human IBD. PMID:25908663

  11. Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.

    Directory of Open Access Journals (Sweden)

    Monique van Velzen

    2013-08-01

    Full Text Available Herpes simplex virus type 1 (HSV-1 infection results in lifelong chronic infection of trigeminal ganglion (TG neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

  12. Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1 and IE2.

    Science.gov (United States)

    Braendstrup, Peter; Mortensen, Bo Kok; Justesen, Sune; Osterby, Thomas; Rasmussen, Michael; Hansen, Andreas Martin; Christiansen, Claus Bohn; Hansen, Morten Bagge; Nielsen, Morten; Vindeløv, Lars; Buus, Søren; Stryhn, Anette

    2014-01-01

    Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.

  13. Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1 and IE2.

    Directory of Open Access Journals (Sweden)

    Peter Braendstrup

    Full Text Available Human cytomegalovirus (HCMV is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2. Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.

  14. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Craig B Wilen

    2011-04-01

    Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

  15. The Development of Plasmodium falciparum-Specific IL10 CD4 T Cells and Protection from Malaria in Children in an Area of High Malaria Transmission.

    Science.gov (United States)

    Boyle, Michelle J; Jagannathan, Prasanna; Bowen, Katherine; McIntyre, Tara I; Vance, Hilary M; Farrington, Lila A; Schwartz, Alanna; Nankya, Felistas; Naluwu, Kate; Wamala, Samuel; Sikyomu, Esther; Rek, John; Greenhouse, Bryan; Arinaitwe, Emmanuel; Dorsey, Grant; Kamya, Moses R; Feeney, Margaret E

    2017-01-01

    Cytokine-producing CD4 T cells have important roles in immunity against Plasmodium falciparum (Pf) malaria. However, the factors influencing functional differentiation of Pf- specific CD4 T cells in naturally exposed children are not well understood. Moreover, it is not known which CD4 T-cell cytokine-producing subsets are most critical for protection. We measured Pf- specific IFNγ-, IL10-, and TNFα-producing CD4 T-cell responses by multi-parametric flow cytometry in 265 children aged 6 months to 10 years enrolled in a longitudinal observational cohort in a high malaria transmission site in Uganda. We found that both age and parasite burden were independently associated with cytokine production by CD4 T cells. IL10 production by IFNγ + CD4 T cells was higher in younger children and in those with high-parasite burden during recent infection. To investigate the role of CD4 T cells in immunity to malaria, we measured associations of Pf -specific CD4 cytokine-producing cells with the prospective risk of Pf infection and clinical malaria, adjusting for household exposure to Pf -infected mosquitos. Overall, the prospective risk of infection was not associated with the total frequency of Pf- specific CD4 T cells, nor of any cytokine-producing CD4 subset. However, the frequency of CD4 cells producing IL10 but not inflammatory cytokines (IFNγ and TNFα) was associated with a decreased risk of clinical malaria once infected. These data suggest that functional polarization of the CD4 T-cell response may modulate the clinical manifestations of malaria and play a role in naturally acquired immunity.

  16. Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

    Science.gov (United States)

    Mascarell, Laurent; Lombardi, Vincent; Louise, Anne; Saint-Lu, Nathalie; Chabre, Henri; Moussu, Hélène; Betbeder, Didier; Balazuc, Anne-Marie; Van Overtvelt, Laurence; Moingeon, Philippe

    2008-09-01

    A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

  17. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    International Nuclear Information System (INIS)

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

    2004-01-01

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

  18. Identification of Rotavirus VP6-Specific CD4+ T Cell Epitopes in a G1P[8] Human Rotavirus-Infected Rhesus Macaque

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    2008-01-01

    Full Text Available A non-human primate model was used to evaluate its potential for identification of rotavirus viral protein 6 (VP6 CD4+ T cell epitopes. Four juvenile rhesus macaques were inoculated with a mixed inoculum (G1P[8] and G9P[8] of human rotaviruses. Infection accompanied by G1P[8] shedding was achieved in the two macaques that had no rotavirus immunoglobulin A (IgA in plasma. To measure the interferon gamma (IFN-γ and tumor necrosis factor (TNF anti-viral cytokines produced by peripheral CD4+ cells that recognize VP6 epitopes, whole blood cells from one infected macaque were stimulated in vitro with VP6 peptides. Stimulation with peptide pools derived from the simian rotavirus VP6 161–395 region revealed reactivity of CD4+ T cells with the VP6 281–331 domain. A VP6 301–315 region was identified as the epitope responsible for IFN-γ production while a broader VP6 293–327 domain was linked to TNF production. These results suggest that human rotavirus-infected macaques can be used for identification of additional epitopes and domains to address specific questions related to the development of pediatric vaccines.

  19. HLA Class II Defects in Burkitt Lymphoma: Bryostatin-1-Induced 17 kDa Protein Restores CD4+ T-Cell Recognition

    Directory of Open Access Journals (Sweden)

    Azim Hossain

    2011-01-01

    Full Text Available While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.

  20. Inhibitory phenotype of HBV-specific CD4+ T-cells is characterized by high PD-1 expression but absent coregulation of multiple inhibitory molecules.

    Directory of Open Access Journals (Sweden)

    Bijan Raziorrouh

    Full Text Available T-cell exhaustion seems to play a critical role in CD8+ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4+ T-cell dysfunction during chronic hepatitis B virus (CHB infection and the role of inhibitory molecules such as programmed death 1 (PD-1 for CD4+ T-cell failure.The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4+ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4+ T-cell proliferation and cytokine production.CD4+ T-cell responses during chronic HBV infection was characterized by reduced Tetramer+CD4+ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4+ T-cell expansion almost in treated patients with viral control.HBV-specific CD4+ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4+ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4+ T-cell functionality with heterogeneous patterns of CD4+ T-cell rejunivation.

  1. Characterisation of the Immunomodulatory Effects of Meningococcal Opa Proteins on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells.

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    Claire Jones

    Full Text Available Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins.

  2. HBV-specific CD4+ cytotoxic T cells in hepatocellular carcinoma are less cytolytic toward tumor cells and suppress CD8+ T cell-mediated antitumor immunity.

    Science.gov (United States)

    Meng, Fanzhi; Zhen, Shoumei; Song, Bin

    2017-08-01

    In East Asia and sub-Saharan Africa, chronic infection is the main cause of the development of hepatocellular carcinoma, an aggressive cancer with low survival rate. Cytotoxic T cell-based immunotherapy is a promising treatment strategy. Here, we investigated the possibility of using HBV-specific CD4 + cytotoxic T cells to eliminate tumor cells. The naturally occurring HBV-specific cytotoxic CD4 + and CD8 + T cells were identified by HBV peptide pool stimulation. We found that in HBV-induced hepatocellular carcinoma patients, the HBV-specific cytotoxic CD4 + T cells and cytotoxic CD8 + T cells were present at similar numbers. But compared to the CD8 + cytotoxic T cells, the CD4 + cytotoxic T cells secreted less cytolytic factors granzyme A (GzmA) and granzyme B (GzmB), and were less effective at eliminating tumor cells. In addition, despite being able to secrete cytolytic factors, CD4 + T cells suppressed the cytotoxicity mediated by CD8 + T cells, even when CD4 + CD25 + regulator T cells were absent. Interestingly, we found that interleukin 10 (IL-10)-secreting Tr1 cells were enriched in the cytotoxic CD4 + T cells. Neutralization of IL-10 abrogated the suppression of CD8 + T cells by CD4 + CD25 - T cells. Neither the frequency nor the absolute number of HBV-specific CD4 + cytotoxic T cells were correlated with the clinical outcome of advanced stage hepatocellular carcinoma patients. Together, this study demonstrated that in HBV-related hepatocellular carcinoma, CD4 + T cell-mediated cytotoxicity was present naturally in the host and had the potential to exert antitumor immunity, but its capacity was limited and was associated with immunoregulatory properties. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  3. Systematic Protein-Protein Docking and Molecular Dynamics Studies of HIV-1 gp120 and CD4: Insights for New Drug Development

    Directory of Open Access Journals (Sweden)

    M. Rizman-Idid

    2011-12-01

    Full Text Available Background and the purpose of the study: The interactions between HIV-1 gp120 and mutated CD4 proteins were investigated in order to identify a lead structure for therapy based on competitive blocking of the HIV binding receptor for human T-cells. Crystal structures of HIV gp120-CD4 complexes reveal a close interaction of the virus receptor with CD4 Phe43, which is embedded in a pocket of the virus protein.Methods: This study applies computer simulations to determine the best binding of amino acid 43 CD4 mutants to HIV gp120. Besides natural CD4, three mutants carrying alternate aromatic residues His, Trp and Tyr at position 43 were investigated. Several docking programs were applied on isolated proteins based on selected crystal structures of gp120-CD4 complexes, as well as a 5 ns molecular dynamics study on the protein complexes. The initial structures were minimized in Gromacs to avoid crystal packing effects, and then subjected to docking experiments using AutoDock4, FireDock, ClusPro and ZDock. In molecular dynamics, the Gibbs free binding energy was calculated for the gp120-CD4 complexes. The docking outputs were analyzed on energy within the respective docking software.Results and conclusion: Visualization and hydrogen bonding analysis were performed using the Swiss-PdbViewer. Strong binding to HIV gp120 can be achieved with an extended aromatic group (Trp. However, the sterical demand of the interaction affects the binding kinetics. In conclusion, a ligand for an efficient blocking of HIV gp120 should involve an extended but conformational flexible aromatic group, i.e. a biphenyl. A docking study on biphenylalanine-43 confirms this expectation

  4. CD4 T Cell Epitope Specificity and Cytokine Potential Are Preserved as Cells Transition from the Lung Vasculature to Lung Tissue following Influenza Virus Infection.

    Science.gov (United States)

    DiPiazza, Anthony; Laniewski, Nathan; Rattan, Ajitanuj; Topham, David J; Miller, Jim; Sant, Andrea J

    2018-07-01

    Pulmonary CD4 T cells are critical in respiratory virus control, both by delivering direct effector function and through coordinating responses of other immune cells. Recent studies have shown that following influenza virus infection, virus-specific CD4 T cells are partitioned between pulmonary vasculature and lung tissue. However, very little is known about the peptide specificity or functional differences of CD4 T cells within these two compartments. Using a mouse model of influenza virus infection in conjunction with intravascular labeling in vivo , the cell surface phenotype, epitope specificity, and functional potential of the endogenous polyclonal CD4 T cell response was examined by tracking nine independent CD4 T cell epitope specificities. These studies revealed that tissue-localized CD4 cells were globally distinct from vascular cells in expression of markers associated with transendothelial migration, residency, and micropositioning. Despite these differences, there was little evidence for remodeling of the viral epitope specificity or cytokine potential as cells transition from vasculature to the highly inflamed lung tissue. Our studies also distinguished cells in the pulmonary vasculature from peripheral circulating CD4 T cells, providing support for the concept that the pulmonary vasculature does not simply reflect circulating cells that are trapped within the narrow confines of capillary vessels but rather is enriched in transitional cells primed in the draining lymph node that have specialized potential to enter the lung tissue. IMPORTANCE CD4 T cells convey a multitude of functions in immunity to influenza, including those delivered in the lymph node and others conveyed by CD4 T cells that leave the lymph node, enter the blood, and extravasate into the lung tissue. Here, we show that the transition of recently primed CD4 cells detected in the lung vasculature undergo profound changes in expression of markers associated with tissue localization as

  5. HIV-1 Infection Is Associated with Depletion and Functional Impairment of Mycobacterium tuberculosis-Specific CD4 T Cells in Individuals with Latent Tuberculosis Infection.

    Science.gov (United States)

    Day, Cheryl L; Abrahams, Deborah A; Harris, Levelle D; van Rooyen, Michele; Stone, Lynnett; de Kock, Marwou; Hanekom, Willem A

    2017-09-15

    Coinfection with HIV is the single greatest risk factor for reactivation of latent Mycobacterium tuberculosis infection (LTBI) and progression to active tuberculosis disease. HIV-associated dysregulation of adaptive immunity by depletion of CD4 Th cells most likely contributes to loss of immune control of LTBI in HIV-infected individuals, although the precise mechanisms whereby HIV infection impedes successful T cell-mediated control of M. tuberculosis have not been well defined. To further delineate mechanisms whereby HIV impairs protective immunity to M. tuberculosis , we evaluated the frequency, phenotype, and functional capacity of M. tuberculosis -specific CD4 T cells in HIV-infected and HIV-uninfected adults with LTBI. HIV infection was associated with a lower total frequency of cytokine-producing M. tuberculosis -specific CD4 T cells, and preferential depletion of a discrete subset of M. tuberculosis -specific IFN-γ + IL-2 - TNF-α + CD4 T cells. M. tuberculosis -specific CD4 T cells in HIV-infected individuals expressed significantly higher levels of Ki67, compared with HIV-uninfected individuals, thus indicating recent activation and turnover of these cells in vivo. The ex vivo proliferative capacity of M. tuberculosis -specific CD4 T cells was markedly impaired in HIV-infected individuals, compared with HIV-uninfected individuals. Moreover, HIV infection was associated with increased M. tuberculosis Ag-induced CD4 T cell death ex vivo, indicating a possible mechanism contributing to impaired proliferative capacity of M. tuberculosis -specific CD4 T cells in HIV-infected individuals. These data provide new insights into the parameters of M. tuberculosis -specific CD4 T cell immunity that are impaired in HIV-infected individuals with LTBI, which may contribute to their increased risk of developing active tuberculosis disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  6. Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

    Science.gov (United States)

    Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Schimmer, Simone; Brandau, Sven; Altenhoff, Petra; Sparwasser, Tim; Dittmer, Ulf

    2013-02-01

    The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

  7. Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Halawani Dalia

    2007-10-01

    Full Text Available Abstract Background HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER-associated protein degradation (ERAD. Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub-proteasome degradative system through an interaction with β-TrCP, a component of the SCFβ-TrCP E3 Ub ligase complex. Results Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFβ-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. Conclusion Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFβ-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

  8. The ThPOK transcription factor differentially affects the development and function of self-specific CD8(+) T cells and regulatory CD4(+) T cells.

    Science.gov (United States)

    Twu, Yuh-Ching; Teh, Hung-Sia

    2014-03-01

    The zinc finger transcription factor ThPOK plays a crucial role in CD4 T-cell development and CD4/CD8 lineage decision. In ThPOK-deficient mice, developing T cells expressing MHC class II-restricted T-cell receptors are redirected into the CD8 T-cell lineage. In this study, we investigated whether the ThPOK transgene affected the development and function of two additional types of T cells, namely self-specific CD8 T cells and CD4(+) FoxP3(+) T regulatory cells. Self-specific CD8 T cells are characterized by high expression of CD44, CD122, Ly6C, 1B11 and proliferation in response to either IL-2 or IL-15. The ThPOK transgene converted these self-specific CD8 T cells into CD4 T cells. The converted CD4(+) T cells are no longer self-reactive, lose the characteristics of self-specific CD8 T cells, acquire the properties of conventional CD4 T cells and survive poorly in peripheral lymphoid organs. By contrast, the ThPOK transgene promoted the development of CD4(+) FoxP3(+) regulatory T cells resulting in an increased recovery of CD4(+) FoxP3(+) regulatory T cells that expressed higher transforming growth factor-β-dependent suppressor activity. These studies indicate that the ThPOK transcription factor differentially affects the development and function of self-specific CD8 T cells and CD4(+) FoxP3(+) regulatory T cells. © 2013 John Wiley & Sons Ltd.

  9. In-Depth Analysis of Citrulline-Specific CD4 T-Cells in Rheumatoid Arthritis

    Science.gov (United States)

    2018-01-01

    player in the activation of lymphoid , myeloid and mast cells , indicating MALT1’s crucial role in innate and adaptive signaling. Therefore, MALT1 is...for RA (IFRA) Program Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell ...Program Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell regulation in RA

  10. In Depth Analysis of Citrulline Specific CD4 T Cells in Rheumatoid Arthritis

    Science.gov (United States)

    2018-01-01

    activation of lymphoid , myeloid and mast cells , indicating MALT1’s crucial role in innate and adaptive signaling. Therefore, MALT1 is regarded a...Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell regulation in RA Jane Buckner...IFRA) Program Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell regulation in

  11. Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

    International Nuclear Information System (INIS)

    Morrow, Peter W.; Tung, H.Y. Lim; Hemmings, Hugh C.

    2004-01-01

    Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A 1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD 4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA

  12. HIV-1 Infection of Primary CD4+ T Cells Regulates the Expression of Specific Human Endogenous Retrovirus HERV-K (HML-2) Elements.

    Science.gov (United States)

    Young, George R; Terry, Sandra N; Manganaro, Lara; Cuesta-Dominguez, Alvaro; Deikus, Gintaras; Bernal-Rubio, Dabeiba; Campisi, Laura; Fernandez-Sesma, Ana; Sebra, Robert; Simon, Viviana; Mulder, Lubbertus C F

    2018-01-01

    Endogenous retroviruses (ERVs) occupy extensive regions of the human genome. Although many of these retroviral elements have lost their ability to replicate, those whose insertion took place more recently, such as the HML-2 group of HERV-K elements, still retain intact open reading frames and the capacity to produce certain viral RNA and/or proteins. Transcription of these ERVs is, however, tightly regulated by dedicated epigenetic control mechanisms. Nonetheless, it has been reported that some pathological states, such as viral infections and certain cancers, coincide with ERV expression, suggesting that transcriptional reawakening is possible. HML-2 elements are reportedly induced during HIV-1 infection, but the conserved nature of these elements has, until recently, rendered their expression profiling problematic. Here, we provide comprehensive HERV-K HML-2 expression profiles specific for productively HIV-1-infected primary human CD4 + T cells. We combined enrichment of HIV-1 infected cells using a reporter virus expressing a surface reporter for gentle and efficient purification with long-read single-molecule real-time sequencing. We show that three HML-2 proviruses-6q25.1, 8q24.3, and 19q13.42-are upregulated on average between 3- and 5-fold in HIV-1-infected CD4 + T cells. One provirus, HML-2 12q24.33, in contrast, was repressed in the presence of active HIV replication. In conclusion, this report identifies the HERV-K HML-2 loci whose expression profiles differ upon HIV-1 infection in primary human CD4 + T cells. These data will help pave the way for further studies on the influence of endogenous retroviruses on HIV-1 replication. IMPORTANCE Endogenous retroviruses inhabit big portions of our genome. Moreover, although they are mainly inert, some of the evolutionarily younger members maintain the ability to express both RNA and proteins. We have developed an approach using long-read single-molecule real-time (SMRT) sequencing that produces long reads that

  13. Green tea epigallocatechin-3-gallate modulates differentiation of naive CD4+ T cells into specific lineage effector cells

    Science.gov (United States)

    CD4+ T helper (Th) subsets Th1, Th9, and Th17 cells are implicated in inducing autoimmunity whereas regulatory T cells (Treg) have a protective effect. We previously showed that epigallocatechin-3-gallate (EGCG) attenuated experimental autoimmune encephalomyelitis (EAE) and altered CD4+ T cell subpo...

  14. In vitro induction of functional allergen-specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity.

    Science.gov (United States)

    Hamza, E; Akdis, C A; Wagner, B; Steinbach, F; Marti, E

    2013-08-01

    Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or tetanus toxoid as control antigen, in the presence of CD4(+) CD25(high) cells. Furthermore, Culicoides-specific CD4(+) CD25(high) regulatory cells were expanded or induced from CD4(+) CD25(-) cells in vitro in the presence of a combination of rIL-2 and rTGF-β1 (rIL-2/rTGF-β1) or of retinoic acid and rapamycin (RetA/Rapa). Proliferation was determined by [(3) H] thymidine incorporation and cytokine production measured by flow cytometry. The ability of Culicoides- but not tetanus-stimulated CD4(+) CD25(high) cells to suppress proliferation of CD4(+) CD25(-) cells was significantly lower in IBH-affected horses (28%) than in healthy controls (86%). The decreased suppression in IBH-affected horses was associated with a significantly higher proportion of IL-4(+) cells and a lower percentage of FoxP3(+) IL-10(+) compared to controls. Addition of rIL-2/rTGF-β1 or of RetA/Rapa to Culicoides-stimulated CD4(+) CD25(high) cells from IBH-affected horses significantly increased the proportion of FoxP3(+) IL-10(+) cells. We also found that RetA/Rapa induced a more significant decrease in the frequency of IL-4(+) cells than rIL-2/rTGF-β1. Moreover, the suppressive activity of Culicoides-stimulated CD4(+) CD25(high) cells was significantly restored by both rIL-2/rTGF-β1and RetA/Rapa, albeit in an antigen-unspecific manner. In contrast, in vitro induced Culicoides-specific CD4(+) CD25(high) cells suppressed

  15. Tuberculosis Therapy Modifies the Cytokine Profile, Maturation State, and Expression of Inhibitory Molecules on Mycobacterium tuberculosis-Specific CD4+ T-Cells.

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    Kapil K Saharia

    Full Text Available Little is known about the expression of inhibitory molecules cytotoxic T-lymphocyte antigen-4 (CTLA-4 and programmed-death-1 (PD-1 on Mycobacterium tuberculosis (Mtb-specific CD4 T-cells and how their expression is impacted by TB treatment.Cryopreserved PBMCs from HIV-TB co-infected and TB mono-infected patients with untreated and treated tuberculosis (TB disease were stimulated for six hours with PPD and stained. Using polychromatic flow cytometry, we characterized the differentiation state, cytokine profile, and inhibitory molecule expression on PPD-specific CD4 T-cells.In our HIV-TB co-infected cohort, TB treatment increased the proportion of PPD-specific CD4 T-cells co-producing IFN-γ+IL-2+TNF-α+ and IFN-γ+IL-2+ (p = 0.0004 and p = 0.0002, respectively while decreasing the proportion of PPD-specific CD4 T-cells co-producing IFN-γ+MIP1-β+TNF-α+ and IFN-γ+MIP1-β+. The proportion of PPD-specific CD4 T-cells expressing an effector memory phenotype decreased (63.6% vs 51.6%, p = 0.0015 while the proportion expressing a central memory phenotype increased (7.8% vs. 21.7%, p = 0.001 following TB treatment. TB treatment reduced the proportion of PPD-specific CD4 T-cells expressing CTLA-4 (72.4% vs. 44.3%, p = 0.0005 and PD-1 (34.5% vs. 29.2%, p = 0.03. Similar trends were noted in our TB mono-infected cohort.TB treatment alters the functional profile of Mtb-specific CD4 T-cells reflecting shifts towards a less differentiated maturational profile and decreases PD-1 and CTLA-4 expression. These could serve as markers of reduced mycobacterial burden. Further study is warranted.

  16. Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium

    Science.gov (United States)

    Begum, Jusnara; Lal, Neeraj; Zuo, Jianmin; Beggs, Andrew; Moss, Paul

    2016-01-01

    Cytomegalovirus (CMV) infection elicits a very strong and sustained intravascular T cell immune response which may contribute towards development of accelerated immune senescence and vascular disease in older people. Virus-specific CD8+ T cell responses have been investigated extensively through the use of HLA-peptide tetramers but much less is known regarding CMV-specific CD4+ T cells. We used a range of HLA class II-peptide tetramers to investigate the phenotypic and transcriptional profile of CMV-specific CD4+ T cells within healthy donors. We show that such cells comprise an average of 0.45% of the CD4+ T cell pool and can reach up to 24% in some individuals (range 0.01–24%). CMV-specific CD4+ T cells display a highly differentiated effector memory phenotype and express a range of cytokines, dominated by dual TNF-α and IFN-γ expression, although substantial populations which express IL-4 were seen in some donors. Microarray analysis and phenotypic expression revealed a profile of unique features. These include the expression of CX3CR1, which would direct cells towards fractalkine on activated endothelium, and the β2-adrenergic receptor, which could permit rapid response to stress. CMV-specific CD4+ T cells display an intense cytotoxic profile with high level expression of granzyme B and perforin, a pattern which increases further during aging. In addition CMV-specific CD4+ T cells demonstrate strong cytotoxic activity against antigen-loaded target cells when isolated directly ex vivo. PD-1 expression is present on 47% of cells but both the intensity and distribution of the inhibitory receptor is reduced in older people. These findings reveal the marked accumulation and unique phenotype of CMV-specific CD4+ T cells and indicate how such T cells may contribute to the vascular complications associated with CMV in older people. PMID:27606804

  17. Acyclovir Therapy Reduces the CD4+ T Cell Response against the Immunodominant pp65 Protein from Cytomegalovirus in Immune Competent Individuals.

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    Annette Pachnio

    Full Text Available Cytomegalovirus (CMV infects the majority of the global population and leads to the development of a strong virus-specific immune response. The CMV-specific CD4+ and CD8+ T cell immune response can comprise between 10 and 50% of the T cell pool within peripheral blood and there is concern that this may impair immunity to other pathogens. Elderly individuals with the highest magnitude of CMV-specific immune response have been demonstrated to be at increased risk of mortality and there is increasing interest in interventions that may serve to moderate this. Acyclovir is an anti-viral drug with activity against a range of herpes viruses and is used as long term treatment to suppress reactivation of herpes simplex virus. We studied the immune response to CMV in patients who were taking acyclovir to assess if therapy could be used to suppress the CMV-specific immune response. The T cell reactivity against the immunodominant late viral protein pp65 was reduced by 53% in people who were taking acyclovir. This effect was seen within one year of therapy and was observed primarily within the CD4+ response. Acyclovir treatment only modestly influenced the immune response to the IE-1 target protein. These data show that low dose acyclovir treatment has the potential to modulate components of the T cell response to CMV antigen proteins and indicate that anti-viral drugs should be further investigated as a means to reduce the magnitude of CMV-specific immune response and potentially improve overall immune function.

  18. The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

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    Hsin-Wei Chen

    Full Text Available Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3 is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4, we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost. A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

  19. Vaccination Expands Antigen-Specific CD4+ Memory T Cells and Mobilizes Bystander Central Memory T Cells

    Science.gov (United States)

    Li Causi, Eleonora; Parikh, Suraj C.; Chudley, Lindsey; Layfield, David M.; Ottensmeier, Christian H.; Stevenson, Freda K.; Di Genova, Gianfranco

    2015-01-01

    CD4+ T helper memory (Thmem) cells influence both natural and vaccine-boosted immunity, but mechanisms for their maintenance remain unclear. Pro-survival signals from the common gamma-chain cytokines, in particular IL-7, appear important. Previously we showed in healthy volunteers that a booster vaccination with tetanus toxoid (TT) expanded peripheral blood TT-specific Thmem cells as expected, but was accompanied by parallel increase of Thmem cells specific for two unrelated and non cross-reactive common recall antigens. Here, in a new cohort of healthy human subjects, we compare blood vaccine-specific and bystander Thmem cells in terms of differentiation stage, function, activation and proliferative status. Both responses peaked 1 week post-vaccination. Vaccine-specific cytokine-producing Thmem cells were predominantly effector memory, whereas bystander cells were mainly of central memory phenotype. Importantly, TT-specific Thmem cells were activated (CD38High HLA-DR+), cycling or recently divided (Ki-67+), and apparently vulnerable to death (IL-7RαLow and Bcl-2 Low). In contrast, bystander Thmem cells were resting (CD38Low HLA-DR- Ki-67-) with high expression of IL-7Rα and Bcl-2. These findings allow a clear distinction between vaccine-specific and bystander Thmem cells, suggesting the latter do not derive from recent proliferation but from cells mobilized from as yet undefined reservoirs. Furthermore, they reveal the interdependent dynamics of specific and bystander T-cell responses which will inform assessments of responses to vaccines. PMID:26332995

  20. Azidothymidine Sensitizes Primary Effusion Lymphoma Cells to Kaposi Sarcoma-Associated Herpesvirus-Specific CD4+ T Cell Control and Inhibits vIRF3 Function.

    Directory of Open Access Journals (Sweden)

    Samantha J Williamson

    2016-11-01

    Full Text Available Kaposi sarcoma-associated herpesvirus (KSHV is linked with the development of Kaposi sarcoma and the B lymphocyte disorders primary effusion lymphoma (PEL and multi-centric Castleman disease. T cell immunity limits KSHV infection and disease, however the virus employs multiple mechanisms to inhibit efficient control by these effectors. Thus KSHV-specific CD4+ T cells poorly recognize most PEL cells and even where they can, they are unable to kill them. To make KSHV-infected cells more sensitive to T cell control we treated PEL cells with the thymidine analogue azidothymidine (AZT, which sensitizes PEL lines to Fas-ligand and TRAIL challenge; effector mechanisms which T cells use. PELs co-cultured with KSHV-specific CD4+ T cells in the absence of AZT showed no control of PEL outgrowth. However in the presence of AZT PEL outgrowth was controlled in an MHC-restricted manner. To investigate how AZT sensitizes PELs to immune control we first examined BJAB cells transduced with individual KSHV-latent genes for their ability to resist apoptosis mediated by stimuli delivered through Fas and TRAIL receptors. This showed that in addition to the previously described vFLIP protein, expression of vIRF3 also inhibited apoptosis delivered by these stimuli. Importantly vIRF3 mediated protection from these apoptotic stimuli was inhibited in the presence of AZT as was a second vIRF3 associated phenotype, the downregulation of surface MHC class II. Although both vFLIP and vIRF3 are expressed in PELs, we propose that inhibiting vIRF3 function with AZT may be sufficient to restore T cell control of these tumor cells.

  1. Activation of nickel-specific CD4+ T lymphocytes in the absence of professional antigen-presenting cells.

    Science.gov (United States)

    Nasorri, Francesca; Sebastiani, Silvia; Mariani, Valentina; De Pità, Ornella; Puddu, Pietro; Girolomoni, Giampiero; Cavani, Andrea

    2002-01-01

    Allergic contact dermatitis ensues from exaggerated T cell responses to haptens. Dendritic cells are required for the initiation of hapten sensitization, but they may not be necessary for disease expression. Here we investigated the antigen-presenting cell requirement of nickel-specific CD4+ lymphocytes isolated from the blood of six allergic individuals. A significant proportion (42 out of 121; 35%) of the T cell clones proliferated in vitro to nickel also in the absence of professional antigen-presenting cells, suggesting a direct T-T hapten presentation. Antigen-presenting-cell-independent T cells showed a predominant T helper 1 phenotype. Nickel recognition by these T cells was major histocompatibility complex class II restricted, not influenced by CD28 triggering, independent from their state of activation, and did not require processing. The capacity of this T cell subset to be directly stimulated by nickel was not due to unique antigen-presenting properties, as both antigen-presenting-cell-dependent and antigen-presenting-cell-independent clones displayed comparable levels of HLA-DR, CD80, and CD86, and were equally capable of presenting nickel to antigen-presenting-cell-independent clones. In contrast, neither T cell types activated antigen-presenting-cell-dependent T lymphocytes. T-T presentation induced T cell receptor downregulation, CD25, CD80, CD86, and HLA-DR upregulation, and interferon-gamma release, although to a lesser extent compared to those induced by dendritic cell-T presentation. Following T-T presentation, the clones did not undergo unresponsiveness and maintained the capacity to respond to dendritic cells pulsed with antigen. In aggregate, our data suggest that antigen-presenting-cell-independent T cell activation can effectively amplify hapten- specific immune responses.

  2. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus

    2011-01-01

    , the functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...... bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized...

  3. Role of 4-1BB receptor in the control played by CD8(+ T cells on IFN-gamma production by Mycobacterium tuberculosis antigen-specific CD4(+ T Cells.

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    Carla Palma

    Full Text Available BACKGROUND: Antigen-specific IFN-gamma producing CD4(+ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-gamma production without affecting protective IFN-gamma is a challenge in tuberculosis research. METHODS AND FINDINGS: Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4(+ T cell-mediated IFN-gamma response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-gamma response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8(+ T cells which suppressed IFN-gamma-secreting CD4(+ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-gamma responses by CD4(+ T cells in protein-boosted mice without affecting the low protective IFN-gamma-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8(+ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-gamma inhibition did not require soluble IL-10, TGF-beta, XCL-1 and MIP-1beta. In vivo Ag85B stimulation induced 4-1BB expression on CD8(+ T cells and in vivo 4-1BB ligation reduced the activation, IFN-gamma production and expansion of Ag85B-specific CD4(+ T cells of DNA-primed and protein-boosted mice. CONCLUSION/SIGNIFICANCE: Antigen-specific suppressor CD8(+ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-gamma-secreting CD4(+ T cells. The selective

  4. Mtb-specific CD27low CD4 T cells as markers of lung tissue destruction during pulmonary tuberculosis in humans.

    Science.gov (United States)

    Nikitina, Irina Yu; Kondratuk, Natalya A; Kosmiadi, George A; Amansahedov, Rasul B; Vasilyeva, Irina A; Ganusov, Vitaly V; Lyadova, Irina V

    2012-01-01

    Effector CD4 T cells represent a key component of the host's anti-tuberculosis immune defense. Successful differentiation and functioning of effector lymphocytes protects the host against severe M. tuberculosis (Mtb) infection. On the other hand, effector T cell differentiation depends on disease severity/activity, as T cell responses are driven by antigenic and inflammatory stimuli released during infection. Thus, tuberculosis (TB) progression and the degree of effector CD4 T cell differentiation are interrelated, but the relationships are complex and not well understood. We have analyzed an association between the degree of Mtb-specific CD4 T cell differentiation and severity/activity of pulmonary TB infection. The degree of CD4 T cell differentiation was assessed by measuring the percentages of highly differentiated CD27(low) cells within a population of Mtb- specific CD4 T lymphocytes ("CD27(low)IFN-γ(+)" cells). The percentages of CD27(low)IFN-γ+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p76%), but varied in blood (12-92%). The major correlate for the accumulation of CD27(low)IFN-γ(+) cells in blood was lung destruction (r = 0.65, p = 2.7 × 10(-7)). A cutoff of 47% of CD27(low)IFN-γ(+) cells discriminated patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27(low)IFN-γ(+)cells following TB therapy correlated with repair and/or reduction of lung destruction (ppulmonary TB. Accumulation of CD27(low)IFN-γ(+) cells in the blood is associated with lung destruction. The findings indicate that there is no deficiency in CD4 T cell differentiation during TB; evaluation of CD27(low)IFN-γ(+) cells provides a valuable means to assess TB activity, lung destruction, and tissue repair following TB therapy.

  5. Poultry Allele-Specific Expression (ASE) of CD4+ T Cells in Response to Marek’s Disease Virus Infection

    Science.gov (United States)

    Marek’s disease (MD) is a T cell lymphoma disease of poultry induced by Marek’s disease virus (MDV), a highly oncogenic alphaherpesvirus. To identify high-confidence candidate genes of MD genetic resistance, transcriptomic data in CD4+ T cells were obtained from MDV infected and non-infected groups ...

  6. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

    Science.gov (United States)

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

  7. The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222

    Science.gov (United States)

    Orecchini, Elisa; Doria, Margherita; Michienzi, Alessandro; Giuliani, Erica; Vassena, Lia; Ciafrè, Silvia Anna; Farace, Maria Giulia; Galardi, Silvia

    2014-01-01

    Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells. PMID:24717285

  8. Establishment and characterization of canine parvovirus-specific murine CD4+ T cell clones and their use for the delineation of T cell epitopes.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); R.W.J. van der Heijden (Roger); E.J. Tijhaar (Edwin); M.C.M. Poelen (Martien); J. Carlson; A.D.M.E. Osterhaus (Albert); F.G.C.M. Uytdehaag (Fons)

    1990-01-01

    textabstractCanine parvovirus (CPV)-specific T cell clones were generated by culturing lymph node cells from CPV-immunized BALB/c mice at limiting dilutions in the presence of CPV antigen and interleukin-2 (IL-2). All isolated T cell clones exhibited the cell surface phenotype Thy1+, CD4+, CD8- and

  9. Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

    Directory of Open Access Journals (Sweden)

    George Q Perrin

    2016-01-01

    Full Text Available The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8 vector expressing cytoplasmic ovalbumin (OVA into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.

  10. Heterogeneity in cytokine profiles of Babesia bovis-specific bovine CD4+ T cells clones activated in vitro.

    OpenAIRE

    Brown, W C; Woods, V M; Dobbelaere, D A; Logan, K S

    1993-01-01

    The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen we...

  11. Upregulation of bacterial-specific Th1 and Th17 responses that are enriched in CXCR5+CD4+ T cells in non-small cell lung cancer.

    Science.gov (United States)

    Ma, Qin-Yun; Huang, Da-Yu; Zhang, Hui-Jun; Wang, Shaohua; Chen, Xiao-Feng

    2017-11-01

    The microbial community in the mucosal surfaces is involved in the development of human cancers, including gastric cancer and colorectal cancer. The respiratory tract in the lung also hosts a distinctive microbial community, but the correlation between this community and lung cancer is largely unknown. Here, we examined the Th1 and Th17 responses toward several bacterial antigens, in CD4 + T cells sourced from the peripheral blood (PB), the lung cancer (LC) tissue, and the gastrointestinal (GI) tract of non-small cell lung cancer (NSCLC) patients. Compared to healthy controls, the NSCLC patients presented significantly higher frequencies of Th1 and Th17 cells reacting to Streptococcus salivarius and S. agalactiae, in the PB, LC, and GI tract. Further investigation showed that the upregulation in anti-bacteria response was likely antigen-specific for two reasons. Firstly, the frequencies of Th1 and Th17 cells reacting to Escherichia coli, a typical GI bacterium, were not upregulated in the PB and the LC of NSCLC patients. Secondly, the S. salivarius and S. agalactiae responses could be partially blocked by Tü39, a MHC class II blocking antibody, suggesting that antigen-specific interaction between CD4 + T cells and antigen-presenting cells was required. We also found that S. salivarius and S. agalactiae could potently activate the monocytes to secrete higher levels of interleukin (IL)-6, IL-12, and tumor necrosis factor, which were Th1- and Th17-skewing cytokines. Interestingly, whereas CXCR5 + CD4 + T cells represented <20% of total CD4 + T cells, they represented 17%-82% of bacteria-specific Th1 or Th17 cells. Together, these data demonstrated that NSCLC patients presented a significant upregulation of bacterial-specific Th1 and Th17 responses that were enriched in CXCR5 + CD4 + T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Retention of Ag-specific memory CD4+ T cells in the draining lymph node indicates lymphoid tissue resident memory populations.

    Science.gov (United States)

    Marriott, Clare L; Dutton, Emma E; Tomura, Michio; Withers, David R

    2017-05-01

    Several different memory T-cell populations have now been described based upon surface receptor expression and migratory capabilities. Here we have assessed murine endogenous memory CD4 + T cells generated within a draining lymph node and their subsequent migration to other secondary lymphoid tissues. Having established a model response targeting a specific peripheral lymph node, we temporally labelled all the cells within draining lymph node using photoconversion. Tracking of photoconverted and non-photoconverted Ag-specific CD4 + T cells revealed the rapid establishment of a circulating memory population in all lymph nodes within days of immunisation. Strikingly, a resident memory CD4 + T cell population became established in the draining lymph node and persisted for several months in the absence of detectable migration to other lymphoid tissue. These cells most closely resembled effector memory T cells, usually associated with circulation through non-lymphoid tissue, but here, these cells were retained in the draining lymph node. These data indicate that lymphoid tissue resident memory CD4 + T-cell populations are generated in peripheral lymph nodes following immunisation. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Identification and HLA-Tetramer-Validation of Human CD4(+) and CD8(+) T Cell Responses against HCMV Proteins IE1 and IE2

    DEFF Research Database (Denmark)

    Braendstrup, Peter; Mortensen, Bo Kok; Justesen, Sune Frederik Lamdahl

    2014-01-01

    tumor development. Both CD4(+) and CD8(+) T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8(+) T cell...

  14. Complexity of type-specific 56 kDa antigen CD4 T-cell epitopes of Orientia tsutsugamushi strains causing scrub typhus in India.

    Directory of Open Access Journals (Sweden)

    Arunachalam Ramaiah

    Full Text Available Orientia tsutsugamushi (Ots is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III and three spacer conserved domains (SVDI, SVDII/III, SVDIII. The total number (152 of antigenic types (amino acid variants varied from 14-36 in the six domains of TSA that we studied. Notably, 55% (787/1435 of the predicted CD4 T-cell epitopes (TCEs from all the six domains had high binding affinities (HBA to at least one of the prevalent Indian human leukocyte antigen (HLA alleles. A surprisingly high proportion (61% of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients.

  15. Glutamic acid decarboxylase-derived epitopes with specific domains expand CD4(+CD25(+ regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Guojiang Chen

    Full Text Available BACKGROUND: CD4(+CD25(+ regulatory T cell (Treg-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used. METHODOLOGY/PRINCIPAL FINDINGS: Splenic lymphocytes from nonobese diabetic (NOD mice were stimulated with different glutamic acid decarboxylase (GAD-derived epitopes for 7-10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524-538-expanded CD4(+CD25(+ T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509-528- or p530 (GAD530-543-expanded CD4(+CD25(+ T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes between the above-mentioned epitopes and MHC class II I-A(g7 were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A(g7, while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A(g7. Furthermore, p524 bound to I-A(g7 more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570-585, p570, which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4(+CD25(+ T cells suppressed the onset of diabetes in NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of

  16. Analysis of the Phenotype of Mycobacterium tuberculosis-Specific CD4+ T Cells to Discriminate Latent from Active Tuberculosis in HIV-Uninfected and HIV-Infected Individuals

    Directory of Open Access Journals (Sweden)

    Catherine Riou

    2017-08-01

    Full Text Available Several immune-based assays have been suggested to differentiate latent from active tuberculosis (TB. However, their relative performance as well as their efficacy in HIV-infected persons, a highly at-risk population, remains unclear. In a study of 81 individuals, divided into four groups based on their HIV-1 status and TB disease activity, we compared the differentiation (CD27 and KLRG1, activation (HLA-DR, homing potential (CCR4, CCR6, CXCR3, and CD161 and functional profiles (IFNγ, IL-2, and TNFα of Mycobacterium tuberculosis (Mtb-specific CD4+ T cells using flow cytometry. Active TB disease induced major changes within the Mtb-responding CD4+ T cell population, promoting memory maturation, elevated activation and increased inflammatory potential when compared to individuals with latent TB infection. Moreover, the functional profile of Mtb-specific CD4+ T cells appeared to be inherently related to their degree of differentiation. While these specific cell features were all capable of discriminating latent from active TB, irrespective of HIV status, HLA-DR expression showed the best performance for TB diagnosis [area-under-the-curve (AUC = 0.92, 95% CI: 0.82–1.01, specificity: 82%, sensitivity: 84% for HIV− and AUC = 0.99, 95% CI: 0.98–1.01, specificity: 94%, sensitivity: 93% for HIV+]. In conclusion, these data support the idea that analysis of T cell phenotype can be diagnostically useful in TB.

  17. Immunomodulatory Effects of Hemagglutinin- (HA- Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4+ T Cells

    Directory of Open Access Journals (Sweden)

    Valentin P. Shichkin

    2014-01-01

    Full Text Available Previously, the mouse A20 B-cell lymphoma engineered to express hemagglutinin (HA antigen (A20HA was used as a systemic tumor model. In this work, we used the A20HA cells as a brain tumor. HA-specific CD4+ T cells were transferred intravenously in a tail vein 5 days after A20HA intracranial inoculation and analyzed on days 2, 9, and 16 after the adoptive transfer by different methods. The transferred cells demonstrated state of activation as early as day 2 after the adoptive transfer and most the of viable HA-specific cells became anergic on day 16. Additionally, symptoms of systemic immunosuppression were observed in mice with massive brain tumors at a late stage of the brain tumor progression (days 20–24 after the A20HA inoculation. Despite that, a deal of HA-specific CD4+ T cells kept the functional activity even at the late stage of A20HA tumor growth. The activated HA-specific CD4+ T cells were found also in the brain of brain-tumor-bearing mice. These cells were still responding to reactivation with HA-peptide in vitro. Our data support an idea about sufficient role of both the tumor-specific and -nonspecific mechanisms inducing immunosuppression in cancer patients.

  18. Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes.

    Science.gov (United States)

    Hanon, E; Hall, S; Taylor, G P; Saito, M; Davis, R; Tanaka, Y; Usuku, K; Osame, M; Weber, J N; Bangham, C R

    2000-02-15

    The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax(11-19)-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo. (Blood. 2000;95:1386-1392)

  19. Humoral and cellular CMV responses in healthy donors; identification of a frequent population of CMV-specific, CD4+ T cells in seronegative donors

    DEFF Research Database (Denmark)

    Loeth, Nina; Assing, Kristian; Madsen, Hans O

    2012-01-01

    .e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV...... protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors.......CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i...

  20. The T-cell accessory molecule CD4 recognizes a monomorphic determinant on isolated Ia

    DEFF Research Database (Denmark)

    Gay, D; Buus, S; Pasternak, J

    1988-01-01

    The membrane protein CD4 is commonly found on mature T cells specific for antigen in association with class II major histocompatibility complex (MHC; Ia) proteins. This correlation has led to the suggestion that CD4 binds to a monomorphic region of the Ia molecule on the antigen-presenting cell...... proteins into a planar membrane system, we show that different Ia molecules can greatly enhance the ability of a CD4+ but not a CD4- variant of this class I-restricted T hybrid to respond to isolated class I molecules. T-cell responses can be strongly augmented by the concurrent expression of CD4 on the T...... cell and any of four different Ia proteins on planar membranes, thus supporting the idea that CD4 binds to a monomorphic region of the Ia molecule and increases the avidity with which the T cell can interact with its target....

  1. Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

    Science.gov (United States)

    Matta, Bharati; Jha, Purushottam; Bora, Puran S; Bora, Nalini S

    2010-02-01

    The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

  2. Microbiota-specific CD4CD8αα Tregs: role in intestinal immune homeostasis and implications for IBD

    Directory of Open Access Journals (Sweden)

    Guillaume eSARRABAYROUSE

    2015-10-01

    Full Text Available In studies in murine models, active suppression by IL-10-secreting Foxp3 regulatory T cells (Tregs has emerged as an essential mechanism in colon homeostasis. However, the role of the equivalent subset in humans remains unclear, leading to suggestions that other subsets and/or mechanisms may substitute for Foxp3 Tregs in the maintenance of colon homeostasis. We recently described a new subset of CD4CD8αα T cells reactive to the gut bacterium Faecalibacterium prausnitzii and endowed with regulatory/suppressive functions. This subset is abundant in the healthy colonic mucosa, but less common in that of patients with irritable bowel disease (IBD. We discuss here the physiological significance and potential role of these Tregs in preventing inflammation of the gut mucosa and the potential applications of these discoveries for IBD management.

  3. Proteomic Profiling of a Primary CD4+ T Cell Model of HIV-1 Latency Identifies Proteins Whose Differential Expression Correlates with Reactivation of Latent HIV-1.

    Science.gov (United States)

    Saha, Jamaluddin Md; Liu, Hongbing; Hu, Pei-Wen; Nikolai, Bryan C; Wu, Hulin; Miao, Hongyu; Rice, Andrew P

    2018-01-01

    The latent HIV-1 reservoir of memory CD4 + T cells that persists during combination antiviral therapy prevents a cure of infection. Insight into mechanisms of latency and viral reactivation are essential for the rational design of strategies to reduce the latent reservoir. In this study, we quantified the levels of >2,600 proteins in the CCL19 primary CD4 + T cell model of HIV-1 latency. We profiled proteins under conditions that promote latent infection and after cells were treated with phorbol 12-myristate 13-acetate (PMA) + ionomycin, which is known to efficiently induce reactivation of latent HIV-1. In an analysis of cells from two healthy blood donors, we identified 61 proteins that were upregulated ≥2-fold, and 36 proteins that were downregulated ≥2-fold under conditions in which latent viruses were reactivated. These differentially expressed proteins are, therefore, candidates for cellular factors that regulate latency or viral reactivation. Two unexpected findings were obtained from the proteomic data: (1) the interactions among the majority of upregulated proteins are largely undetermined in published protein-protein interaction networks and (2) downregulated proteins are strongly associated with Gene Ontology terms related to mitochondrial protein synthesis. This proteomic data set provides a useful resource for future mechanistic studies of HIV-1 latency.

  4. ACVP-03: Novel CD4+ T Cell Specific Immunohistochemistry Detection and Analysis Utilizing Masking of Not-T Cell CD4 in Fixed Tissues from Virally Infected and Uninfected Specimens | Frederick National Laboratory for Cancer Research

    Science.gov (United States)

    The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. CD4 (DAB) and CD68/CD163 (FastRed) double immunohistochemistry will be performed, in whic

  5. The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD.

    Science.gov (United States)

    Martins, Marcia Valéria B S; Lima, Mônica Cristina B S; Duppre, Nadia C; Matos, Haroldo J; Spencer, John S; Brennan, Patrick J; Sarno, Euzenir N; Fonseca, Leila; Pereira, Geraldo M B; Pessolani, Maria Cristina V

    2007-05-01

    There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome

  6. The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

    Science.gov (United States)

    Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

    2012-11-01

    The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

  7. HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

    Science.gov (United States)

    Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

    1999-06-18

    To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.

  8. HIV controllers exhibit enhanced frequencies of major histocompatibility complex class II tetramer+ Gag-specific CD4+ T cells in chronic clade C HIV-1 infection

    DEFF Research Database (Denmark)

    Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos

    2017-01-01

    Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibilit...

  9. Human CD4 restores normal T cell development and function in mice deficient in murine CD4

    OpenAIRE

    1994-01-01

    The ability of a human coreceptor to function in mice was investigated by generating human CD4 (hCD4)-expressing transgenic mice on a mouse CD4-deficient (mCD4-/-) background. From developing thymocyte to matured T lymphocyte functions, hCD4 was shown to be physiologically active. By examining the expansion and deletion of specific V beta T cell families in mutated mice with and without hCD4, it was found that hCD4 can participate in positive and negative selection. Mature hCD4 single positiv...

  10. A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

    OpenAIRE

    Paquette, Y; Doyon, L; Laperrière, A; Hanna, Z; Ball, J; Sekaly, R P; Jolicoeur, P

    1992-01-01

    The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to...

  11. Identification of CD4+ T-cell Epitopes on Mycobacterium Tuberculosis- Secreted MPB51 Protein in C57BL/6 Mice

    Directory of Open Access Journals (Sweden)

    A.R. Rafiei

    2006-01-01

    Full Text Available Introduction & Objective: Both CD4+ type 1 helper (Th1 cells and CD8+ T cells play effective roles in protection against Mycobacterium tuberculosis infection. DNA vaccine encoding MPB51 can induce Th1-type immune responses and protective immunity upon challenge with M.tuberculosis. This study address to identify T-cell immunodominant epitopes on MPB51 in C57BL/6 mice.Materials & Methods : We cloned DNA encoding MPB51 molecule in pCI plasmid. After constructing MPB51 DNA-covered gold cartridge, C57BL/6 mice were immunized by using a gene gun system. Two weeks after the last immunization, the immune spleen cells were cultured in the presence of a synthetic overlapping library peptides covering the mature MPB51 sequence or medium alone. Intracellular and cell culture supernatant gamma interferon (IFN- production was analyzed using flow cytometry and ELISA, respectively.Results : Mapping of T-cell epitopes on MPB51 molecule was performed in the spleen lymphocytes restimulated by 20-mer overlapping synthetic peptides of mature MPB51 sequence. Flow cytometric analysis with intracellular IFN- and the T-cell phenotype revealed that P171-190 and P191-210 peptides contain immunodominant CD4+ T-cell epitopes. Further analysis by using T-cell subset depletion and serial peptide dilution revealed that P171 and p191 are H2-Ab-restricted dominant and subdominant CD4+ T cell epitopes, respectively. Conclusion: This study proved that vaccination with plasmid DNA encoding M. tuberculosis-secreted MPB51 protein not only induce CD4+ T cells immune response but also is an appropriate method for identifying immunogenic peptides.

  12. CD4+ T Cells Mediate Aspergillosis Vaccine Protection.

    Science.gov (United States)

    Diaz-Arevalo, Diana; Kalkum, Markus

    2017-01-01

    Adaptive effector CD4 + T cells play essential roles in the defense against fungal infections, especially against invasive aspergillosis (IA). Such protective CD4 + T cells can be generated through immunization with specialized antifungal vaccines, as has been demonstrated for pulmonary Aspergillus fumigatus infections in mouse experiments. Adaptive transfer of fungal antigen-specific CD4 + T cells conferred protection onto non-immunized naive mice, an experimental approach that could potentially become a future treatment option for immunosuppressed IA patients, focusing on the ultimate goal to improve their otherwise dim chances for survival. Here, we describe the different techniques to analyze CD4 + T cell immune responses after immunization with a recombinant fungal protein. We present three major methods that are used to analyze the role of CD4 + T cells in protection against A. fumigatus challenge. They include (1) transplantation of CD4 + T cells from vaccinated mice into immunosuppressed naive mice, observing increasing protection of the cell recipients, (2) depletion of CD4 + T cells from vaccinated mice, which abolishes vaccine protection, and (3) T cell proliferation studies following stimulation with overlapping synthetic peptides or an intact protein vaccine. The latter can be used to validate immunization status and to identify protective T cell epitopes in vaccine antigens. In the methods detailed here, we used versions of the well-studied Asp f3 protein expressed in a bacterial host, either as the intact full length protein or its N-terminally truncated version, comprised of residues 15-168. However, these methods are generally applicable and can well be adapted to study other protein-based subunit vaccines.

  13. Grass-specific CD4+ T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy

    Science.gov (United States)

    Archila, LD; DeLong, JH; Wambre, E; James, EA; Robinson, DM; Kwok, WW

    2014-01-01

    Background Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity amongst grass pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. Objectives We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4+ T cell level amongst DR04:01 restricted Pooideae grass pollen T-cell epitopes. Methods After In vitro culture of blood mononucleated cells from Grass-pollen allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labeled DR04:01/Phleum pratense tetramers and PE-labeled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity amongst allergen-specific DR04:01-restricted T-cells in 6 subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. Results T-cells with various degree of cross reactive profiles could be detected. Poa p 1 97-116, Lol p 1 221-240, Lol p 5a 199-218, and Poa p 5a 199-218 were identified as minimally-cross-reactive T-cell epitopes that do not show cross reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally-cross reactive T-cell epitopes are present in Grass-pollen allergic subjects. Conclusions Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells, suggest that a multiple allergen system should be considered for immunotherapy instead of a mono allergen system. PMID:24708411

  14. Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

    Science.gov (United States)

    Archila, L D; DeLong, J H; Wambre, E; James, E A; Robinson, D M; Kwok, W W

    2014-07-01

    Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity among grass-pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4(+) T cell level among DR04:01 restricted Pooideae grass-pollen T-cell epitopes. After in vitro culture of blood mono-nucleated cells from grass-pollen-allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labelled DR04:01/Phleum pratense tetramers and PE-labelled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity among allergen-specific DR04:01-restricted T-cells in six subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass-pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. T-cells with various degrees of cross-reactive profiles could be detected. Poa p 1 97-116 , Lol p 1 221-240 , Lol p 5a 199-218 , and Poa p 5a 199-218 were identified as minimally cross-reactive T-cell epitopes that do not show cross-reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally cross-reactive T-cell epitopes are present in Grass-pollen-allergic subjects. Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross-reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells suggest that a multiple allergen system should be considered for immunotherapy instead of a mono-allergen system. © 2014 John Wiley & Sons Ltd.

  15. Binding of the mannose-specific lectin, Griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available development. Anti- microb. Agents Chemother. 41:1521?1530. 7. Buchacher, A., et al. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization...

  16. Characteristics of Prevotella intermedia-specific CD4+ T cell clones from peripheral blood of a chronic adult periodontitis patient

    NARCIS (Netherlands)

    Wassenaar, A.; Reinhardus, C.; Abraham-Inpijn, L.; Snijders, A.; Kievits, F.

    1998-01-01

    Periodontitis is a chronic destructive inflammatory disease associated with periodontopathic bacteria. In addition, autoantigens such as collagen and heat shock proteins (hsp) have been suggested to play a role. Established periodontal lesions are characterized by dense infiltrations of immune cells

  17. Slow desensitization of imatinib-induced nonimmediate reactions and dynamic changes of drug-specific CD4+CD25+CD134+ lymphocytes.

    Science.gov (United States)

    Klaewsongkram, Jettanong; Thantiworasit, Pattarawat; Sodsai, Pimpayao; Buranapraditkun, Supranee; Mongkolpathumrat, Pungjai

    2016-11-01

    Imatinib is a tyrosine kinase inhibitor indicated for the treatment of gastrointestinal stromal tumors (GISTs) and certain neoplastic diseases; however, nonimmediate adverse reactions are common. To describe the process of imatinib slow desensitization in patients who experienced nonimmediate reactions to imatinib and the dynamic change in drug-specific CD4 + CD25 + CD134 + T-lymphocyte percentages. Five patients diagnosed as having GISTs and with a recent history of imatinib-induced nonimmediate reactions (maculopapular exanthema with eosinophilia, exfoliative dermatitis, palmar-plantar erythrodysesthesia, and drug rash with eosinophilia and systemic symptoms) were desensitized using a slow desensitization protocol. The reintroduced imatinib dosage was stepped up every week starting from 10 mg/d and increasing to 25, 50, 75, 100, 150, 200, and 300 mg/d until the target dose of 400 mg/d was achieved. Prednisolone of up to 30 mg/d was allowed if allergic reactions recurred. The percentages of CD4 + CD25 + CD134 + T cells present after incubating peripheral blood mononuclear cells with imatinib, at baseline and after successful desensitization, were analyzed using flow cytometric analysis. By using a slow desensitization technique, all patients were able to receive 400 mg/d of imatinib, and prednisolone was gradually tapered off. The percentages of imatinib-induced CD4 + CD25 + CD134 + T cells decreased from a mean (SD) of 11.3% (6.5%) and 13.4% (7.3%) at baseline to 3.2% (0.7%) and 3.0% (1.1%) after successful desensitization, when stimulating peripheral blood mononuclear cells with 1 and 2 μM of imatinib, respectively. Slow desensitization is a helpful procedure in treating patients with imatinib-induced nonimmediate reactions other than simple maculopapular exanthema. The reduced percentages of imatinib-induced CD4 + CD25 + CD134 + T cells in these patients may be associated with immune tolerance. Copyright © 2016 American College of Allergy, Asthma & Immunology

  18. The effect of beta-interferon therapy on myelin basic protein-elicited CD4+ T cell proliferation and cytokine production in multiple sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Chris J; Krakauer, Martin; Bendtzen, Klaus

    2008-01-01

    Interferon (IFN)-beta therapy has well-established clinical benefits in multiple sclerosis (MS), but the underlying modulation of cytokine responses to myelin self-antigens remains poorly understood. We analysed the CD4+ T cell proliferation and cytokine responses elicited by myelin basic protein...... (MBP) and a foreign recall antigen, tetanus toxoid (TT), in mononuclear cell cultures from fourteen MS patients undergoing IFN-beta therapy. The MBP-elicited IFN-gamma-, TNF-alpha- and IL-10 production decreased during therapy (p...

  19. Cellular responses to Plasmodium falciparum erythrocyte membrane protein-1: use of relatively conserved synthetic peptide pools to determine CD4 T cell responses in malaria-exposed individuals in Benin, West Africa

    Directory of Open Access Journals (Sweden)

    Sanni Ambaliou

    2002-04-01

    Full Text Available Abstract Background Plasmodium falciparum erythrocyte membrane protein-1, a variant antigen of the malaria parasite, is potentially a target for the immune response. It would be important to determine whether there are CD4 T cells that recognise conserved regions. However, within the relatively conserved region, there is variation. It is not possible to test T cell responses from small field samples with all possible peptides. Methods We have aligned sequences that are relatively conserved between several PfEMP1 molecules, and chosen a representative sequence similar to most of the PfEMP1 variants. Using these peptides as pools representing CIDRα, CIDRβ and DBLβ-δ domains, DBLα domain, and EXON 2 domain of PfEMP1, we measured the CD4 T cell responses of malaria-exposed donors from Benin, West Africa by a FACS based assay. Results All the three peptide pools elicited a CD4 T cell response in a proportion of malaria-exposed and non-exposed donors. CD4 T cell proliferation occurs at a relatively higher magnitude to peptide pools from the DBLα and EXON 2 in the malaria-exposed donors living in Benin than in the UK malaria-unexposed donors. Conclusions These findings suggest that an immunological recall response to conserved peptides of a variant antigen can be measured. Further testing of individual peptides in a positive pool will allow us to determine those conserved sequences recognised by many individuals. These types of assays may provide information on conserved peptides of PfEMP1 which could be useful for stimulating T cells to provide help to P. falciparum specific B cells.

  20. Secreted protein acidic and rich in cysteine functions in colitis via IL17A regulation in mucosal CD4+ T cells.

    Science.gov (United States)

    Tanaka, Makoto; Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Hotta, Yuma; Toyokawa, Yuki; Ushiroda, Chihiro; Hirai, Yasuko; Aoi, Wataru; Higashimura, Yasuki; Mizushima, Katsura; Okayama, Tetsuya; Katada, Kazuhiro; Kamada, Kazuhiro; Ishikawa, Takeshi; Handa, Osamu; Itoh, Yoshito

    2018-03-01

    Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycol that regulates cell proliferation, tissue repair, and tumorigenesis. Despite evidence linking SPARC to inflammation, the mechanisms are unclear. Accordingly, the role of SPARC in intestinal inflammation was investigated. Colitis was induced in wild-type (WT) and SPARC knockout (KO) mice using trinitrobenzene sulfonic acid (TNBS). Colons were assessed for damage; leukocyte infiltration; Tnf, Ifng, Il17a, and Il10 mRNA expression; and histology. Cytokine profiling of colonic lamina propria mononuclear cells (LPMCs) was performed by flow cytometry. Naïve CD4 + T cells were isolated from WT and SPARC KO mouse spleens, and the effect of SPARC on Th17 cell differentiation was examined. Recombination activating gene 1 knockout (RAG1 KO) mice reconstituted with T cells from either WT or SPARC KO mice were investigated. Trinitrobenzene sulfonic acid exposure significantly reduced bodyweight and increased mucosal inflammation, leukocyte infiltration, and Il17a mRNA expression in WT relative to SPARC KO mice. The percentage of IL17A-producing CD4 + T cells among LPMCs from KO mice was lower than that in WT mice when both groups were exposed to TNBS. Th17 cell differentiation was suppressed in cells from SPARC KO mice. In the T cell transfer colitis model, RAG1 KO mice receiving T cells from WT mice were more severely affected than those reconstituted with cells from SPARC KO mice. Secreted protein acidic and rich in cysteine accelerates colonic mucosal inflammation via modulation of IL17A-producing CD4 + T cells. SPARC is a potential therapeutic target for conditions involving intestinal inflammation. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  1. CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model

    Directory of Open Access Journals (Sweden)

    Prabitha Natarajan

    2017-08-01

    Full Text Available Approximately 3–10% of human red blood cell (RBC transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that “non-responders” may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken

  2. Tumor Immunology meets…Immunology: Modified cancer cells as professional APC for priming naïve tumor-specific CD4+ T cells.

    Science.gov (United States)

    Bou Nasser Eddine, Farah; Ramia, Elise; Tosi, Giovanna; Forlani, Greta; Accolla, Roberto S

    2017-01-01

    Although recent therapeutic approaches have revitalized the enthusiasm of the immunological way to combat cancer, still the comprehension of immunity against tumors is largely incomplete. Due to their specific function, CD8+ T cells with cytolytic activity (CTL) have attracted the attention of most investigators because CTL are considered the main effectors against tumor cells. Nevertheless, CTL activity and persistence is largely dependent on the action of CD4+ T helper cells (TH). Thus establishment of tumor-specific TH cell response is key to the optimal response against cancer. Here we describe emerging new strategies to increase the TH cell recognition of tumor antigens. In particular, we review recent data indicating that tumor cells themselves can act as surrogate antigen presenting cells for triggering TH response and how these findings can help in constructing immunotherapeutic protocols for anti-cancer vaccine development.

  3. Glucocorticoid-induced TNF receptor family related protein ligand [GITR-L] is requisite for optimal functioning of regulatory CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Gongxian eLiao

    2014-02-01

    Full Text Available Glucocorticoid-Induced Tumor necrosis factor Receptor family-related protein (GITR, TNFRSF18, CD357 is constitutively expressed on regulatory T cells (Tregs and is inducible on effector T cells (Teffs. In this report, we examine the role of GITR-Ligand (GITR-L, which is expressed by antigen presenting cells, on the development and expansion of Tregs. We found that GITR-L is dispensable for the development of naturally occurring FoxP3+ Treg cells in the thymus. However, the expansion of Treg in GITR-L-/- mice is impaired after injection of the dendritic cells (DCs inducing factor Flt3 ligand. Furthermore, DCs from the liver of GITR-L-/- mice were less efficient in inducing proliferation of antigen-specific Treg cells in vitro than the same cells from WT littermates. Upon gene transfer of ovalbumin into hepatocytes of GITR-L-/- FoxP3(GFP reporter mice using adeno-associated virus (AAV8-OVA the number of antigen-specific Treg in liver and spleen is reduced. The reduced number of Tregs resulted in an increase in the number of ovalbumin specific CD8+ T effector cells. This is highly significant because proliferation of antigen specific CD8+ cells itself is dependent on the presence of GITR-L, as shown by in vitro experiments and by adoptive transfers into GITR-L-/-Rag-/- and Rag-/- mice that had received AAV8-OVA. Surprisingly, administering αCD3 significantly reduced the numbers of FoxP3+ Treg cells in the liver and spleen of GITR-L-/- but not WT mice. Because soluble Fc-GITR-L partially rescues αCD3 induced in vitro depletion of the CD103+ subset of FoxP3+CD4+ Treg cells, we conclude that expression of GITR-L by antigen presenting cells is requisite for optimal Treg-mediated regulation of immune responses including those in response during gene transfer.

  4. Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

    Directory of Open Access Journals (Sweden)

    Kuo-Ching Sheng

    2013-01-01

    Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

  5. Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Th17 cells and polyfunctional HIV-specific T-cell responses.

    Science.gov (United States)

    Macal, M; Sankaran, S; Chun, T-W; Reay, E; Flamm, J; Prindiville, T J; Dandekar, S

    2008-11-01

    Human immunodeficiency virus (HIV) infection leads to severe CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) that persists despite the initiation of highly active antiretroviral therapy (HAART). It is not known whether restoration of gut mucosal CD4+ T cells and their functions is feasible during therapy and how that relates to immune correlates and viral reservoirs. Intestinal biopsies and peripheral blood samples from HIV-infected patients who were either HAART naive or on long-term HAART were evaluated. Our data demonstrated that gut CD4+ T-cell restoration ranged from modest (50%), compared with uninfected controls. Despite persistent CD4+ T-cell proviral burden and residual immune activation in GALT during HAART, effective CD4+ T-cell restoration (>50%) was achieved, which was associated with enhanced Th17 CD4+ T-cell accumulation and polyfunctional anti-HIV cellular responses. Our findings suggest that a threshold of>50% CD4+ T-cell restoration may be sufficient for polyfunctional HIV-specific T cells with implications in the evaluation of vaccines and therapeutics.

  6. Infection of CD4+ T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: Evidence using antibodies specific to the receptor's large extracellular domain

    International Nuclear Information System (INIS)

    Jin, Qingwen; Agrawal, Lokesh; VanHorn-Ali, Zainab; Alkhatib, Ghalib

    2006-01-01

    To analyze HTLV-1 cytotropism, we developed a highly sensitive vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. We used this system in a functional cDNA screening to isolate and confirm that the glucose transporter protein 1 (GLUT-1) is a receptor for HTLV-1. GLUT-1 is a ubiquitously expressed plasma membrane glycoprotein with 12 transmembrane domains and 6 extracellular loops (ECL). We demonstrate for the first time that peptide antibodies (GLUT-IgY) raised in chicken to the large extracellular loop (ECL1) detect GLUT-1 at the cell surface and inhibit envelope (Env)-mediated fusion and infection. Efficient GLUT-IgY staining was detected with peripheral blood CD4 + lymphocytes purified by positive selection. Further, GLUT-IgY caused efficient inhibition of Env-mediated fusion and infection of CD4 + T and significantly lower inhibition of CD8 + T lymphocytes. The specificity of GLUT-IgY antibodies to GLUT-1 was demonstrated by ECL1 peptide competition studies. Grafting ECL1 of GLUT-1 onto the receptor-negative GLUT-3 conferred significant receptor activity. In contrast, grafting ECL1 of GLUT-3 onto GLUT-1 resulted in a significant loss of the receptor activity. The ECL1-mediated receptor activity was efficiently blocked with four different human monoclonal antibody (HMab) to HTLV-1 Env. The ECL1-derived peptide blocked HTLV-1 Env-mediated fusion with several nonhuman mammalian cell lines. The results demonstrate the utilization of cell surface GLUT-1 in HTLV-1 infection of CD4 + T lymphocytes and implicate a critical role for the ECL1 region in viral tropism

  7. Dynamic Response Genes in CD4+ T Cells Reveal a Network of Interactive Proteins that Classifies Disease Activity in Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Sandra Hellberg

    2016-09-01

    Full Text Available Multiple sclerosis (MS is a chronic inflammatory disease of the CNS and has a varying disease course as well as variable response to treatment. Biomarkers may therefore aid personalized treatment. We tested whether in vitro activation of MS patient-derived CD4+ T cells could reveal potential biomarkers. The dynamic gene expression response to activation was dysregulated in patient-derived CD4+ T cells. By integrating our findings with genome-wide association studies, we constructed a highly connected MS gene module, disclosing cell activation and chemotaxis as central components. Changes in several module genes were associated with differences in protein levels, which were measurable in cerebrospinal fluid and were used to classify patients from control individuals. In addition, these measurements could predict disease activity after 2 years and distinguish low and high responders to treatment in two additional, independent cohorts. While further validation is needed in larger cohorts prior to clinical implementation, we have uncovered a set of potentially promising biomarkers.

  8. Glutamine or whey-protein supplementation on alloxan-induced diabetic rats. Effects on CD4+ and CD8+ lymphocytes Efeitos da oferta de glutamina ou de proteína do soro de leite sobre os linfócitos CD4+ e CD8+ em ratos diabéticos aloxano induzidos

    Directory of Open Access Journals (Sweden)

    Renato Motta Neto

    2007-06-01

    Full Text Available PURPOSE: To evaluate the effects of glutamine (L-Gln or whey-protein supplementation on CD4+ and CD8+ lymphocytes in alloxan-induced diabetic rats. METHODS: Thirty-two healthy male Wistar rats were used in the experiment. Eight rats served as baseline controls (G-1. The remaining 24 animals received alloxan 150mg/Kg intraperitonially dissolved in buffer solution and were equally distributed in 3 subgroups, upon induction of diabetes mellitus, and treated as follows: (G2: saline, 2.0ml; (G3: glutamine solution (0.7g/kg, 2.0 ml; and (G4: whey-protein (WPS solution (0.7g/kg, 2.0 ml. All solutions were administered by daily 7:00 AM gavages during 30 days. Next, arterial blood samples (3.0 ml were collected from anesthetized rats for CD4+ and CD8+ lymphocyte count through flow cytometry technology. RESULTS: CD4+ and CD8+ counts decreased significantly in all groups compared with baseline values (G1. G2 rats CD4+/CD8+ ratio decreased significantly compared with G1. CD4+/CD8+ ratio increased significantly (>260% in L-Gln treated group (G3 compared with saline-treated rats (G2. There were no statistical differences in lymphocyte counts (CD4+ and CD8+ between L-Gln (G3 and saline-treated (G2 groups. There was a significant reduction in CD8+ cell count compared with CD4+ cell count in L-Gln treated rats (G3. CONCLUSION: The offer of L-Gln to experimental diabetic rats enhances the immunologic response to infection.OBJETIVO: Avaliar os efeitos da suplementação de glutamina ou proteína do soro de leite ( PSL sobre os linfócitos CD4+ e CD8+ em ratos diabéticos aloxano induzidos. MÉTODOS: Trinta e dois ratos Wistar machos, saudáveis, foram utilizados no estudo. Oito ratos foram usados como controles basais (G1. Os 24 animais remanescentes foram equitativamente distribuídos em 3 subgrupos, após indução do diabetes mellitus por injeção intraperitonial de aloxano (150mg/Kg e tratados como se segue: (G2: salina; (G3: 2,0 ml de solução de glutamina

  9. Immunotherapy of non-Hodgkin lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells

    Science.gov (United States)

    Turtle, Cameron J.; Hanafi, Laïla-Aïcha; Berger, Carolina; Hudecek, Michael; Pender, Barbara; Robinson, Emily; Hawkins, Reed; Chaney, Colette; Cherian, Sindhu; Chen, Xueyan; Soma, Lorinda; Wood, Brent; Li, Daniel; Heimfeld, Shelly; Riddell, Stanley R.; Maloney, David G.

    2016-01-01

    CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that impact toxicity and efficacy have been difficult to define because of differences in lymphodepletion regimens and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4+:CD8+ ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had markedly increased CAR-T cell expansion and persistence, and higher response rates (50% CR, 72% ORR, n=20) than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR, n=12). The complete response (CR) rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR, n=11). Cy/Flu minimized the effects of an immune response to the murine scFv component of the CAR, which limited CAR-T cell expansion, persistence, and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥ 3 neurotoxicity were observed in 13% and 28% of all patients, respectively. Serum biomarkers one day after CAR-T cell infusion correlated with subsequent development of sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4+:CD8+ ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of a lymphodepletion regimen that improved disease response and overall and progression-free survival. PMID:27605551

  10. Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(posCD25(high T cells for immunotherapy.

    Directory of Open Access Journals (Sweden)

    Jorieke H Peters

    Full Text Available BACKGROUND: Regulatory T cell (Treg based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4(posCD25(high Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent. CONCLUSIONS/SIGNIFICANCE: The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

  11. Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

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    Kristin Kassler

    2011-01-01

    Full Text Available The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4. Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120.

  12. The colocalization potential of HIV-specific CD8+ and CD4+ T-cells is mediated by integrin β7 but not CCR6 and regulated by retinoic acid.

    Directory of Open Access Journals (Sweden)

    Vanessa Sue Wacleche

    Full Text Available CD4(+ T-cells from gut-associated lymphoid tissues (GALT are major targets for HIV-1 infection. Recruitment of excess effector CD8(+ T-cells in the proximity of target cells is critical for the control of viral replication. Here, we investigated the colocalization potential of HIV-specific CD8(+ and CD4(+ T-cells into the GALT and explored the role of retinoic acid (RA in regulating this process in a cohort of HIV-infected subjects with slow disease progression. The expression of the gut-homing molecules integrin β7, CCR6, and CXCR3 was identified as a "signature" for HIV-specific but not CMV-specific CD4(+ T-cells thus providing a new explanation for their enhanced permissiveness to infection in vivo. HIV-specific CD8(+ T-cells also expressed high levels of integrin β7 and CXCR3; however CCR6 was detected at superior levels on HIV-specific CD4(+ versus CD8(+ T-cells. All trans RA (ATRA upregulated the expression of integrin β7 but not CCR6 on HIV-specific T-cells. Together, these results suggest that HIV-specific CD8(+ T-cells may colocalize in excess with CD4(+ T-cells into the GALT via integrin β7 and CXCR3, but not via CCR6. Considering our previous findings that CCR6(+CD4(+ T-cells are major cellular targets for HIV-DNA integration in vivo, a limited ability of CD8(+ T-cells to migrate in the vicinity of CCR6(+CD4(+ T-cells may facilitate HIV replication and dissemination at mucosal sites.

  13. CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

    Science.gov (United States)

    Brown, W C; Davis, W C; Dobbelaere, D A; Rice-Ficht, A C

    1994-01-01

    The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61

  14. Specific assay measuring binding of /sup 125/I-Gp 120 from HIV to T4/sup +//CD4/sup +/ cells

    Energy Technology Data Exchange (ETDEWEB)

    Lundin, K.; Nygren, A.; Ramstedt, U.; Gidlund, M.; Wigzell, H.; Arthur, L.O.; Robey, W.G.; Morein, B.

    1987-02-26

    The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4/sup +/ (T4/sup +/) cells. /sup 125/I-Gp120 could be shown to selectively bind to CD4/sup +/ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4/sup +/ cells. Monoclonal antibodies to other cell surface components do not interfere with /sup 125/I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block /sup 125/I-GP120 binding, though with variable titers. The authors believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding. (Auth.). 24 refs.; 2 figs.; 8 tabs.

  15. Potential contribution of a novel Tax epitope-specific CD4+ T cells to graft-versus-Tax effect in adult T cell leukemia patients after allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Tamai, Yotaro; Hasegawa, Atsuhiko; Takamori, Ayako; Sasada, Amane; Tanosaki, Ryuji; Choi, Ilseung; Utsunomiya, Atae; Maeda, Yasuhiro; Yamano, Yoshihisa; Eto, Tetsuya; Koh, Ki-Ryang; Nakamae, Hirohisa; Suehiro, Youko; Kato, Koji; Takemoto, Shigeki; Okamura, Jun; Uike, Naokuni; Kannagi, Mari

    2013-04-15

    Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for adult T cell leukemia/lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). We previously reported that Tax-specific CD8(+) cytotoxic T lymphocyte (CTL) contributed to graft-versus-ATL effects in ATL patients after allo-HSCT. However, the role of HTLV-1-specific CD4(+) T cells in the effects remains unclear. In this study, we showed that Tax-specific CD4(+) as well as CD8(+) T cell responses were induced in some ATL patients following allo-HSCT. To further analyze HTLV-1-specific CD4(+) T cell responses, we identified a novel HLA-DRB1*0101-restricted epitope, Tax155-167, recognized by HTLV-1-specific CD4(+) Th1-like cells, a major population of HTLV-1-specific CD4(+) T cell line, which was established from an ATL patient at 180 d after allo-HSCT from an unrelated seronegative donor by in vitro stimulation with HTLV-1-infected cells from the same patient. Costimulation of PBMCs with both the identified epitope (Tax155-167) and known CTL epitope peptides markedly enhanced the expansion of Tax-specific CD8(+) T cells in PBMCs compared with stimulation with CTL epitope peptide alone in all three HLA-DRB1*0101(+) patients post-allo-HSCT tested. In addition, direct detection using newly generated HLA-DRB1*0101/Tax155-167 tetramers revealed that Tax155-167-specific CD4(+) T cells were present in all HTLV-1-infected individuals tested, regardless of HSCT. These results suggest that Tax155-167 may be the dominant epitope recognized by HTLV-1-specific CD4(+) T cells in HLA-DRB1*0101(+)-infected individuals and that Tax-specific CD4(+) T cells may augment the graft-versus-Tax effects via efficient induction of Tax-specific CD8(+) T cell responses.

  16. Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

    Science.gov (United States)

    Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

    2006-04-01

    BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

  17. Loss of memory CD4+ T-cells in semi-wild mandrills (Mandrillus sphinx) naturally infected with species-specific simian immunodeficiency virus SIVmnd-1.

    Science.gov (United States)

    Greenwood, Edward J D; Schmidt, Fabian; Liégeois, Florian; Kondova, Ivanela; Herbert, Anaïs; Ngoubangoye, Barthelemy; Rouet, François; Heeney, Jonathan L

    2014-01-01

    Simian immunodeficiency virus (SIV) infection is found in a number of African primate species and is thought to be generally non-pathogenic. However, studies of wild primates are limited to two species, with SIV infection appearing to have a considerably different outcome in each. Further examination of SIV-infected primates exposed to their natural environment is therefore warranted. We performed a large cross-sectional study of a cohort of semi-wild mandrills with naturally occurring SIV infection, including 39 SIV-negative and 33 species-specific SIVmnd-1-infected animals. This study was distinguished from previous reports by considerably greater sample size, examination of exclusively naturally infected animals in semi-wild conditions and consideration of simian T-lymphotropic virus (STLV) status in addition to SIVmnd-1 infection. We found that SIVmnd-1 infection was associated with a significant and progressive loss of memory CD4(+) T-cells. Limited but significant increases in markers of immune activation in the T-cell populations, significant increases in plasma neopterin and changes to B-cell subsets were also observed in SIV-infected animals. However, no increase in plasma soluble CD14 was observed. Histological examination of peripheral lymph nodes suggested that SIVmnd-1 infection was not associated with a significant disruption of the lymph node architecture. Whilst this species has evolved numerous strategies to resist the development of AIDS, significant effects of SIV infection could be observed when examined in a natural environment. STLVmnd-1 infection also had significant effects on some markers relevant to understanding SIV infection and thus should be considered in studies of SIV infection of African primates where present.

  18. CD4 cell count and viral load-specific rates of AIDS, non-AIDS and deaths according to current antiretroviral use

    DEFF Research Database (Denmark)

    Mocroft, Amanda; Phillips, Andrew N; Gatell, Jose

    2013-01-01

    CD4 cell count and viral loads are used in clinical trials as surrogate endpoints for assessing efficacy of newly available antiretrovirals. If antiretrovirals act through other pathways or increase the risk of disease this would not be identified prior to licensing. The aim of this study...

  19. Evaluation of immunological cross-reactivity between clade A9 high-risk human papillomavirus types on the basis of E6-Specific CD4+ memory T cell responses

    NARCIS (Netherlands)

    van den Hende, Muriel; Redeker, Anke; Kwappenberg, Kitty M. C.; Franken, Kees L. M. C.; Drijfhout, Jan W.; Oostendorp, Jaap; Valentijn, A. Rob P. M.; Fathers, Loraine M.; Welters, Marij J. P.; Melief, Cornelis J. M.; Kenter, Gemma G.; van der Burg, Sjoerd H.; Offringa, Rienk

    2010-01-01

    CD4(+) T cell responses against the E6 oncoprotein of human papillomavirus (HPV) type 16 and 5 closely related members of clade A9 (HPV31, 33, 35, 52, and 58) were charted in peripheral blood mononuclear cell cultures from healthy subjects and patients who underwent HPV16 E6/E7-specific vaccination.

  20. Ag85A-specific CD4+ T cell lines derived after boosting BCG-vaccinated cattle with Ad5-85A possess both mycobacterial growth inhibition and anti-inflammatory properties.

    Science.gov (United States)

    Metcalfe, Hannah J; Biffar, Lucia; Steinbach, Sabine; Guzman, Efrain; Connelley, Tim; Morrison, Ivan; Vordermeier, H Martin; Villarreal-Ramos, Bernardo

    2018-05-11

    There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4 + T cells post-boosting. Here, the capacity of Ag85A-specific CD4 + T cell lines - derived before and after viral boosting - to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4 + T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1β, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4 + T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology. Copyright © 2018 Department for Environment Food and Rural Affairs. Published by Elsevier Ltd.. All rights reserved.

  1. HuMax-CD4

    DEFF Research Database (Denmark)

    Skov, Lone; Kragballe, Knud; Zachariae, Claus

    2003-01-01

    BACKGROUND: Psoriasis is characterized by infiltration with mononuclear cells. Especially activated memory CD4+ T cells are critical in the pathogenesis. Interaction between the CD4 receptor and the major histocompatibility complex class II molecule is important for T-cell activation. OBJECTIVE......: To test safety and efficacy of a fully human monoclonal anti-CD4 antibody (HuMax-CD4) in the treatment of psoriasis. DESIGN: Multicenter, double-blind, placebo-controlled, randomized clinical trial. Patients Eighty-five patients with moderate to severe psoriasis. INTERVENTIONS: Subcutaneous infusions...... dose level, 6 (38%) of 16 patients obtained more than 25% reduction of PASI and 3 (19%) obtained more than 50% reduction of PASI. A dose-dependent decrease in total lymphocyte count was seen and was parallel to a dose-dependent decrease in CD4+ T cells. This decrease was due to a decrease in the memory...

  2. Toll-like receptor 3 signalling up-regulates expression of the HIV co-receptor G-protein coupled receptor 15 on human CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Miriam Kiene

    Full Text Available BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF and was more prominent on gut-homing compared to lymph node-homing CD4(+ T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+ T cells to HIV infection and target cell availability in the gut in some infected individuals.

  3. CD4+/CD8+ double-positive T cells

    DEFF Research Database (Denmark)

    Overgaard, Nana H; Jung, Ji-Won; Steptoe, Raymond J

    2015-01-01

    CD4(+)/CD8(+) DP thymocytes are a well-described T cell developmental stage within the thymus. However, once differentiated, the CD4(+) lineage or the CD8(+) lineage is generally considered to be fixed. Nevertheless, mature CD4(+)/CD8(+) DP T cells have been described in the blood and peripheral...... cells, CD4(+)/CD8(+) T cell populations, outside of the thymus, have recently been described to express concurrently ThPOK and Runx3. Considerable heterogeneity exists within the CD4(+)/CD8(+) DP T cell pool, and the function of CD4(+)/CD8(+) T cell populations remains controversial, with conflicting...... reports describing cytotoxic or suppressive roles for these cells. In this review, we describe how transcriptional regulation, lineage of origin, heterogeneity of CD4 and CD8 expression, age, species, and specific disease settings influence the functionality of this rarely studied T cell population....

  4. Characterization of the antigen-specific CD4+ T cell response induced by prime-boost strategies with CAF01 and CpG adjuvants administered by the intranasal and subcutaneous routes

    Directory of Open Access Journals (Sweden)

    Annalisa eCiabattini

    2015-08-01

    Full Text Available The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1821 adjuvants, administered by the parenteral and nasal routes. By using peptide-MHC class II tetramers, antigen-specific CD4+ T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4+ tetramer-positive T cells in the spleen, however only parenterally primed cells responded to booster immunization. Subcutaneous priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4+ tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4+ T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that subcutaneous priming is fundamental for eliciting CD4+ T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses.

  5. Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

    Science.gov (United States)

    Abbott, Jeffrey R; Palmer, Guy H; Howard, Chris J; Hope, Jayne C; Brown, Wendy C

    2004-12-01

    Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

  6. Old and New World arenaviruses share a highly conserved epitope in the fusion domain of the glycoprotein 2, which is recognized by Lassa virus-specific human CD4+ T-cell clones

    International Nuclear Information System (INIS)

    Meulen, Jan ter; Badusche, Marlis; Satoguina, Judith; Strecker, Thomas; Lenz, Oliver; Loeliger, Cornelius; Sakho, Mohamed; Koulemou, Kekoura; Koivogui, Lamine; Hoerauf, Achim

    2004-01-01

    Data from human studies and animal experiments indicate a dominant role of T-cells over antibodies in controlling acute Lassa virus infection and providing immunity to reinfection. Knowledge of the epitopes recognized by T-cells may therefore be crucial to the development of a recombinant Lassa virus vaccine. In order to study human T-cell reactivity to the most conserved structural protein of Lassa virus, the glycoprotein 2 (GP2), seven GP2-specific CD4+ T-cell clones (TCCs) were generated from the lymphocytes of a Lassa antibody positive individual. All TCC displayed high specific proliferation, showed DR-restriction, and produced IFN-γ upon stimulation with recombinant GP2. The epitope of four of the clones was localized to a short stretch of 13 amino acids located in the N-terminal part of GP2 (aa 289-301, numbering according to sequence of GPC). This epitope is conserved in all strains of Lassa virus and lymphocytic choriomeningitis virus (LCMV), shows >90% similarity in all New World arenaviruses of clade B, and overlaps with the proposed fusion domain of GP2. Peptides with conservative aa exchanges, as they naturally occur in the epitope 289-301 of the Old World arenavirus Mopeia and some New World arenaviruses, continued to effectively stimulate the Lassa-GP2-specific T-cell clones tested. The finding of a human T-helper cell epitope, which is highly conserved between Old and New World arenaviruses, is of importance for the design of arenavirus vaccines

  7. A Sensitive Method for Detecting Peptide-specific CD4+ T Cell Responses in Peripheral Blood from Patients with Myasthenia Gravis

    Science.gov (United States)

    Sharma, Sapna; Malmeström, Clas; Lindberg, Christopher; Meisel, Sarah; Schön, Karin; Verolin, Martina; Lycke, Nils Yngve

    2017-01-01

    Myasthenia gravis (MG) is an autoimmune neurological disorder typified by skeletal muscle fatigue and most often production of autoantibodies against the nicotinic acetylcholine receptor (AChR). The present study was undertaken to assess the extent of AChR-peptide recognition in MG patients using co-culturing (DC:TC) of autologous monocyte-derived dendritic cells (moDCs) and highly enriched CD4+ T cells from the blood as compared to the traditional whole peripheral blood mononuclear cell (PBMC) cultures. We found that the DC:TC cultures were highly superior to the PBMC cultures for detection of reactivity toward HLA-DQ/DR-restricted AChR-peptides. In fact, whereas DC:TC cultures identified recognition in all MG patients the PBMC cultures failed to detect responsiveness in around 40% of the patients. Furthermore, reactivity to multiple peptides was evident in DC:TC cultures, while PBMC cultures mostly exhibited reactivity to a single peptide. No healthy control (HC) CD4+ T cells responded to the peptides in either culture system. Interestingly, whereas spontaneous production of IFNγ and IL-17 was observed in the DC:TC cultures from MG patients, recall responses to peptides enhanced IL-10 production in 9/13 MG patients, while little increase in IFNγ and IL-17 was seen. HCs did not produce cytokines to peptide stimulations. We conclude that the DC: TC culture system is significantly more sensitive and better identifies the extent of responsiveness in MG patients to AChR-peptides than traditional PBMC cultures. PMID:29114250

  8. Effects of peritoneal fluid from endometriosis patients on interferon-gamma-induced protein-10 (CXCL10) and interleukin-8 (CXCL8) released by neutrophils and CD4+ T cells.

    Science.gov (United States)

    Kim, Ji-Yeon; Lee, Dong-Hyung; Joo, Jong-Kil; Jin, Jun-O; Wang, Ji-Won; Hong, Young-Seoub; Kwak, Jong-Young; Lee, Kyu-Sup

    2009-09-01

    Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-gamma-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4(+) T cells, and monocytes. Neutrophils, CD4(+) T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay. The addition of ePF to cultures of CD4(+) T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF (R = 0.89, P = 0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4(+) T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils (R = 0.89, P ePF significantly enhanced the interferon-gamma-induced release of IP-10 by nuetrophils and CD4(+) T cells. These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.

  9. Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells.

    Directory of Open Access Journals (Sweden)

    Ilgiz A Mufazalov

    Full Text Available Interleukin-1 (IL-1 plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1. To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment.

  10. Calcineurin B in CD4+ T Cells Prevents Autoimmune Colitis by Negatively Regulating the JAK/STAT Pathway.

    Science.gov (United States)

    Mencarelli, Andrea; Vacca, Maurizio; Khameneh, Hanif Javanmard; Acerbi, Enzo; Tay, Alicia; Zolezzi, Francesca; Poidinger, Michael; Mortellaro, Alessandra

    2018-01-01

    Calcineurin (Cn) is a protein phosphatase that regulates the activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors, which are key regulators of T-cell development and function. Here, we generated a conditional Cnb1 mouse model in which Cnb1 was specifically deleted in CD4 + T cells (Cnb1 CD4 mice) to delineate the role of the Cn-NFAT pathway in immune homeostasis of the intestine. The Cnb1 CD4 mice developed severe, spontaneous colitis characterized at the molecular level by an increased T helper-1-cell response but an unaltered regulatory T-cell compartment. Antibiotic treatment ameliorated the intestinal inflammation observed in Cnb1 CD4 mice, suggesting that the microbiota contributes to the onset of colitis. CD4 + T cells isolated from Cnb1 CD4 mice produced high levels of IFNγ due to increased activation of the JAK2/STAT4 pathway induced by IL-12. Our data highlight that Cn signaling in CD4 + T cells is critical for intestinal immune homeostasis in part by inhibiting IL-12 responsiveness of CD4 + T cells.

  11. Low-dose temozolomide before dendritic-cell vaccination reduces (specifically) CD4+CD25++Foxp3+ regulatory T-cells in advanced melanoma patients.

    Science.gov (United States)

    Ridolfi, Laura; Petrini, Massimiliano; Granato, Anna Maria; Gentilcore, Giusy; Simeone, Ester; Ascierto, Paolo Antonio; Pancisi, Elena; Ancarani, Valentina; Fiammenghi, Laura; Guidoboni, Massimo; de Rosa, Francesco; Valmorri, Linda; Scarpi, Emanuela; Nicoletti, Stefania Vittoria Luisa; Baravelli, Stefano; Riccobon, Angela; Ridolfi, Ruggero

    2013-05-31

    In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

  12. Rat eosinophils stimulate the expansion of Cryptococcus neoformans-specific CD4+ and CD8+ T cells with a T-helper 1 profile

    Science.gov (United States)

    Garro, Ana P; Chiapello, Laura S; Baronetti, José L; Masih, Diana T

    2011-01-01

    Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, revealing a strong granulomatous response and a low susceptibility to dissemination. Moreover, it has been shown that eosinophils are components of the inflammatory response to C. neoformans infections. In this in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, and that the phenomenon involves the engagement of FcγRII and CD18. Moreover, our results showed that the phagocytosis of opsonized C. neoformans triggers eosinophil activation, as indicated by (i) the up-regulation of major histocompatibility complex (MHC) class I, MHC class II and costimulatory molecules, and (ii) an increase in interleukin (IL)-12, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production. However, nitric oxide (NO) and hydrogen peroxide (H2O2) synthesis by eosinophils was down-regulated after interaction with C. neoformans. Furthermore, this work demonstrated that CD4+ and CD8+ T lymphocytes isolated from spleens of infected rats and cultured with C. neoformans-pulsed eosinophils proliferate in an MHC class II- and class I-dependent manner, respectively, and produce important amounts of T-helper 1 (Th1) type cytokines, such as TNF-α and IFN-γ, in the absence of T-helper 2 (Th2) cytokine synthesis. In summary, the present study demonstrates that eosinophils act as fungal antigen-presenting cells and suggests that C. neoformans-loaded eosinophils might participate in the adaptive immune response. PMID:21039463

  13. GM-CSF production allows the identification of immunoprevalent antigens recognized by human CD4+ T cells following smallpox vaccination.

    Directory of Open Access Journals (Sweden)

    Valeria Judkowski

    Full Text Available The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a "T cell-driven" methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.

  14. CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury.

    Science.gov (United States)

    Mathes, Denise; Weirather, Johannes; Nordbeck, Peter; Arias-Loza, Anahi-Paula; Burkard, Matthias; Pachel, Christina; Kerkau, Thomas; Beyersdorf, Niklas; Frantz, Stefan; Hofmann, Ulrich

    2016-12-01

    The present study analyzed the effect of CD4 + Forkhead box protein 3 negative (Foxp3 - ) T-cells and Foxp3 + CD4 + T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. We examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4 + T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4 + T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4 + T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4 + T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4 + T-cells in CD4 KO mice increased the infarct size only when including the Foxp3 + CD25 + subset. Depletion of CD4 + Foxp3 + T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. CD4 + Foxp3 + T-cells enhance myocardial ischemia-reperfusion injury. CD4 + T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

    Science.gov (United States)

    David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

    2005-04-01

    Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

  16. Disease specific protein corona

    Science.gov (United States)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  17. Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1.

    Science.gov (United States)

    Maywald, Martina; Rink, Lothar

    2017-08-01

    The essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms. Cell proliferation and pro-inflammatory cytokine production were monitored by [ 3 H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression. Zinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4 + CD25 + Foxp3 + and CD4 + CD25 + CTLA-4 + T cells (p zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1. In summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.

  18. CD4~+Foxp3~+ regulatory T cells converted by rapamycin from peripheral CD4~+ CD25~-naive T cells display more potent regulatory ability in vitro

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-fei; GAO Jie; ZHANG Dong; WANG Zi-han; ZHU Ji-ye

    2010-01-01

    Background Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells. Methods Peripheral CD4~+CD25~- naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4~+CD25~- T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4~+CD25~+CD127~- subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4~+CD25~+ T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1×10~5 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4~+CD25~- T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25×10~5/well) in 96-well round-bottom plates for 6 days. Then 1×10~5 or 0.25×10~5 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4~+CD25~- T cells using flow cytometry. Data were analyzed using CellQuest software.Results We found that RAPA can convert peripheral CD4~+CD25~- naive T Cells to CD4~+Foxp3~+ Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity. Conclusion Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and

  19. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn

    2007-01-01

    -linked lymphocytic choriomeningitis virus (LCMV)-derived epitopes was long-lived and protective. Notably, in contrast to full-length protein, the response elicited with the beta(2)-microglobulin-linked LCMV-derived epitope was CD4(+) T-cell independent. Furthermore, virus-specific CD8(+) T cells primed...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T-cell...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin...

  20. Tumor infiltrating BRAFV600E-specific CD4 T cells correlated with complete clinical response in melanoma. | Office of Cancer Genomics

    Science.gov (United States)

    T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who obtained a complete response following adoptive transfer of tumor infiltrating lymphocytes (TIL).

  1. Inhibition of protein geranylgeranylation and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells.

    Science.gov (United States)

    Hechinger, Anne-Kathrin; Maas, Kristina; Dürr, Christoph; Leonhardt, Franziska; Prinz, Gabriele; Marks, Reinhard; Gerlach, Ulrike; Hofmann, Maike; Fisch, Paul; Finke, Jürgen; Pircher, Hanspeter; Zeiser, Robert

    2013-01-01

    Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without

  2. A Quantitative Analysis of Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans.

    Directory of Open Access Journals (Sweden)

    Cecilia S Lindestam Arlehamn

    2016-07-01

    Full Text Available We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb, in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50-75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total. The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive "megapool" of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

  3. Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells.

    Science.gov (United States)

    Nishida, Atsushi; Nagahama, Kiyotaka; Imaeda, Hirotsugu; Ogawa, Atsuhiro; Lau, Cindy W; Kobayashi, Taku; Hisamatsu, Tadakazu; Preffer, Frederic I; Mizoguchi, Emiko; Ikeuchi, Hiroki; Hibi, Toshifumi; Fukuda, Minoru; Andoh, Akira; Blumberg, Richard S; Mizoguchi, Atsushi

    2012-12-17

    Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

  4. Neonatal colonisation expands a specific intestinal antigen-presenting cell subset prior to CD4 T-cell expansion, without altering T-cell repertoire.

    Directory of Open Access Journals (Sweden)

    Charlotte F Inman

    Full Text Available Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα(+ antigen-presenting cell subset, whilst SIRPα(-CD11R1(+ antigen-presenting cells (APCs are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα(+ antigen-presenting cells as orchestrators of early-life mucosal immune development.

  5. Necroptosis takes place in human immunodeficiency virus type-1 (HIV-1-infected CD4+ T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Ting Pan

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection is characterized by progressive depletion of CD4+ T lymphocytes and dysfunction of the immune system. The numbers of CD4+ T lymphocytes in the human body are maintained constantly by homeostatic mechanisms that failed during HIV-1 infection, resulting in progressive loss of CD4+ T cells mainly via apoptosis. Recently, a non-apoptotic form of necrotic programmed cell death, named necroptosis, has been investigated in many biological and pathological processes. We then determine whether HIV-1-infected cells also undergo necroptosis. In this report, we demonstrate that HIV-1 not only induces apoptosis, but also mediates necroptosis in the infected primary CD4+ T lymphocytes and CD4+ T-cell lines. Necroptosis-dependent cytopathic effects are significantly increased in HIV-1-infected Jurkat cells that is lack of Fas-associated protein-containing death domain (FADD, indicating that necroptosis occurs as an alternative cell death mechanism in the absence of apoptosis. Unlike apoptosis, necroptosis mainly occurs in HIV-infected cells and spares bystander damage. Treatment with necrostatin-1(Nec-1, a RIP1 inhibitor that specifically blocks the necroptosis pathway, potently restrains HIV-1-induced cytopathic effect and interestingly, inhibits the formation of HIV-induced syncytia in CD4+ T-cell lines. This suggests that syncytia formation is mediated, at least partially, by necroptosis-related processes. Furthermore, we also found that the HIV-1 infection-augmented tumor necrosis factor-alpha (TNF-α plays a key role in inducing necroptosis and HIV-1 Envelope and Tat proteins function as its co-factors. Taken together,necroptosis can function as an alternative cell death pathway in lieu of apoptosis during HIV-1 infection, thereby also contributing to HIV-1-induced cytopathic effects. Our results reveal that in addition to apoptosis, necroptosis also plays an important role in HIV-1-induced pathogenesis.

  6. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M. [Department of Microbiology, University of Washington, Seattle, WA (United States); Wolf-Yadlin, Alejandro [Department of Genome Sciences, University of Washington, Seattle, WA (United States); Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C. [Department of Microbiology, University of Washington, Seattle, WA (United States); Jahan, Tahmina A. [Proteomics Resource, UW Medicine at South Lake Union, Seattle, WA (United States); Krasnoselsky, Alexei L.; Palermo, Robert E. [Department of Microbiology, University of Washington, Seattle, WA (United States); Katze, Michael G., E-mail: honey@uw.edu [Department of Microbiology, University of Washington, Seattle, WA (United States); Washington National Primate Research Center, University of Washington, Seattle, WA (United States)

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

  7. Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I-associated neuroimmunological disease.

    Science.gov (United States)

    Yamano, Yoshihisa; Takenouchi, Norihiro; Li, Hong-Chuan; Tomaru, Utano; Yao, Karen; Grant, Christian W; Maric, Dragan A; Jacobson, Steven

    2005-05-01

    CD4(+)CD25(+) Tregs are important in the maintenance of immunological self tolerance and in the prevention of autoimmune diseases. As the CD4(+)CD25(+) T cell population in patients with human T cell lymphotropic virus type I-associated (HTLV-I-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) has been shown to be a major reservoir for this virus, it was of interest to determine whether the frequency and function of CD4(+)CD25(+) Tregs in HAM/TSP patients might be affected. In these cells, both mRNA and protein expression of the forkhead transcription factor Foxp3, a specific marker of Tregs, were lower than those in CD4(+)CD25(+) T cells from healthy individuals. The virus-encoded transactivating HTLV-I tax gene was demonstrated to have a direct inhibitory effect on Foxp3 expression and function of CD4(+)CD25(+) T cells. This is the first report to our knowledge demonstrating the role of a specific viral gene product (HTLV-I Tax) on the expression of genes associated with Tregs (in particular, foxp3) resulting in inhibition of Treg function. These results suggest that direct human retroviral infection of CD4(+)CD25(+) T cells may be associated with the pathogenesis of HTLV-I-associated neurologic disease.

  8. Identifying immunogenic CD4+ T-cell epitopes of Myeloid cell leukemia 1 using overlapping 20-mer peptides spanning the whole protein

    DEFF Research Database (Denmark)

    Woodworth, Joshua S.; Agger, Else Marie; Hansen, Paul Robert

    2015-01-01

    ) small-molecule inhibitors [6] and (iii) peptide inhibitors [7]. In recent years, therapeutic vaccination with synthetic peptides derived from anti-apoptotic proteins such as Mcl-1 has emerged as a promising strategy against hematological cancers. In this study, 34 overlapping 20-mer peptides, spanning...

  9. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    Energy Technology Data Exchange (ETDEWEB)

    Ohnuma, Kei [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Uchiyama, Masahiko [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Computational Intelligence and System Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Hatano, Ryo; Takasawa, Wataru; Endo, Yuko [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Dang, Nam H. [Department of Hematologic Malignancies, Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Morimoto, Chikao, E-mail: morimoto@ims.u-tokyo.ac.jp [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan)

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  10. Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells

    Science.gov (United States)

    Carrette, Florent; Henriquez, Monique L.; Fujita, Yu

    2018-01-01

    T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus–specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7−/− effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7−/− CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells. PMID:29491007

  11. CD4-regulatory cells in COPD patients

    DEFF Research Database (Denmark)

    Smyth, Lucy J C; Starkey, Cerys; Vestbo, Jørgen

    2007-01-01

    BACKGROUND: The numbers of airway CD8 and B lymphocytes are increased in COPD patients, suggesting an autoimmune process. CD4-regulatory T cells control autoimmunity but have not been studied in patients with COPD. OBJECTIVE: To compare T-regulatory cell numbers in the BAL from COPD patients......, smokers with normal lung function, and healthy nonsmokers (HNS). METHODS: BAL and peripheral blood mononuclear cell (PBMC) samples were obtained from 26 COPD patients, 19 smokers, and 8 HNS. Flow cytometry was performed for regulatory phenotypic markers. RESULTS: COPD patients had increased BAL CD8...... numbers compared to smokers and HNS. CD4 numbers were similar between groups. There was increased BAL CD4CD25(bright) expression in smokers (median 28.8%) and COPD patients (median 23.1%) compared to HNS (median 0%). Increased FoxP3 expression was confirmed in BAL CD4CD25(bright) cells. BAL CD4CD25 cells...

  12. A general approach for controlling transcription and probing epigenetic mechanisms: application to the CD4 locus.

    Science.gov (United States)

    Wan, Mimi; Kaundal, Ravinder; Huang, Haichang; Zhao, Jiugang; Yang, Xiaojun; Chaiyachati, Barbara H; Li, Sicong; Chi, Tian

    2013-01-15

    Synthetic regulatory proteins such as tetracycline (tet)-controlled transcription factors are potentially useful for repression as well as ectopic activation of endogenous genes and also for probing their regulatory mechanisms, which would offer a versatile genetic tool advantageous over conventional gene targeting methods. In this study, we provide evidence supporting this concept using Cd4 as a model. CD4 is expressed in double-positive and CD4 cells but irreversibly silenced in CD8 cells. The silencing is mediated by heterochromatin established during CD8 lineage development via transient action of the Cd4 silencer; once established, the heterochromatin becomes self-perpetuating independently of the Cd4 silencer. Using a tet-sensitive Cd4 allele harboring a removable Cd4 silencer, we found that a tet-controlled repressor recapitulated the phenotype of Cd4-deficient mice, inhibited Cd4 expression in a reversible and dose-dependent manner, and could surprisingly replace the Cd4 silencer to induce irreversible Cd4 silencing in CD8 cells, thus suggesting the Cd4 silencer is not the (only) determinant of heterochromatin formation. In contrast, a tet-controlled activator reversibly disrupted Cd4 silencing in CD8 cells. The Cd4 silencer impeded this disruption but was not essential for its reversal, which revealed a continuous role of the silencer in mature CD8 cells while exposing a remarkable intrinsic self-regenerative ability of heterochromatin after forced disruption. These data demonstrate an effective approach for gene manipulation and provide insights into the epigenetic Cd4 regulatory mechanisms that are otherwise difficult to obtain.

  13. [CD4 lymphocytopenia in systemic lupus erythematosus].

    Science.gov (United States)

    Ferreira, Sofia; Vasconcelos, Júlia; Marinho, António; Farinha, Fátima; Almeida, Isabel; Correia, João; Barbosa, Paulo; Mendonça, Teresa; Vasconcelos, Carlos

    2009-01-01

    Systemic Lupus Erythematosus (SLE) is an inflammatory chronic disease characterized by the presence of autoantibodies, immunocomplex production and organ injury. Several alterations of the immune system have been described, namely of CD4 T cells, with particular focus on regulatory subgroup. Quantify peripheral CD4 T cells in a population of patients with SLE and correlate it with lupus activity, affected organs, therapeutics and infections. Retrospective study involving all SLE patients seen in the clinical immunology outpatient clinic of the Hospital Geral Santo António, Porto that has done some peripheral blood flow cytometry study. Twenty-nine patients have been evaluated, 16 were taking glucocorticoids and six immunossupressors. The mean SLEDAI at the study time was nine and the ECLAM was three. Thirty-one percent of the patients had leukopenia, 76% lymphocytopenia and the same number CD4 depletion. Fifty-five percent of the patients had CD4 levels lower than 500/mm3, 31% lower than 200/mm3. All patients with SLEDAI > or = 20 and ECLAM > or = 4 had CD4 counts inferior to 500/mm3 and all patients with inactive disease had CD4 superior to 500/mm3. There have been three opportunistic infections: cryptococcal meningitis, pulmonary aspergilosis, Pneumocystis jirovecii pneumonia, all in patients with CD4 counts lower than 500/mm3. Decreased CD4 T cells counts have been very common in this study population. There is an inverse relation between CD4 cells counts and disease activity. Opportunistic infections occurred in patients with severe CD4 depletion.

  14. Novel teleost CD4-bearing cell populations provide insights into the evolutionary origins and primordial roles of CD4+ lymphocytes and CD4+ macrophages

    OpenAIRE

    Takizawa, Fumio; Magadan, Susana; Parra, David; Xu, Zhen; Koryt����, Tom����; Boudinot, Pierre; Sunyer, J. Oriol

    2016-01-01

    Tetrapods contain a single CD4 co-receptor with four immunoglobulin domains that likely arose from a primordial two-domain ancestor. Notably, teleost fish contain two CD4 genes. Like tetrapod CD4, CD4-1 of rainbow trout includes four immunoglobulin domains while CD4-2 contains only two. Since CD4-2 is reminiscent of the prototypic two-domain CD4 co-receptor, we hypothesized that by characterizing the cell types bearing CD4-1 and CD4-2, we would shed light into the evolution and primordial rol...

  15. Dysregulated cytokine expression by CD4+ T cells from post-septic mice modulates both Th1 and Th2-mediated granulomatous lung inflammation.

    Directory of Open Access Journals (Sweden)

    William F Carson

    Full Text Available Previous epidemiological studies in humans and experimental studies in animals indicate that survivors of severe sepsis exhibit deficiencies in the activation and effector function of immune cells. In particular, CD4+ T lymphocytes can exhibit reduced proliferative capacity and improper cytokine responses following sepsis. To further investigate the cell-intrinsic defects of CD4+ T cells following sepsis, splenic CD4+ T cells from sham surgery and post-septic mice were transferred into lymphopenic mice. These recipient mice were then subjected to both TH1-(purified protein derivative and TH2-(Schistosoma mansoni egg antigen driven models of granulomatous lung inflammation. Post-septic CD4+ T cells mediated smaller TH1 and larger TH2 lung granulomas as compared to mice receiving CD4+ T cells from sham surgery donors. However, cytokine production by lymph node cells in antigen restimulation assays indicated increased pan-specific cytokine expression by post-septic CD4+ T cell recipient mice in both TH1 and TH2 granuloma models. These include increased production of T(H2 cytokines in TH1 inflammation, and increased production of T(H1 cytokines in TH2 inflammation. These results suggest that cell-intrinsic defects in CD4+ T cell effector function can have deleterious effects on inflammatory processes post-sepsis, due to a defect in the proper regulation of TH-specific cytokine expression.

  16. Changes in Reactivity In Vitro of CD4+CD25+ and CD4+CD25− T Cell Subsets in Transplant Tolerance

    Science.gov (United States)

    Hall, Bruce M.; Robinson, Catherine M.; Plain, Karren M.; Verma, Nirupama D.; Tran, Giang T.; Nomura, Masaru; Carter, Nicole; Boyd, Rochelle; Hodgkinson, Suzanne J.

    2017-01-01

    Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4+CD25+ T cells, yet in many models, proliferation of CD4+ T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4+, CD4+CD25+, and CD4+CD25− T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG) cardiac allografts and from naïve DA rats were examined. Proliferation of CD4+ T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4+CD25− T cells was greater than naïve CD4+ T cells. In contrast, proliferation of CD4+CD25− T cells from tolerant hosts to specific-donor PVG was not greater than CD4+ T cells, whereas their response to Lewis and self-DA was greater than CD4+ T cells. Paradoxically, CD4+CD25+ T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4+CD25+ T cells proliferate to both PVG and Lewis but not to self-DA. CD4+CD25+ T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN)-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4+CD25+ T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4+CD25+ T cells that mediate transplant tolerance depend on IFN−γ or IL-5 from alloactivated Th1 and Th2 cells. PMID:28878770

  17. CD4 and CD8

    Science.gov (United States)

    ... PF4 Antibody Hepatitis A Testing Hepatitis B Testing Hepatitis C Testing HER2/neu Herpes Testing High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV Antiretroviral Drug Resistance Testing, Genotypic HIV Viral Load HLA Testing HLA- ...

  18. Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells.

    Science.gov (United States)

    Boudousquie, Caroline; Bossi, Giovanna; Hurst, Jacob M; Rygiel, Karolina A; Jakobsen, Bent K; Hassan, Namir J

    2017-11-01

    The success of immune system-based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti-CD3 scFv antibody) were previously shown to redirect CD8 + and CD4 + T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma-specific protein, gp100, presented by HLA-A*0201) efficiently redirects and activates effector and memory cells from both CD8 + and CD4 + repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8 + T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4 + effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8 + and CD4 + repertoires secrete key pro-inflammatory cytokines (tumour necrosis factor-α, interferon-γ, interleukin-6) and chemokines (macrophage inflammatory protein-1α-β, interferon-γ-inducible protein-10, monocyte chemoattractant protein-1). At an individual cell level, IMCgp100-redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti-cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8 + T cell-mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

  19. Comparative magnitude and kinetics of human cytomegalovirus-specific CD4⁺ and CD8⁺ T-cell responses in pregnant women with primary versus remote infection and in transmitting versus non-transmitting mothers: Its utility for dating primary infection in pregnancy.

    Science.gov (United States)

    Fornara, Chiara; Furione, Milena; Arossa, Alessia; Gerna, Giuseppe; Lilleri, Daniele

    2016-07-01

    To discriminate between primary (PI) and remote (RI) human cytomegalovirus (HCMV) infection, several immunological parameters were monitored for a 2-year period in 53 pregnant women with PI, and 33 pregnant women experiencing HCMV PI at least 5 years prior. Cytokine (IFN-γ and IL-2) production by and phenotype (effector/memory CD45RA(+)) of HCMV-specific CD4(+) and CD8(+) T-cells as well as the lymphoproliferative responses (LPR) were evaluated, with special reference to the comparison between a group of women transmitting (T) and a group of non-transmitting (NT) the infection to fetus. While HCMV-specific CD4(+) T-cells reached at 90 days post-infection (p.i.) values comparable to RI, CD8(+) T-cells reached at 60 days p.i. levels significantly higher and persisting throughout the entire follow-up. Instead, IL-2 production and lymphoproliferative responses were lower in PI than RI for the entire follow-up period. Effector memory CD45RA(+) CD4(+) and CD8(+) HCMV-specific T-cells increased until 90 days p.i., reaching and maintaining levels higher than RI. The comparison between T and NT women showed that, at 30 days p.i., in NT women there was a significantly higher IL-2 production by HCMV-specific CD4(+) T-cells, and at 60 days p.i. a significantly higher frequency of both specific CD4(+) and CD8(+) CD45RA(+) T-cells. HCMV T-cell response appears to correlate with virus transmission to fetus and some parameters (CD4(+) lymphoproliferation, and frequency of HCMV-specific CD8(+) IL2(+) T-cells) may help in dating PI during pregnancy. © 2015 Wiley Periodicals, Inc.

  20. Role of LAP+CD4+ T cells in the tumor microenvironment of colorectal cancer.

    Science.gov (United States)

    Zhong, Wu; Jiang, Zhi-Yuan; Zhang, Lei; Huang, Jia-Hao; Wang, Shi-Jun; Liao, Cun; Cai, Bin; Chen, Li-Sheng; Zhang, Sen; Guo, Yun; Cao, Yun-Fei; Gao, Feng

    2017-01-21

    To investigate the abundance and potential functions of LAP + CD4 + T cells in colorectal cancer (CRC). Proportions of LAP + CD4 + T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box (Fox)p3, cytotoxic T-lymphocyte-associated protein (CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP - CD4 + and LAP + CD4 + T cells were isolated using a magnetic cell-sorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β. The proportion of LAP + CD4 + T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P CD4 + T cells was significantly higher in tumor tissues (11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P CD4 + T cells and TNM stage ( P cell sorting gave an overall enrichment of LAP + CD4 + T cells (95.02% ± 2.87%), which was similar for LAP - CD4 + T cells (94.75% ± 2.76%). In contrast to LAP - CD4 + T cells, LAP + CD4 + T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5 ( P CD4 + T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAP - CD4 + T cells. LAP + CD4 + T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.

  1. Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

    Directory of Open Access Journals (Sweden)

    Jodi L McGill

    Full Text Available Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

  2. Enteroantigen-presenting B cells efficiently stimulate CD4(+) T cells in vitro

    DEFF Research Database (Denmark)

    Schmidt, Esben Gjerløff Wedebye; Kristensen, Nanna Ny; Claesson, Mogens Helweg

    2011-01-01

    Presentation of enterobacterial antigens by antigen-presenting cells and activation of enteroantigen-specific CD4(+) T cells are considered crucial steps in inflammatory bowel disease (IBD) pathology. The detrimental effects of such CD4(+) T cells have been thoroughly demonstrated in models...... of colitis. Also, we have previously established an in vitro assay where murine enteroantigen-specific colitogenic CD4(+) CD25(-) T cells are activated by splenocytes pulsed with an enterobacterial extract....

  3. Chronic exposure to water pollutant trichloroethylene increased epigenetic drift in CD4(+) T cells.

    Science.gov (United States)

    Gilbert, Kathleen M; Blossom, Sarah J; Erickson, Stephen W; Reisfeld, Brad; Zurlinden, Todd J; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Cooney, Craig A

    2016-05-01

    Autoimmune disease and CD4(+) T-cell alterations are induced in mice exposed to the water pollutant trichloroethylene (TCE). We examined here whether TCE altered gene-specific DNA methylation in CD4(+) T cells as a possible mechanism of immunotoxicity. Naive and effector/memory CD4(+) T cells from mice exposed to TCE (0.5 mg/ml in drinking water) for 40 weeks were examined by bisulfite next-generation DNA sequencing. A probabilistic model calculated from multiple genes showed that TCE decreased methylation control in CD4(+) T cells. Data from individual genes fitted to a quadratic regression model showed that TCE increased gene-specific methylation variance in both CD4 subsets. TCE increased epigenetic drift of specific CpG sites in CD4(+) T cells.

  4. Chronic exposure to water pollutant trichloroethylene increased epigenetic drift in CD4+ T cells

    Science.gov (United States)

    Gilbert, Kathleen M; Blossom, Sarah J; Erickson, Stephen W; Reisfeld, Brad; Zurlinden, Todd J; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Cooney, Craig A

    2016-01-01

    Aim: Autoimmune disease and CD4+ T-cell alterations are induced in mice exposed to the water pollutant trichloroethylene (TCE). We examined here whether TCE altered gene-specific DNA methylation in CD4+ T cells as a possible mechanism of immunotoxicity. Materials & methods: Naive and effector/memory CD4+ T cells from mice exposed to TCE (0.5 mg/ml in drinking water) for 40 weeks were examined by bisulfite next-generation DNA sequencing. Results: A probabilistic model calculated from multiple genes showed that TCE decreased methylation control in CD4+ T cells. Data from individual genes fitted to a quadratic regression model showed that TCE increased gene-specific methylation variance in both CD4 subsets. Conclusion: TCE increased epigenetic drift of specific CpG sites in CD4+ T cells. PMID:27092578

  5. CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response

    Science.gov (United States)

    Barinov, Aleksandr; Galgano, Alessia; Krenn, Gerald; Tanchot, Corinne; Vasseur, Florence

    2017-01-01

    CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation. PMID:28686740

  6. CD4(+)and CD8(+)T-cell reactions against leukemia-associated- or minor-histocompatibility-antigens in AML-patients after allogeneic SCT.

    Science.gov (United States)

    Steger, Brigitte; Milosevic, Slavoljub; Doessinger, Georg; Reuther, Susanne; Liepert, Anja; Braeu, Marion; Schick, Julia; Vogt, Valentin; Schuster, Friedhelm; Kroell, Tanja; Busch, Dirk H; Borkhardt, Arndt; Kolb, Hans-Jochem; Tischer, Johanna; Buhmann, Raymund; Schmetzer, Helga

    2014-04-01

    T-cells play an important role in the remission-maintenance in AML-patients (pts) after SCT, however the role of LAA- (WT1, PR1, PRAME) or minor-histocompatibility (mHag, HA1) antigen-specific CD4(+) and CD8(+)T-cells is not defined. A LAA/HA1-peptide/protein stimulation, cloning and monitoring strategy for specific CD8(+)/CD4(+)T-cells in AML-pts after SCT is given. Our results show that (1) LAA-peptide-specific CD8+T-cells are detectable in every AML-pt after SCT. CD8(+)T-cells, recognizing two different antigens detectable in 5 of 7 cases correlate with long-lasting remissions. Clonal TCR-Vβ-restriction exemplarily proven by spectratyping in PRAME-specific CD8(+)T-cells; high PRAME-peptide-reactivity was CD4(+)-associated, as shown by IFN-γ-release. (2) Two types of antigen-presenting cells (APCs) were tested for presentation of LAA/HA1-proteins to CD4(+)T-cells: miniEBV-transduced lymphoblastoid cells (B-cell-source) and CD4-depleted MNC (source for B-cell/monocyte/DC). We provide a refined cloning-system for proliferating, CD40L(+)CD4(+)T-cells after LAA/HA1-stimulation. CD4(+)T-cells produced cytokines (GM-CSF, IFN-γ) upon exposure to LAA/HA1-stimulation until after at least 7 restimulations and demonstrated cytotoxic activity against naive blasts, but not fibroblasts. Antileukemic activity of unstimulated, stimulated or cloned CD4(+)T-cells correlated with defined T-cell-subtypes and the clinical course of the disease. In conclusion we provide immunological tools to enrich and monitor LAA/HA1-CD4(+)- and CD8(+)T-cells in AML-pts after SCT and generate data with relevant prognostic value. We were able to demonstrate the presence of LAA-peptide-specific CD8(+)T-cell clones in AML-pts after SCT. In addition, we were also able to enrich specific antileukemic reactive CD4(+)T-cells without GvH-reactivity upon repeated LAA/HA1-protein stimulation and limiting dilution cloning. Copyright © 2013 Elsevier GmbH. All rights reserved.

  7. Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

    Science.gov (United States)

    Guittard, Geoffrey; Gallardo, Devorah L; Li, Wenmei; Melis, Nicolas; Lui, Julian C; Kortum, Robert L; Shakarishvili, Nicholas G; Huh, Sunmee; Baron, Jeffrey; Weigert, Roberto; Kramer, Joshua A; Samelson, Lawrence E; Sommers, Connie L

    2017-01-01

    RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1 f/f Sos2 -/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.

  8. Functional Heterogeneity in the CD4+ T Cell Response to Murine γ-Herpesvirus 68

    Science.gov (United States)

    Hu, Zhuting; Blackman, Marcia A.; Kaye, Kenneth M.; Usherwood, Edward J.

    2015-01-01

    CD4+ T cells are critical for the control of virus infections, T cell memory and immune surveillance. Here we studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)-specific CD4+ T cells using gp150-specific TCR transgenic mice. This allowed a more detailed study of the characteristics of the CD4+ T cell response than previously available approaches for this virus. Most gp150-specific CD4+ T cells expressed T-bet and produced IFN-γ, indicating MHV-68 infection triggered differentiation of CD4+ T cells largely into the Th1 subset, whereas some became TFH and Foxp3+ regulatory T cells. These CD4+ T cells were protective against MHV-68 infection, in the absence of CD8+ T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4+ T cells, based on Ly6C expression. Ly6C expression positively correlated with IFN-γ, TNF-α and granzyme B production, T-bet and KLRG1 expression, proliferation and CD4+ T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression and secondary expansion potential. Ly6C+ and Ly6C− gp150-specific CD4+ T cells were able to interconvert in a bidirectional manner upon secondary antigen exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4+ T cells, but inversely correlated with memory potential. Interconversion between Ly6C+ and Ly6C− cells may maintain a balance between the two antigen-specific CD4+ T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4+ T cells during persistent virus infection. PMID:25662997

  9. Notch controls the survival of memory CD4+ T cells by regulating glucose uptake.

    Science.gov (United States)

    Maekawa, Yoichi; Ishifune, Chieko; Tsukumo, Shin-ichi; Hozumi, Katsuto; Yagita, Hideo; Yasutomo, Koji

    2015-01-01

    CD4+ T cells differentiate into memory T cells that protect the host from subsequent infection. In contrast, autoreactive memory CD4+ T cells harm the body by persisting in the tissues. The underlying pathways controlling the maintenance of memory CD4+ T cells remain undefined. We show here that memory CD4+ T cell survival is impaired in the absence of the Notch signaling protein known as recombination signal binding protein for immunoglobulin κ J region (Rbpj). Treatment of mice with a Notch inhibitor reduced memory CD4+ T cell numbers and prevented the recurrent induction of experimental autoimmune encephalomyelitis. Rbpj-deficient CD4+ memory T cells exhibit reduced glucose uptake due to impaired AKT phosphorylation, resulting in low Glut1 expression. Treating mice with pyruvic acid, which bypasses glucose uptake and supplies the metabolite downstream of glucose uptake, inhibited the decrease of autoimmune memory CD4+ T cells in the absence of Notch signaling, suggesting memory CD4+ T cell survival relies on glucose metabolism. Together, these data define a central role for Notch signaling in maintaining memory CD4+ T cells through the regulation of glucose uptake.

  10. The exhausted CD4+CXCR5+ T cells involve the pathogenesis of human tuberculosis disease.

    Science.gov (United States)

    Bosco, Munyemana Jean; Wei, Ming; Hou, Hongyan; Yu, Jing; Lin, Qun; Luo, Ying; Sun, Ziyong; Wang, Feng

    2018-06-21

    The CD4 + CXCR5 + T cells have been previously established. However, their decreased frequency during tuberculosis (TB) disease is partially understood. The aim of this study was to explore the depletion of CD4 + CXCR5 + T cells in human TB. The frequency and function of CD4 + CXCR5 + T cells were evaluated in active TB (ATB) patients and healthy control (HC) individuals. The function of CD4 + CXCR5 + T cells was determined after blockade of inhibitory receptors. The frequency of CD4 + CXCR5 + T cells was decreased in ATB patients. The expression of activation markers (HLA-DR and ICOS) and inhibitory receptors (Tim-3 and PD-1) on CD4 + CXCR5 + T cells was increased in ATB group. TB-specific antigen stimulation induced higher expression of inhibitory receptors than phytohemagglutinin stimulation in ATB group. In contrast, TB antigen stimulation did not induce a significantly increased expression of IL-21 and Ki-67 on CD4 + CXCR5 + T cells. However, blockade of inhibitory receptors Tim-3 and PD-1 not only increased the frequency of CD4 + CXCR5 + T cells, but also restored their proliferation and cytokine secretion potential. An increased expression of inhibitory receptors involves the depletion of CD4 + CXCR5 + T cells, and blockade of inhibitory receptors can restore the function of CD4 + CXCR5 + T cells in ATB patients. Copyright © 2018. Published by Elsevier Ltd.

  11. Regulation of CD4 T cells and their effects on immunopathological inflammation following viral infection.

    Science.gov (United States)

    Bhattacharyya, Mitra; Madden, Patrick; Henning, Nathan; Gregory, Shana; Aid, Malika; Martinot, Amanda J; Barouch, Dan H; Penaloza-MacMaster, Pablo

    2017-10-01

    CD4 T cells help immune responses, but knowledge of how memory CD4 T cells are regulated and how they regulate adaptive immune responses and induce immunopathology is limited. Using adoptive transfer of virus-specific CD4 T cells, we show that naive CD4 T cells undergo substantial expansion following infection, but can induce lethal T helper type 1-driven inflammation. In contrast, memory CD4 T cells exhibit a biased proliferation of T follicular helper cell subsets and were able to improve adaptive immune responses in the context of minimal tissue damage. Our analyses revealed that type I interferon regulates the expansion of primary CD4 T cells, but does not seem to play a critical role in regulating the expansion of secondary CD4 T cells. Strikingly, blockade of type I interferon abrogated lethal inflammation by primary CD4 T cells following viral infection, despite that this treatment increased the numbers of primary CD4 T-cell responses. Altogether, these data demonstrate important aspects of how primary and secondary CD4 T cells are regulated in vivo, and how they contribute to immune protection and immunopathology. These findings are important for rational vaccine design and for improving adoptive T-cell therapies against persistent antigens. © 2017 John Wiley & Sons Ltd.

  12. Genetic subspecies diversity of the chimpanzee CD4 virus-receptor gene

    DEFF Research Database (Denmark)

    Hvilsom, Christina; Carlsen, Frands; Siegismund, Hans R

    2008-01-01

    six among the subspecies of chimpanzees. We found the CD4 receptor to be conserved in individuals belonging to the P. t. verus subspecies and divergent from the other three subspecies, which harbored highly variable CD4 receptors. The CD4 receptor of chimpanzees differed from that of humans. We...... question whether the observed diversity can explain the species-specific differences in susceptibility to and pathogenicity of SIV/HIV....

  13. The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

    Science.gov (United States)

    Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

    2016-10-17

    Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  14. Cd4As2Br3

    Directory of Open Access Journals (Sweden)

    Mohammed Kars

    2014-03-01

    Full Text Available Single crystals of Cd4As2Br3 (tetracadmium biarsenide tribromide were grown by a chemical transport reaction. The structure is isotypic with the members of the cadmium and mercury pnictidohalides family with general formula M4A2X3 (M = Cd, Hg; A = P, As, Sb; X = Cl, Br, I and contains two independent As atoms on special positions with site symmetry -3 and two independent Cd atoms, of which one is on a special position with site symmetry -3. The Cd4As2Br3 structure consists of AsCd4 tetrahedra sharing vertices with isolated As2Cd6 octahedra that contain As–As dumbbells in the centre of the octahedron. The Br atoms are located in the voids of this three-dimensional arrangement and bridge the different polyhedra through Cd...Br contacts.

  15. The Decrease of Peripheral Blood CD4+ T Cells Indicates Abdominal Compartment Syndrome in Severe Acute Pancreatitis.

    Directory of Open Access Journals (Sweden)

    Yao Liu

    Full Text Available Few data are available on the role of T lymphocytes and inflammatory cytokines in abdominal compartment syndrome (ACS in severe acute pancreatitis (SAP. We conducted a retrospective study to assess the risk factors associated with ACS in SAP.A total of 76 SAP patients who were admitted within 24 hours after symptom onset in our study. There were 36 patients suffering from ACS and 40 from intra-abdominal hypertension (IAH. On the 1st, 3rd and 7th days after hospital admission, the following variables were assessed: serum value of C-reactive protein (CRP, and the proportions of peripheral CD4+ and CD8+ T lymphocytes. Acute Physiology and Chronic Health Evaluation II (APACHE II score, and computed tomography severity index (CTSI score were assessed on days 1 and 7 after hospitalization.Compared with the patients with IAH, ACS patients showed statistically higher CRP value on 7th day after hospital admission, proportions of CD4+ T cells on days 1, 3, 7 and CD4+/CD8+ ratio on day 1 were significantly lower (P < 0.05, respectively. A CD4+ T cell proportion of 30.3% on the 1st day indicated ACS with an area under the curve (AUC of 0.774, a sensitivity with 82.5% and specificity with 72.0%, respectively. Sensitivity/specificity for predicting ACS in SAP patients on day 1 was 70.0%/68.0% for CD4+/CD8+ ratio, 72.2%/65.0% for APACHE II score.The reduction of peripheral blood CD4+ T lymphocytes is associated with ACS in SAP, and may act as a potential predictor of ACS in SAP.

  16. Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Bartholdy, C; Wodarz, D

    2001-01-01

    investigated whether CD4(+) Th cells are required to establish and maintain this new equilibrium. The absence of IFN-gamma does not impair the generation of IL-2-producing CD4(+) cells, and depletion of these cells precipitates severe CD8(+) T cell-mediated immunopathology in IFN-gamma(-/-) mice, indicating...... an important role of CD4(+) T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-gamma(-/-) mice following CD4(+) cell depletion. Initially the antiviral CTL response did not require CD4(+) cells, but with time an impaired reactivity toward...... especially the glycoprotein 33--41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33--41 and nucleoprotein 396--404) CD8(+) T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4...

  17. Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

    OpenAIRE

    Desbarats, J; Freed, J H; Campbell, P A; Newell, M K

    1996-01-01

    The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investig...

  18. CD4+ T-cell lines used to evaluate a Mycobacterium avium subsp. paratuberculosis (MAP) peptide vaccine

    DEFF Research Database (Denmark)

    Lybeck, Kari; Sjurseth, Siri K.; Al-Touama, Zainab

    The aim of the study was to establish a protocol for generation of MAP-specific T-cell lines and to use these lines for evaluation of a peptide vaccine. A protocol for culturing T-cell lines from peripheral blood of goats naturally infected with MAP was established. CD4+ T cells were positively...... selected using an anti CD4 mAb and Dynabeads. Sorted CD4+ cells were cultivated with purified protein derivative from MAP (PPDj) or E. coli sonicate, IL-2, and IL-15. After two cultivation cycles, T cells were tested for recall responses in a proliferative T-cell assay. T-cell line responses were...... in average 92 % for PPDj, and -3 % for E. coli sonicate. CD4+ T-cell lines stimulated with PPDj showed a 6 fold increase in IFN- γ production compared to controls. These results indicated that the T-cell lines were MAP-specific. The protocol was subsequently used to evaluate MAP-specific peptides as vaccine...

  19. Cytotoxic human CD4(+) T cells

    NARCIS (Netherlands)

    van de Berg, Pablo J.; van Leeuwen, Ester M.; ten Berge, Ineke J.; van Lier, Rene

    2008-01-01

    The induction of adaptive immune responses critically depends on helper signals provided by CD4(+) T cells. These signals not only license antigen presenting cells (APC) to activate naïve CD8(+) T cells leading to the formation of vast numbers of cytotoxic T lymphocytes but also support the

  20. CD4+ T-cell epitope prediction using antigen processing constraints.

    Science.gov (United States)

    Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

    2016-05-01

    T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Reactivity of naive CD4+CD25- T cells against gut microflora in healthy mice

    DEFF Research Database (Denmark)

    Gad, Monika; Lundsgaard, Dorthe; Kjellev, Stine

    2006-01-01

    We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under...

  2. The frequencies of IFNγ+IL2+TNFα+ PPD-specific CD4+CD45RO+ T-cells correlate with the magnitude of the QuantiFERON® gold in-tube response in a prospective study of healthy indian adolescents.

    Science.gov (United States)

    Jenum, Synne; Grewal, Harleen M S; Hokey, David A; Kenneth, John; Vaz, Mario; Doherty, Timothy Mark; Jahnsen, Frode Lars

    2014-01-01

    QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced. To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity. Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls. There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response. Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load.

  3. Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

    Science.gov (United States)

    Niu, Qingli; Hou, Wei; Churchyard, Gavin; Nitayaphan, Sorachai; Pitisuthithum, Punnee; Rerks-Ngarm, Supachai; Franchini, Genoveffa

    2018-01-01

    The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1β) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination. PMID:29474461

  4. PKM2-dependent metabolic reprogramming in CD4+ T cells is crucial for hyperhomocysteinemia-accelerated atherosclerosis.

    Science.gov (United States)

    Lü, Silin; Deng, Jiacheng; Liu, Huiying; Liu, Bo; Yang, Juan; Miao, Yutong; Li, Jing; Wang, Nan; Jiang, Changtao; Xu, Qingbo; Wang, Xian; Feng, Juan

    2018-06-01

    Inflammation mediated by activated T cells plays an important role in the initiation and progression of hyperhomocysteinemia (HHcy)-accelerated atherosclerosis in ApoE -/- mice. Homocysteine (Hcy) activates T cells to secrete proinflammatory cytokines, especially interferon (IFN)-γ; however, the precise mechanisms remain unclear. Metabolic reprogramming is critical for T cell inflammatory activation and effector functions. Our previous study demonstrated that Hcy regulates T cell mitochondrial reprogramming by enhancing endoplasmic reticulum (ER)-mitochondria coupling. In this study, we further explored the important role of glycolysis-mediated metabolic reprogramming in Hcy-activated CD4 + T cells. Mechanistically, Hcy-activated CD4 + T cell increased the protein expression and activity of pyruvate kinase muscle isozyme 2 (PKM2), the final rate-limiting enzyme in glycolysis, via the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin signaling pathway. Knockdown of PKM2 by small interfering RNA reduced Hcy-induced CD4 + T cell IFN-γ secretion. Furthermore, we generated T cell-specific PKM2 knockout mice by crossing LckCre transgenic mice with PKM2 fl/fl mice and observed that Hcy-induced glycolysis and oxidative phosphorylation were both diminished in PKM2-deficient CD4 + T cells with reduced glucose and lipid metabolites, and subsequently reduced IFN-γ secretion. T cell-depleted apolipoprotein E-deficient (ApoE -/- ) mice adoptively transferred with PKM2-deficient CD4 + T cells, compared to mice transferred with control cells, showed significantly decreased HHcy-accelerated early atherosclerotic lesion formation. In conclusion, this work indicates that the PKM2-dependent glycolytic-lipogenic axis, a novel mechanism of metabolic regulation, is crucial for HHcy-induced CD4 + T cell activation to accelerate early atherosclerosis in ApoE -/- mice. Metabolic reprogramming is crucial for Hcy-induced CD4 + T cell inflammatory activation. Hcy activates

  5. CD4 T-Cell Memory Generation and Maintenance

    Science.gov (United States)

    Gasper, David J.; Tejera, Melba Marie; Suresh, M.

    2014-01-01

    Immunologic memory is the adaptive immune system's powerful ability to remember a previous antigen encounter and react with accelerated vigor upon antigen re-exposure. It provides durable protection against reinfection with pathogens and is the foundation for vaccine-induced immunity. Unlike the relatively restricted immunologic purview of memory B cells and CD8 T cells, the field of CD4 T-cell memory must account for multiple distinct lineages with diverse effector functions, the issue of lineage commitment and plasticity, and the variable distribution of memory cells within each lineage. Here, we discuss the evidence for lineage-specific CD4 T-cell memory and summarize the known factors contributing to memory-cell generation, plasticity, and long-term maintenance. PMID:24940912

  6. Chronic exposure to trichloroethylene increases DNA methylation of the Ifng promoter in CD4+ T cells.

    Science.gov (United States)

    Gilbert, Kathleen M; Blossom, Sarah J; Erickson, Stephen W; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Li, Jingyun; Cooney, Craig A

    2016-10-17

    CD4 + T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4 + T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4 + T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4 + T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4 + T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4 + T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4 + T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4 + T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. DJ-1/Park7 Sensitive Na+ /H+ Exchanger 1 (NHE1) in CD4+ T Cells.

    Science.gov (United States)

    Zhou, Yuetao; Shi, Xiaolong; Chen, Hong; Zhang, Shaqiu; Salker, Madhuri S; Mack, Andreas F; Föller, Michael; Mak, Tak W; Singh, Yogesh; Lang, Florian

    2017-11-01

    DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na + /H + exchanger 1 (NHE1). ROS formation in CD4 + T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4 + T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pH i ) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4 + T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4 + T cells from DJ-1 deficient mice than in CD4 + T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4 + T cells, and blunted the difference between DJ-1 -/- and DJ-1 +/+ CD4 + T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1 -/- CD4 + T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4 + T cells. J. Cell. Physiol. 232: 3050-3059, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Retinol as a micronutrients related to cervical local immunity: The expression of tumor necrosis factor-alpha specifically stimulated with E6 epitope of human papillomavirus type-16 and ratio of CD4+/CD8+ T cell in natural history of cervical cancer

    Science.gov (United States)

    Utami, T. W.; Aziz, M. F.; Ibrahim, F.; Andrijono

    2017-08-01

    Retinol is one of the antioxidant micronutrients that plays essential roles in the immune system, by preventing the persistence of modulating CD4+ and CD8+ T cells and cytokines production. Tumor Necrosis Factor-Alpha (TNF-α) is an acute pro-inflammatory cytokine which has many crucial roles in controlling HPV. In contrast, when persistent infection occurs, TNF-α induces carcinogenesis. The ratio of CD4+ cells to CD8+ T cells and adequate TNF-α production in acute HPV infection are key points for clearance. The aim of this research is to analyze the sufficiency level of retinol deposit, the expression of TNF-α, and the ratio of CD4+: CD8+ T cells in a normal cervix, clearance and persistent HPV subclinical infection, and cervical cancer group. The sufficiency level of retinol deposit was analyzed from peripheral blood using the ELISA method. The cervico-vaginal secretions, which were incubated for 24 hours, were stimulated specifically by E6 epitope HPV type-16, measuring TNF-α expression semi-quantitatively by the ELISpot method and CD4+/CD8+ T cells quantitatively by flowcytometry method. The sufficient level of retinol deposit in a normal cervix, clearance HPV subclinical infection, persistent, and cervical cancer group was 85%, 75% (OR 1.89), 33.3% (OR 11.33), and 75% (OR 1.89), respectively. The expression of TNF-α in normal cervix group was 10%, while for cervical cancer it was 75% (OR 27.00; p CD4+: CD8+ T cells in the normal cervix and cervical cancer group was 10% and 25% (OR 0.33). There was no high ratio of CD4+: CD8+ T cells in clearance (OR 1.22) and persistent (OR 0.95) HPV subclinical infection groups. This study was able to prove that the normal cervix group has the highest retinol deposit sufficiency level and the cervical cancer group has the highest TNF-α expression (OR 27; p < 0.001). The lowest of retinol deposit sufficiency level was not in cervical cancer, but in the persistent HPV subclinical infection group (OR 11.33). There was

  9. Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

    Directory of Open Access Journals (Sweden)

    Karen A.O. Martins

    2016-01-01

    Full Text Available Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol, MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.

  10. Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity.

    Science.gov (United States)

    Martins, Karen A O; Cooper, Christopher L; Stronsky, Sabrina M; Norris, Sarah L W; Kwilas, Steven A; Steffens, Jesse T; Benko, Jacqueline G; van Tongeren, Sean A; Bavari, Sina

    2016-01-01

    Protein-based vaccines offer a safer alternative to live-attenuated or inactivated vaccines but have limited immunogenicity. The identification of adjuvants that augment immunogenicity, specifically in a manner that is durable and antigen-specific, is therefore critical for advanced development. In this study, we use the filovirus virus-like particle (VLP) as a model protein-based vaccine in order to evaluate the impact of four candidate vaccine adjuvants on enhancing long term protection from Ebola virus challenge. Adjuvants tested include poly-ICLC (Hiltonol), MPLA, CpG 2395, and alhydrogel. We compared and contrasted antibody responses, neutralizing antibody responses, effector T cell responses, and T follicular helper (Tfh) cell frequencies with each adjuvant's impact on durable protection. We demonstrate that in this system, the most effective adjuvant elicits a Th1-skewed antibody response and strong CD4 T cell responses, including an increase in Tfh frequency. Using immune-deficient animals and adoptive transfer of serum and cells from vaccinated animals into naïve animals, we further demonstrate that serum and CD4 T cells play a critical role in conferring protection within effective vaccination regimens. These studies inform on the requirements of long term immune protection, which can potentially be used to guide screening of clinical-grade adjuvants for vaccine clinical development.

  11. Preparation of anti-CD4 monoclonal antibody-conjugated magnetic poly(glycidyl methacrylate) particles and their application on CD4+ lymphocyte separation.

    Science.gov (United States)

    Pimpha, Nuttaporn; Chaleawlert-umpon, Saowaluk; Chruewkamlow, Nuttapol; Kasinrerk, Watchara

    2011-03-15

    Novel immunomagnetic particles have been prepared for separation of CD4(+) lymphocytes. The magnetic nanoparticles with a diameter of approximately 5-6 nm were first synthesized by co-precipitation from ferrous and ferric iron solutions and subsequently encapsulated with poly(glycidyl methacrylate) (PGMA) by precipitation polymerization. Monoclonal antibody specific to CD4 molecules expressed on CD4(+) lymphocytes was conjugated to the surface of magnetic PGMA particles through covalent bonding between epoxide functional groups on the particle surface and primary amine groups of the antibodies. The generated immunomagnetic particles have successfully separated CD4(+) lymphocytes from whole blood with over 95% purity. The results indicated that these particles can be employed for cell separation and provide a strong potential to be applied in various biomedical applications including diagnosis, and monitoring of human diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. eCD4-Ig variants that more potently neutralize HIV-1.

    Science.gov (United States)

    Fetzer, Ina; Gardner, Matthew R; Davis-Gardner, Meredith E; Prasad, Neha R; Alfant, Barnett; Weber, Jesse A; Farzan, Michael

    2018-03-28

    The HIV-1 entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralizes all HIV-1, HIV-2 and SIV isolates that it has been tested against, suggesting that it may be useful in clinical settings where antibody escape is a concern. Here we characterize three new eCD4-Ig variants, each with different architectures and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig, and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications. IMPORTANCE HIV-1 bNAbs have properties different from antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral lifecycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the

  13. CD4 T cell activation and disease activity at onset of multiple sclerosis

    DEFF Research Database (Denmark)

    Jensen, J; Langkilde, Annika Reynberg; Fenst, C

    2004-01-01

    We studied CD4 T cell activation in patients with clinically isolated syndromes (CIS) suggesting an initial attack of multiple sclerosis. The percentage of blood CD26+ CD4 T cells was increased in these patients, and correlated with magnetic resonance imaging disease activity and clinical disease...... severity. In contrast, the percentage of CD25+ CD4 T cells in cerebrospinal fluid correlated negatively with the cerebrospinal fluid concentration of myelin basic protein and the presence of IgG oligoclonal bands. These results suggest that distinct systemic and intrathecal T cell activation states...

  14. Effect of memory CD4+ T cells' signal transducer and activator of transcription (STATs) functional shift on cytokine-releasing properties in asthma.

    Science.gov (United States)

    Chen, Zhihong; Pan, Jue; Jia, Yi; Li, Dandan; Min, Zhihui; Su, Xiaoqiong; Yuan, Honglei; Shen, Geng; Cao, Shengxuan; Zhu, Lei; Wang, Xiangdong

    2017-02-01

    Recent data have demonstrated that long-lived memory T cells are present in the human lung and can play significant roles in the pathogenesis of specific allergic and autoimmune diseases. However, most evidence has been obtained from mouse studies, and the potential roles of memory T cells in human allergic diseases, such as asthma, remain largely unknown. Thirty-three asthmatics, 26 chronic obstructive pulmonary disease (COPD) patients, and 22 healthy volunteers were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood, and cell surface staining (CD4, CD45RO, CRTH2, CD62L, and CCR7) was performed for the detection of memory CD4 + T cells in blood. After stimulation with interleukin-27 (IL-27) or IL-4 for 15 min, the STAT1/STAT6 phosphorylation of memory CD4 + T cells was measured separately by flow cytometric techniques. The cytokine-releasing profiles after 6 days of culture under neutralization, T H 2, T H 2 + lipopolysaccharide (LPS), and T H 2 + house dust mite (HDM) conditions were detected by intracellular protein (IL-5, IL-17, and interferon (IFN)-γ) staining. Correlation analyses between the profile of memory CD4 + T cells and clinical characteristics of asthma were performed. The number of circulating memory CD4 + T (CD4 + Tm) cells in asthmatics was increased compared with that in the healthy subjects (48 ± 5.7 % vs. 32 ± 4.1 %, p T H 2 memory cells but not non-T H 2 memory cells in blood. The cytokine-releasing profiles of asthmatics was unique, specifically IL-5 high , IL-17 high , and IFN-r low , compared with those of COPD patients and healthy subjects. The IL-17 production levels in CD4 + Tm cells are associated with disease severity and positively correlated with medication consumption in asthma. The long-lived, antigen-specific memory CD4 + T cells, rather than PBMCs or peripheral lymphocytes, might be the ideal T cell subset candidates for analyzing the endotype of asthma

  15. A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses.

    Directory of Open Access Journals (Sweden)

    Daniela Santoro Rosa

    Full Text Available T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+ T cells are important for the generation and maintenance of functional CD8(+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18, capable of eliciting broad CD4(+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+/CD8(+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+ and CD8(+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2 simultaneously in response to HIV-1 peptides. For CD4(+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2. The vaccine also generated long-lived central and effector memory CD4(+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+ T cells and antibody responses- elicited by other HIV immunogens.

  16. Habitual physical activity levels are positively correlated with CD4 ...

    African Journals Online (AJOL)

    Habitual physical activity levels are positively correlated with CD4 counts in an ... per month) and functional independence as assessed from the responses to the ... and between CD4 cell counts and total habitual physical activity levels (p ...

  17. Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Kara G Lassen

    2006-07-01

    Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

  18. World Health Organization guidelines should not change the CD4 ...

    African Journals Online (AJOL)

    2013-03-02

    Mar 2, 2013 ... The World Health Organization (WHO) currently recommends that HIV-positive adults start antiretroviral therapy (ART) at. CD4 counts <350 cells/µl. Several countries have changed their guidelines to recommend ART irrespective of CD4 count or at a threshold of 500 CD4 cells/µl. Consequently, WHO is ...

  19. World Health Organization guidelines should not change the CD4 ...

    African Journals Online (AJOL)

    The World Health Organization (WHO) currently recommends that HIV-positive adults start antiretroviral therapy (ART) at CD4 counts <350 cells/μl. Several countries have changed their guidelines to recommend ART irrespective of CD4 count or at a threshold of 500 CD4 cells/μl. Consequently, WHO is currently revising its ...

  20. Baseline CD4 lymphocyte count among HIV patients in Kano ...

    African Journals Online (AJOL)

    kemrilib

    macrophages), the devastating effect of HIV on the immune system is not surprising. Given that HIV induced immunodeficiency is largely due to infection and gradual depletion of. CD4+ T-helper cells, CD4 count has become a useful indicator of immune function in infected patients. Hence CD4 count along with viral load ...

  1. Task-shifting of CD4 T cell count monitoring by the touchscreen-based Muse™ Auto CD4/CD4% single-platform system for CD4 T cell numeration: Implication for decentralization in resource-constrained settings.

    Science.gov (United States)

    Kouabosso, André; Mossoro-Kpinde, Christian Diamant; Bouassa, Ralph-Sydney Mboumba; Longo, Jean De Dieu; Mbeko Simaleko, Marcel; Grésenguet, Gérard; Bélec, Laurent

    2018-04-01

    The accuracy of CD4 T cell monitoring by the recently developed flow cytometry-based CD4 T cell counting Muse™ Auto CD4/CD4% Assay analyzer (EMD Millipore Corporation, Merck Life Sciences, KGaA, Darmstadt, Germany) was evaluated in trained lay providers against laboratory technicians. After 2 days of training on the Muse™ Auto CD4/CD4% analyzer, EDTA-blood samples from 6 HIV-positive and 4 HIV-negative individuals were used for CD4 T cell counting in triplicate in parallel by 12 trained lay providers as compared to 10 lab technicians. Mean number of CD4 T cells in absolute number was 829 ± 380 cells/μl by lay providers and 794 ± 409 cells/μl by technicians (P > 0.05); and in percentage 36.2 ± 14.8%CD4 by lay providers and 36.1 ± 15.0%CD4 by laboratory technician (P > 0.05). The unweighted linear regression and Passing-Bablok regression analyses on CD4 T cell results expressed in absolute count revealed moderate correlation between CD4 T cell counts obtained by lay providers and lab technicians. The mean absolute bias measured by Bland-Altman analysis between CD4 T cell/μl obtained by lay providers and lab technicians was -3.41 cells/μl. Intra-assay coefficient of variance (CV) of Muse™ Auto CD4/CD4% in absolute number was 10.1% by lay providers and 8.5% by lab technicians (P > 0.05), and in percentage 5.5% by lay providers and 4.4% by lab technicians (P > 0.05). The inter-assay CV of Muse™ Auto CD4/CD4% in absolute number was 13.4% by lay providers and 10.3% by lab technicians (P > 0.05), and in percentage 7.8% by lay providers and 6.9% by lab technicians (P > 0.05). The study demonstrates the feasibility of CD4 T cell counting using the alternative flow cytometer Muse™ Auto CD4/CD4% analyzer by trained lay providers and therefore the practical possibility of decentralization CD4 T cell counting to health community centers. Copyright © 2018. Published by Elsevier B.V.

  2. Biomarkers of Progression after HIV Acute/Early Infection: Nothing Compares to CD4+ T-cell Count?

    Directory of Open Access Journals (Sweden)

    Gabriela Turk

    2018-01-01

    Full Text Available Progression of HIV infection is variable among individuals, and definition disease progression biomarkers is still needed. Here, we aimed to categorize the predictive potential of several variables using feature selection methods and decision trees. A total of seventy-five treatment-naïve subjects were enrolled during acute/early HIV infection. CD4+ T-cell counts (CD4TC and viral load (VL levels were determined at enrollment and for one year. Immune activation, HIV-specific immune response, Human Leukocyte Antigen (HLA and C-C chemokine receptor type 5 (CCR5 genotypes, and plasma levels of 39 cytokines were determined. Data were analyzed by machine learning and non-parametric methods. Variable hierarchization was performed by Weka correlation-based feature selection and J48 decision tree. Plasma interleukin (IL-10, interferon gamma-induced protein (IP-10, soluble IL-2 receptor alpha (sIL-2Rα and tumor necrosis factor alpha (TNF-α levels correlated directly with baseline VL, whereas IL-2, TNF-α, fibroblast growth factor (FGF-2 and macrophage inflammatory protein (MIP-1β correlated directly with CD4+ T-cell activation (p < 0.05. However, none of these cytokines had good predictive values to distinguish “progressors” from “non-progressors”. Similarly, immune activation, HIV-specific immune responses and HLA/CCR5 genotypes had low discrimination power. Baseline CD4TC was the most potent discerning variable with a cut-off of 438 cells/μL (accuracy = 0.93, κ-Cohen = 0.85. Limited discerning power of the other factors might be related to frequency, variability and/or sampling time. Future studies based on decision trees to identify biomarkers of post-treatment control are warrantied.

  3. Specificity and affinity quantification of protein-protein interactions.

    Science.gov (United States)

    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  4. Immune regulation in Chandipura virus infection: characterization of CD4+ T regulatory cells from infected mice

    Directory of Open Access Journals (Sweden)

    Shahir Prajakta

    2011-05-01

    Full Text Available Abstract Back ground Chandipura virus produces acute infection in mice. During infection drastic reduction of CD4+, CD8+ and CD19 + cell was noticed. Depletion of lymphocytes also noticed in spleen. The reduction may be due to the regulatory mechanism of immune system to prevent the bystander host tissue injury. There are several mechanisms like generation of regulatory cells, activation induced cell death (ACID etc were indicated to control the activation and maintain cellular homeostasis. Role of regulatory cells in homeostasis has been described in several viral diseases. This study was undertaken to characterize CD4+T regulatory cells from the infected mice. Method In this study we purified the CD4+ T cells from Chandipura virus infected susceptible Balb/c mice. CD4+ T regulatory cells were identified by expression of cell surface markers CD25, CD127 and CTLA-4 and intracellular markers Foxp3, IL-10 and TGF-beta. Antigen specificity and ability to suppress the proliferation of other lymphocytes were studied in vitro by purified CD4+CD25+T regulatory cells from infected mice. The proliferation was calculated by proliferation module of Flow Jo software. Expression of death receptors on regulatory cells were studied by flowcytometer. Results The CD4+ T cells isolated from infected mice expressed characteristic markers of regulatory phenotype at all post infective hours tested. The CD4+ T regulatory cells were proliferated when stimulated with Chandipura virus antigen. The regulatory cells did not suppress the proliferation of splenocytes stimulated with anti CD3 antibody when co cultured with them. Interesting observation was, while purification of CD4+ T cells by negative selection, the population of cells negative for CD4 also co purified along with CD4+ T cell. Flow cytometry analysis and light microscopy revealed that CD4 negative cells were of different size and shape (atypical compared to the normal lymphocytes. Greater percentage of

  5. Pulmonary CCR2+CD4+ T cells are immune regulatory and attenuate lung fibrosis development.

    Science.gov (United States)

    Milger, Katrin; Yu, Yingyan; Brudy, Eva; Irmler, Martin; Skapenko, Alla; Mayinger, Michael; Lehmann, Mareike; Beckers, Johannes; Reichenberger, Frank; Behr, Jürgen; Eickelberg, Oliver; Königshoff, Melanie; Krauss-Etschmann, Susanne

    2017-11-01

    Animal models have suggested that CCR2-dependent signalling contributes to the pathogenesis of pulmonary fibrosis, but global blockade of CCL2 failed to improve the clinical course of patients with lung fibrosis. However, as levels of CCR2 + CD4 + T cells in paediatric lung fibrosis had previously been found to be increased, correlating with clinical symptoms, we hypothesised that distinct CCR2 + cell populations might either increase or decrease disease pathogenesis depending on their subtype. To investigate the role of CCR2 + CD4 + T cells in experimental lung fibrosis and in patients with idiopathic pulmonary fibrosis and other fibrosis. Pulmonary CCR2 + CD4 + T cells were analysed using flow cytometry and mRNA profiling, followed by in silico pathway analysis, in vitro assays and adoptive transfer experiments. Frequencies of CCR2 + CD4 + T cells were increased in experimental fibrosis-specifically the CD62L - CD44 + effector memory T cell phenotype, displaying a distinct chemokine receptor profile. mRNA profiling of isolated CCR2 + CD4 + T cells from fibrotic lungs suggested immune regulatory functions, a finding that was confirmed in vitro using suppressor assays. Importantly, adoptive transfer of CCR2 + CD4 + T cells attenuated fibrosis development. The results were partly corroborated in patients with lung fibrosis, by showing higher percentages of Foxp3 + CD25 + cells within bronchoalveolar lavage fluid CCR2 + CD4 + T cells as compared with CCR2 - CD4 + T cells. Pulmonary CCR2 + CD4 + T cells are immunosuppressive, and could attenuate lung inflammation and fibrosis. Therapeutic strategies completely abrogating CCR2-dependent signalling will therefore also eliminate cell populations with protective roles in fibrotic lung disease. This emphasises the need for a detailed understanding of the functions of immune cell subsets in fibrotic lung disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights

  6. Memory T follicular helper CD4 T cells

    Directory of Open Access Journals (Sweden)

    J. Scott eHale

    2015-02-01

    Full Text Available T follicular helper (Tfh cells are the subset of CD4 T helper cells that are required for generation and maintenance of germinal center reactions and the generation of long-lived humoral immunity. This specialized T helper subset provides help to cognate B cells via their expression of CD40 ligand, IL-21, IL-4, and other molecules. Tfh cells are characterized by their expression of the chemokine receptor CXCR5, expression of the transcriptional repressor Bcl6, and their capacity to migrate to the follicle and promote germinal center B cell responses. Until recently, it remained unclear whether Tfh cells differentiated into memory cells and whether they maintain their Tfh commitment at the memory phase. This review will highlight several recent studies that support the idea of Tfh-committed CD4 T cells at the memory stage of the immune response. The implication of these findings is that memory Tfh cells retain their capacity to recall their Tfh-specific effector functions upon reactivation to provide help for B cell responses and play an important role in prime and boost vaccination or during recall responses to infection. The markers that are useful for distinguishing Tfh effector and memory cells, as well as the limitations of using these markers will be discussed. Tfh effector and memory generation, lineage maintenance, and plasticity relative to other T helper lineages (Th1, Th2, Th17, etc will also be discussed. Ongoing discoveries regarding the maintenance and lineage stability versus plasticity of memory Tfh cells will improve strategies that utilize CD4 T cell memory to modulate antibody responses during prime and boost vaccination.

  7. Clonal expansion of CD4+ Cytotoxic T Lymphocytes in IgG4-related disease

    Science.gov (United States)

    Mattoo, Hamid; Mahajan, Vinay S.; Maehara, Takashi; Deshpande, Vikram; Della-Torre, Emanuel; Wallace, Zachary S.; Kulikova, Maria; Drijvers, Jefte M.; Daccache, Joe; Carruthers, Mollie N.; Castellino, Flavia; Stone, James R.; Stone, John H.; Pillai, Shiv

    2016-01-01

    Background IgG4-related disease (IgG4-RD) is a systemic condition of unknown etiology, characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4+ T cells constitute the major inflammatory cell population in IgG4-RD lesions. Objective We used an unbiased approach to characterize CD4+ T cell subsets in IgG4-RD subjects based on their clonal expansion and their ability to infiltrate affected tissue sites. Methods We used flow cytometry to identify CD4+ effector/memory T cells (TEM) in a cohort of 101 IgG4-related disease (IgG4-RD) patients. These expanded cells were characterized by gene expression analysis and flow cytometry. Next-generation sequencing of the T cell receptor β chain gene was performed on CD4+SLAMF7+ CTLs and CD4+GATA3+ TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined using quantitative multi-color imaging. Results CD4+ effector/memory T cells with a cytolytic phenotype were expanded in IgG4-RD patients. Next-generation sequencing revealed prominent clonal expansions of these CD4+CTLs but not CD4+GATA3+ memory TH2 cells in subjects with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally-expanded CD4+CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B cell depletion was associated with a reduction in disease-associated CD4+ CTLs Conclusions IgG4-RD is prominently linked to clonally-expanded, IL-1β, and TGF- β1 secreting, CD4+ CTLs in peripheral blood as well as in inflammatory tissue lesions. These active, terminally-differentiated, cytokine-secreting effector CD4+ T cells are now linked to a human disease characterized by chronic inflammation and fibrosis. PMID:26971690

  8. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    International Nuclear Information System (INIS)

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael; Ellett, Anne; Farrugia, William; Wesselingh, Steven L.; Cunningham, Anthony L.; Ramsland, Paul A.; Gorry, Paul R.

    2011-01-01

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

  9. Fighting Viral Infections and Virus-Driven Tumors with Cytotoxic CD4+ T Cells

    Science.gov (United States)

    Muraro, Elena; Merlo, Anna; Martorelli, Debora; Cangemi, Michela; Dalla Santa, Silvia; Dolcetti, Riccardo; Rosato, Antonio

    2017-01-01

    CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors. PMID:28289418

  10. CD4 T Cell Responses in Latent and Chronic Viral Infections

    Science.gov (United States)

    Walton, Senta; Mandaric, Sanja; Oxenius, Annette

    2013-01-01

    The spectrum of tasks which is fulfilled by CD4 T cells in the setting of viral infections is large, ranging from support of CD8 T cells and humoral immunity to exertion of direct antiviral effector functions. While our knowledge about the differentiation pathways, plasticity, and memory of CD4 T cell responses upon acute infections or immunizations has significantly increased during the past years, much less is still known about CD4 T cell differentiation and their beneficial or pathological functions during persistent viral infections. In this review we summarize current knowledge about the differentiation, direct or indirect antiviral effector functions, and the regulation of virus-specific CD4 T cells in the setting of persistent latent or active chronic viral infections with a particular emphasis on herpes virus infections for the former and chronic lymphocytic choriomeningitis virus infection for the latter. PMID:23717308

  11. Different thresholds of T cell activation regulate FIV infection of CD4+CD25+ and CD4+CD25- cells

    International Nuclear Information System (INIS)

    Joshi, Anjali; Garg, Himanshu; Tompkins, Mary B.; Tompkins, Wayne A.

    2005-01-01

    Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4 + CD25 + T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4 + CD25 + and CD4 + CD25 - cells under different stimulation conditions. Both CD4 + CD25 + and CD4 + CD25 - cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4 + CD25 + cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4 + CD25 - T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4 + CD25 - cells to replicate FIV, it induces apoptosis in a high percentage of CD4 + CD25 + T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4 + CD25 - cells. These results suggest that CD4 + CD25 + and CD4 + CD25 - T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4 + CD25 + and CD4 + CD25 - cells to serve as potential reservoirs of a productive and latent FIV infection

  12. CD4 cells can be more efficient at tumor rejection than CD8 cells.

    Science.gov (United States)

    Perez-Diez, Ainhoa; Joncker, Nathalie T; Choi, Kyungho; Chan, William F N; Anderson, Colin C; Lantz, Olivier; Matzinger, Polly

    2007-06-15

    Researchers designing antitumor treatments have long focused on eliciting tumor-specific CD8 cytotoxic T lymphocytes (CTL) because of their potent killing activity and their ability to reject transplanted organs. The resulting treatments, however, have generally been surprisingly poor at inducing complete tumor rejection, both in experimental models and in the clinic. Although a few scattered studies suggested that CD4 T "helper" cells might also serve as antitumor effectors, they have generally been studied mostly for their ability to enhance the activity of CTL. In this mouse study, we compared monoclonal populations of tumor-specific CD4 and CD8 T cells as effectors against several different tumors, and found that CD4 T cells eliminated tumors that were resistant to CD8-mediated rejection, even in cases where the tumors expressed major histocompatibility complex (MHC) class I molecules but not MHC class II. MHC class II expression on host tissues was critical, suggesting that the CD4 T cells act indirectly. Indeed, the CD4 T cells partnered with NK cells to obtain the maximal antitumor effect. These findings suggest that CD4 T cells can be powerful antitumor effector cells that can, in some cases, outperform CD8 T cells, which are the current "gold standard" effector cell in tumor immunotherapy.

  13. Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy

    International Nuclear Information System (INIS)

    Hu Mingqian; Wang Jiongkun; Cai Jiye; Wu Yangzhe; Wang Xiaoping

    2008-01-01

    To date, nanoscale imaging of the morphological changes and adhesion force of CD4 + T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4 + T cells. The AFM images revealed that the volume of activated CD4 + T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4 + T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity

  14. Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

    Science.gov (United States)

    Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

    2008-09-12

    To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

  15. A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation.

    Science.gov (United States)

    Graessel, Anke; Hauck, Stefanie M; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B; Suttner, Kathrin

    2015-08-01

    Naive CD4(+) T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4(+) T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4(+) T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4(+) T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4(+) T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Casp8p41: The Protean Mediator of Death in CD4 T-cells that Replicate HIV

    Directory of Open Access Journals (Sweden)

    Rahul Sampath

    2016-01-01

    Full Text Available HIV cure is now the focus of intense research after Timothy Ray Brown (the Berlin patient set the precedent of being the first and only person cured. A major barrier to achieving this goal on a meaningful scale is an elimination of the latent reservoir, which is thought to comprise CD4-positive cells that harbor integrated, replication-competent HIV provirus. These cells do not express viral proteins, are indistinguishable from uninfected CD4 cells, and are thought to be responsible for HIV viral rebound–-that occurs within weeks of combination anti retroviral therapy (cART interruption. Modalities to engineer transcriptional stimulation (reactivation of this dormant integrated HIV provirus, leading to expression of cytotoxic viral proteins, are thought to be a specific way to eradicate the latently infected CD4 pool and are becoming increasingly relevant in the era of HIV cure. HIV protease is one such protein produced after HIV reactivation that cleaves procaspase-8 to generate a novel protein Casp8p41. Casp8p41 then binds to the BH3 domain of BAK, leading to BAK oligomerization, mitochondrial depolarization, and apoptosis. In central memory T cells (TCMs from HIV-infected patients, an elevated Bcl-2/procaspase-8 ratio was observed, and Casp8p41 binding to Bcl-2 was associated with a lack of reactivation-induced cell death. This was reversed by priming cells with a specific Bcl-2 antagonist prior to reactivation, resulting in increased cell death and decreased HIV DNA in a Casp8p41-dependent pathway. This review describes the biology, clinical relevance, and implications of Casp8p41 for a potential cure.

  17. Phenotypic and functional profiling of CD4 T cell compartment in distinct populations of healthy adults with different antigenic exposure.

    Directory of Open Access Journals (Sweden)

    Sophie Roetynck

    Full Text Available Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully.We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for "Th1" type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures.These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.

  18. A single amino-acid change in a highly conserved motif of gp41 elicits HIV-1 neutralization and protects against CD4 depletion.

    Science.gov (United States)

    Petitdemange, Caroline; Achour, Abla; Dispinseri, Stefania; Malet, Isabelle; Sennepin, Alexis; Ho Tsong Fang, Raphaël; Crouzet, Joël; Marcelin, Anne-Geneviève; Calvez, Vincent; Scarlatti, Gabriella; Debré, Patrice; Vieillard, Vincent

    2013-09-01

    The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

  19. Reduced Hepatitis B Virus (HBV)-Specific CD4+ T-Cell Responses in Human Immunodeficiency Virus Type 1-HBV-Coinfected Individuals Receiving HBV-Active Antiretroviral Therapy

    OpenAIRE

    Chang, J. Judy; Wightman, Fiona; Bartholomeusz, Angeline; Ayres, Anna; Kent, Stephen J.; Sasadeusz, Joseph; Lewin, Sharon R.

    2005-01-01

    Functional hepatitis B virus (HBV)-specific T cells are significantly diminished in individuals chronically infected with HBV compared to individuals with self-limiting HBV infection or those on anti-HBV therapy. In individuals infected with human immunodeficiency virus type 1 (HIV-1), coinfection with HBV is associated with an increased risk of worsening liver function following antiviral therapy and of more rapid HBV disease progression. Total HBV-specific T-cell responses in subjects with ...

  20. CD4+ T cell effects on CD8+ T cell location defined using bioluminescence.

    Directory of Open Access Journals (Sweden)

    Mitra Azadniv

    2011-01-01

    Full Text Available T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

  1. Lymphoid tissue inducer cells: pivotal cells in the evolution of CD4 immunity and tolerance?

    Directory of Open Access Journals (Sweden)

    Peter John Lane

    2012-02-01

    Full Text Available Phylogeny suggests that the evolution of placentation in mammals was accompanied by substantial changes in the mammalian immune system: in particular lymph nodes and CD4 high affinity memory antibody responses co-evolved during the same period. Lymphoid tissue inducer cells (LTi are members of an emerging family of innate lymphoid cells (ILCs that are crucial for lymph node development, but our studies have indicated that they also play a pivotal role in the long-term maintenance of memory CD4 T cells in adult mammals through their expression of the tumor necrosis family members, OX40- and CD30-ligands. Additionally, our studies have shown that these two molecules are also key operators in CD4 effector function, as their absence obviates the need for the FoxP3-dependent regulatory T cells (Tregs that prevent CD4 driven autoimmune responses. In this perspective article, we summarize findings from our group over the last 10 years, and focus specifically on the role of LTi in thymus. We suggest that like memory CD4 T cells, LTi also play a role in the selection and maintenance of the Tregs that under normal circumstances are absolutely required to regulate CD4 effector cells.

  2. Validation of Microcapillary Flow Cytometry for Community-Based CD4+ T Lymphocyte Enumeration in Remote Burkina Faso

    Science.gov (United States)

    Renault, Cybèle A; Traore, Arouna; Machekano, Rhoderick N; Israelski, Dennis M

    2010-01-01

    Background: CD4+ T lymphocyte enumeration plays a critical role in the initiation and monitoring of HIV-infected patients on antiretroviral therapy. There is an urgent need for low-cost CD4+ enumeration technologies, particularly for use in dry, dusty climates characteristic of many small cities in Sub-Saharan Africa. Design: Cross-sectional study. Methods: Blood samples from 98 HIV-infected patients followed in a community HIV clinic in Ouahigouya, Burkina Faso were obtained for routine CD4+ T lymphocyte count monitoring. The blood samples were divided into two aliquots, on which parallel CD4+ measurements were performed using microcapillary (Guava EasyCD4) and dedicated (Becton Dickinson FACSCount) CD4+ enumeration systems. Spearman rank correlation coefficient was calculated, and the sensitivity, specificity and positive predictive value (PPV) for EasyCD4 <200 cells/µL were determined compared to the reference standard FACSCount CD4 <200 cells/µL. Results: Mean CD4 counts for the EasyCD4 and FACSCount were 313.75 cells/µL and 303.47 cells/µL, respectively. The Spearman rank correlation coefficient was 0.92 (p<0.001). Median values using EasyCD4 were higher than those with the FACSCount (p=0.004). For a CD4<350 cells/uL, sensitivity of the EasyCD4 was 93.9% (95%CI 85.2-98.3%), specificity was 90.6% (95% CI 75.0-98.0%), and PPV was 95.4% (95%CI 87.1-99.0%). Conclusion: Use of the EasyCD4 system was feasible and highly accurate in the harsh conditions of this remote city in Sub-Saharan Africa, demonstrating acceptable sensitivity and specificity compared to a standard operating system. Microcapillary flow cytometry offers a cost-effective alternative for community-based, point-of-care CD4+ testing and could play a substantial role in scaling up HIV care in remote, resource-limited settings. PMID:21253463

  3. Involvement of CD244 in regulating CD4+ T cell immunity in patients with active tuberculosis.

    Directory of Open Access Journals (Sweden)

    Bingfen Yang

    Full Text Available CD244 (2B4 is a member of the signaling lymphocyte activation molecule (SLAM family of immune cell receptors and it plays an important role in modulating NK cell and CD8(+ T cell immunity. In this study, we investigated the expression and function of CD244/2B4 on CD4(+ T cells from active TB patients and latent infection individuals. Active TB patients had significantly elevated CD244/2B4 expression on M. tuberculosis antigen-specific CD4(+ T cells compared with latent infection individuals. The frequencies of CD244/2B4-expressing antigen-specific CD4(+ T cells were significantly higher in retreatment active TB patients than in new active TB patients. Compared with CD244/2B4-dull and -middle CD4(+ T cells, CD244/2B4-bright CD4(+ T cell subset had significantly reduced expression of IFN-γ, suggesting that CD244/2B4 expression may modulate IFN-γ production in M. tuberculosis antigen-responsive CD4(+ T cells. Activation of CD244/2B4 signaling by cross-linking led to significantly decreased production of IFN-γ. Blockage of CD244/2B4 signaling pathway of T cells from patients with active TB resulted in significantly increased production of IFN-γ, compared with isotype antibody control. In conclusion, CD244/2B4 signaling pathway has an inhibitory role on M. tuberculosis antigen-specific CD4(+ T cell function.

  4. Korelasi Kadar Feritin dengan Jumlah CD4, CD8, dan Rasio CD4/CD8 pada Penyandang Talasemia Mayor Anak

    Directory of Open Access Journals (Sweden)

    Bonnie Arseno

    2017-11-01

    Hasil. Didapatkan jumlah CD4 absolut, CD4%, CD8 absolut dan rasio CD4/CD8 menurun. Selain itu, terdapat jumlah CD4 absolut, CD8 absolut dan CD8% meningkat. Pada kelompok usia ≤5 tahun, korelasi kadar feritin dengan CD8 absolut, CD8%, dan rasio CD4/CD8 berturut-turut menghasilkan koefisien korelasi 0,691, 0,557, -0,680, dan p<0,05. Sementara pada kelompok lama terapi ≤5 tahun korelasi kadar feritin dengan CD8 absolut, CD8%, dan rasio CD4/CD8 menghasilkan koefisien korelasi 0,709, 0,571, -0,726 dengan p<0,05. Kesimpulan. Tidak terdapat korelasi antara kadar feritin dengan jumlah CD4, CD8, rasio CD4/CD8. Peningkatan kadar feritin akan diikuti dengan peningkatan jumlah CD8 absolut dan CD8%, serta penurunan rasio CD4/CD8 pada penyandang talasemia mayor anak berdasar atas usia dan lama terapi ≤5 tahun.

  5. Downregulation of cathepsin G reduces the activation of CD4+ T cells in murine autoimmune diabetes.

    Science.gov (United States)

    Zou, Fang; Lai, Xiaoyang; Li, Jing; Lei, Shuihong; Hu, Lei

    2017-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease due to progressive injury of islet cells mediated by T lymphocytes (T cells). Our previous studies have shown that only cathepsin G (CatG), not other proteases, is involved in the antigen presentation of proinsulin, and if the presentation is inhibited, the activation of CD4+ T cells induced by proinsulin is alleviated in T1DM patients, and CatG-specific inhibitor reduces the activation of CD4+ cells induced by proinsulin in T1DM patients. Therefore, we hypothesize that CatG may play an important role in the activation of CD4+ T cells in T1DM. To this end, mouse studies were conducted to demonstrate that CatG impacts the activation of CD4+ T cells in non-obese diabetic (NOD) mice. CatG gene expression and the activation of CD4+ T cells were examined in NOD mice. The effect of CatG inhibitor was investigated in NOD mice on the activation of CD4+ T cells, islet β cell function, islet inflammation and β-cell apoptosis. Furthermore, NOD mice were injected with CatG siRNA in early stage to observe the effect of CatG knockdown on the activation status of CD4+ T cells and the progression of diabetes. During the pathogenesis of diabetes, the expression level of CatG in NOD mice gradually increased and the CD4+ T cells were gradually activated, resulting in more TH1 cells and less TH2 and Treg cells. Treatment with CatG-specific inhibitor reduced the blood glucose level, improved the function of islet β cells and reduced the activation of CD4+ T cells. Early application of CatG siRNA improved the function of islet β cells, reduced islet inflammation and β cell apoptosis, and lowered the activation level of CD4+ T cells, thus slowing down the progression of diabetes.

  6. Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers

    Directory of Open Access Journals (Sweden)

    Kuszynski Charles

    2011-07-01

    Full Text Available Abstract Background Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent. Results The utility of MHC dextramers was evaluated in three autoimmune disease models: 1 proteolipid protein (PLP 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s mice; 2 myelin oligodendrocyte glycoprotein (MOG 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b mice; and 3 cardiac myosin heavy chain (Myhc-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo. Conclusions The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.

  7. CD4 + CELL RESPONSE TO ANTI-RETROVIRAL THERAPY (ARTs ...

    African Journals Online (AJOL)

    enhances immunity by sustained HIV- viral suppression, increase in CD4+ cell count and immune restoration. ... Seventy three (70.9%) of patients still had immune depletion with low CD4+ cell counts at one year of receiving HAART. ..... homeostasis and function in advanced HIV disease. Science 1997; 277: 112- 116. 7.

  8. Immunophenotypic enumeration of CD4 T-lymphocyte values in ...

    African Journals Online (AJOL)

    McRoy

    lymphocytes play a central role in regulation of immune response.[2] These ..... influence of sex hormones on lymphocyte subpopulations. ... Friedland GH. Early treatment for HIV-The Time. Has Come. N Engl J Med 1990;322:1000-1002. 7. Gebo KA, Gallant JE, Keruly JC, Moore RD. Absolute CD4 vs. CD4 percentage for ...

  9. Preferential susceptibility of Th9 and Th2 CD4+ T cells to X4-tropic HIV-1 infection.

    Science.gov (United States)

    Orlova-Fink, Nina; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

    2017-10-23

    The functional polarization of CD4 T cells determines their antimicrobial effector profile, but may also impact the susceptibility to infection with HIV-1. Here, we analyzed the susceptibility of CD4 T cells with different functional polarization to infection with X4 and R5-tropic HIV-1. CD4 T cells with a Th1, Th2, Th17, and Th9 polarization were subjected to in-vitro infection assays with X4, R5, or vesicular stomatitis virus-G protein-pseudotyped HIV-1. In addition, we sorted differentially polarized CD4 T-cell subsets from individuals treated with antiretroviral therapy and analyzed the tropism of viral env sequences. Th9-polarized CD4 T cells and, to a lesser extent, Th2-polarized CD4 T cells expressed higher surface levels of CXCR4, and are more permissive to X4-tropic infection in vitro. In contrast, Th1 and Th17 CD4 T cells exhibited stronger surface expression of CCR5, and were more susceptible to infection with R5-tropic viruses. Correspondingly, the distribution of X4-tropic viral sequences in antiretroviral therapy-treated HIV-1-infected patients was biased toward Th9/Th2 cells, whereas R5-tropic sequences were more frequently observed in Th17 cells. CD4 T-cell polarization is associated with a distinct susceptibility to X4 and R5-tropic HIV-1 infection.

  10. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    Science.gov (United States)

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  11. Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells

    DEFF Research Database (Denmark)

    Petersen, C M; Christensen, E I; Andresen, B S

    1992-01-01

    degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation...... occasional labeling was seen in endosomes but whole cell CD4 decreased dramatically. However, methylamine-treated PMA blasts showed accumulation of CD4 in lysosomes and accordingly, pulse-chase experiments in biolabeled cell cultures suggested a manifest reduction of CD4 half-life in response to PMA. Despite...... in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal...

  12. Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

    International Nuclear Information System (INIS)

    Pham, Son; Tabarin, Thibault; Garvey, Megan; Pade, Corinna; Rossy, Jérémie; Monaghan, Paul; Hyatt, Alex; Böcking, Till; Leis, Andrew; Gaus, Katharina; Mak, Johnson

    2015-01-01

    Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.

  13. Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Pham, Son [Deakin University, Victoria 3216 (Australia); CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Tabarin, Thibault [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Garvey, Megan; Pade, Corinna [Deakin University, Victoria 3216 (Australia); CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Rossy, Jérémie [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Monaghan, Paul; Hyatt, Alex [CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Böcking, Till [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Leis, Andrew [CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia); Gaus, Katharina, E-mail: k.gaus@unsw.edu.au [ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, New South Wales 3220 (Australia); Mak, Johnson, E-mail: j.mak@deakin.edu.au [Deakin University, Victoria 3216 (Australia); CSIRO Australian Animal Health Laboratory, Victoria 3220 (Australia)

    2015-12-15

    Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.

  14. The clinical and economic impact of point-of-care CD4 testing in mozambique and other resource-limited settings: a cost-effectiveness analysis.

    Directory of Open Access Journals (Sweden)

    Emily P Hyle

    2014-09-01

    Full Text Available Point-of-care CD4 tests at HIV diagnosis could improve linkage to care in resource-limited settings. Our objective is to evaluate the clinical and economic impact of point-of-care CD4 tests compared to laboratory-based tests in Mozambique.We use a validated model of HIV testing, linkage, and treatment (CEPAC-International to examine two strategies of immunological staging in Mozambique: (1 laboratory-based CD4 testing (LAB-CD4 and (2 point-of-care CD4 testing (POC-CD4. Model outcomes include 5-y survival, life expectancy, lifetime costs, and incremental cost-effectiveness ratios (ICERs. Input parameters include linkage to care (LAB-CD4, 34%; POC-CD4, 61%, probability of correctly detecting antiretroviral therapy (ART eligibility (sensitivity: LAB-CD4, 100%; POC-CD4, 90% or ART ineligibility (specificity: LAB-CD4, 100%; POC-CD4, 85%, and test cost (LAB-CD4, US$10; POC-CD4, US$24. In sensitivity analyses, we vary POC-CD4-specific parameters, as well as cohort and setting parameters to reflect a range of scenarios in sub-Saharan Africa. We consider ICERs less than three times the per capita gross domestic product in Mozambique (US$570 to be cost-effective, and ICERs less than one times the per capita gross domestic product in Mozambique to be very cost-effective. Projected 5-y survival in HIV-infected persons with LAB-CD4 is 60.9% (95% CI, 60.9%-61.0%, increasing to 65.0% (95% CI, 64.9%-65.1% with POC-CD4. Discounted life expectancy and per person lifetime costs with LAB-CD4 are 9.6 y (95% CI, 9.6-9.6 y and US$2,440 (95% CI, US$2,440-US$2,450 and increase with POC-CD4 to 10.3 y (95% CI, 10.3-10.3 y and US$2,800 (95% CI, US$2,790-US$2,800; the ICER of POC-CD4 compared to LAB-CD4 is US$500/year of life saved (YLS (95% CI, US$480-US$520/YLS. POC-CD4 improves clinical outcomes and remains near the very cost-effective threshold in sensitivity analyses, even if point-of-care CD4 tests have lower sensitivity/specificity and higher cost than published

  15. Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection

    Directory of Open Access Journals (Sweden)

    Janice K. P. Lam

    2018-03-01

    Full Text Available Long term carriers were shown to generate robust polyfunctional T cell (PFC responses against lytic and latent antigens of Epstein-Barr virus (EBV. However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM] and asymptomatic (AS EBV infection. Evaluation of IFN-γ secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-γ, TNF-α, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6–12 months. In conclusion, EBV antigen-specific CD4+ and CD

  16. Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

    Science.gov (United States)

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

    2013-03-01

    Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Mitofusin 2 Promotes Apoptosis of CD4+ T Cells by Inhibiting Autophagy in Sepsis

    Directory of Open Access Journals (Sweden)

    Lan Ying

    2017-01-01

    Full Text Available Apoptosis of CD4+ T cells is a primary pathophysiological mechanism of immune dysfunction in the pathogenesis of sepsis. Mitofusin 2 (Mfn2, an integral mitochondrial outer membrane protein, has been confirmed to be associated with cellular metabolism, proliferation, and apoptosis. The function of Mfn2 in CD4+ T cell apoptosis in sepsis is poorly understood. Here, we discovered increased in vivo Mfn2 expression, autophagy deficiency, and elevated cell apoptosis in murine splenic CD4+ T cells after cecal ligation and puncture (CLP. We also observed almost identical results in splenic CD4+ T cells upon lipopolysaccharide (LPS stimulation in vitro. Furthermore, overexpression of Mfn2 resulted in impaired autophagy and increased apoptosis in Jurkat cells. Pharmacological inhibition of autophagy with 3-methyladenine enhanced Mfn2 overexpression-induced cell apoptosis. In addition, overexpression of Mfn2 downregulated phorbol myristate acetate (PMA/ionomycin-, rapamycin- and starvation-induced autophagy in Jurkat T cells. Taken together, these data indicate a critical role of Mfn2 in CD4+ T cell apoptosis in sepsis and the underlying mechanism of autophagy deficiency.

  18. Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells

    Science.gov (United States)

    Li, Peilin; Fujimoto, Katsuya; Bourguingnon, Lilly; Yukl, Steven; Deeks, Steven; Wong, Joseph K

    2014-01-01

    Preventing mucosal transmission of HIV is critical to halting the HIV epidemic. Novel approaches to preventing mucosal transmission are needed. Hyaluronic acid (HA) is a major extracellular component of mucosa and the primary ligand for the cell surface receptor CD44. CD44 enhances HIV infection of CD4+ T cells, but the role of HA in this process is not clear. To study this, virions were generated with CD44 (HIVCD44) or without CD44 (HIVmock). Exogenous HA reduced HIV infection of unstimulated CD4+ T cells in a CD44-dependent manner. Conversely, hyaluronidase-mediated reduction of endogenous HA on the cell surface enhanced HIV binding to and infection of unstimulated CD4+ T cells. Exogenous HA treatment reduced activation of protein kinase C alpha via CD44 on CD4+ T cells during infection with HIVCD44. These results reveal new roles for HA during the interaction of HIV with CD4+ T cells that may be relevant to mucosal HIV transmission and could be exploitable as a future strategy to prevent HIV infection. PMID:24957217

  19. Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

    Science.gov (United States)

    Jensen, Helle; Folkersen, Lasse; Skov, Søren

    2012-01-01

    NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

  20. Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

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    Helle Jensen

    Full Text Available NKG2D is a stimulatory receptor expressed by natural killer (NK cells, CD8(+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+ T-cells, however recently a subset of NKG2D(+ CD4(+ T-cells has been found, which is specific for human cytomegalovirus (HCMV. This particular subset of HCMV-specific NKG2D(+ CD4(+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+ CD4(+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA to investigate the gene expression profile of NKG2D(+ CD4(+ T-cells, generated from HCMV-primed CD4(+ T-cells. We show that the HCMV-primed NKG2D(+ CD4(+ T-cells possess a higher differentiated phenotype than the NKG2D(- CD4(+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+ T-cells, whereas it is produced de novo in resting CD4(+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+ CD4(+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression.

  1. Professional memory CD4+ T lymphocytes preferentially reside and rest in the bone marrow.

    Science.gov (United States)

    Tokoyoda, Koji; Zehentmeier, Sandra; Hegazy, Ahmed N; Albrecht, Inka; Grün, Joachim R; Löhning, Max; Radbruch, Andreas

    2009-05-01

    CD4(+) T lymphocytes are key to immunological memory. Here we show that in the memory phase of specific immune responses, most of the memory CD4(+) T lymphocytes had relocated into the bone marrow (BM) within 3-8 weeks after their generation-a process involving integrin alpha2. Antigen-specific memory CD4(+) T lymphocytes highly expressed Ly-6C, unlike most splenic CD44(hi)CD62L(-) CD4(+) T lymphocytes. In adult mice, more than 80% of Ly-6C(hi)CD44(hi)CD62L(-) memory CD4(+) T lymphocytes were in the BM. In the BM, they associated to IL-7-expressing VCAM-1(+) stroma cells. Gene expression and proliferation were downregulated, indicating a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and efficiently induced the production of high-affinity antibodies by B lymphocytes. Thus, in the memory phase of immunity, memory helper T cells are maintained in BM as resting but highly reactive cells in survival niches defined by IL-7-expressing stroma cells.

  2. Saturation Mutagenesis of the HIV-1 Envelope CD4 Binding Loop Reveals Residues Controlling Distinct Trimer Conformations.

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    Maria Duenas-Decamp

    2016-11-01

    Full Text Available The conformation of HIV-1 envelope (Env glycoprotein trimers is key in ensuring protection against waves of neutralizing antibodies generated during infection, while maintaining sufficient exposure of the CD4 binding site (CD4bs for viral entry. The CD4 binding loop on Env is an early contact site for CD4 while penetration of a proximal cavity by CD4 triggers Env conformational changes for entry. The role of residues in the CD4 binding loop in regulating the conformation of the trimer and trimer association domain (TAD was investigated using a novel saturation mutagenesis approach. Single mutations identified, resulted in distinct trimer conformations affecting CD4bs exposure, the glycan shield and the TAD across diverse HIV-1 clades. Importantly, mutations that improve access to the CD4bs without exposing the immunodominant V3 loop were identified. The different trimer conformations identified will affect the specificity and breadth of nabs elicited in vivo and are important to consider in design of Env immunogens for vaccines.

  3. Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes.

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    Markus Fehrholz

    Full Text Available Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP- B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes.Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®. Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were

  4. Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

    Science.gov (United States)

    Tremblay-McLean, Alexandra; Bruneau, Julie; Lebouché, Bertrand; Lisovsky, Irene; Song, Rujun; Bernard, Nicole F

    2017-10-12

    Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4 - ) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4 - ) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

  5. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    International Nuclear Information System (INIS)

    Kim, Kyung-Chang; Kim, Hyeon Guk; Roh, Tae-Young; Park, Jihwan; Jung, Kyung-Min; Lee, Joo-Shil; Choi, Sang-Yun; Kim, Sung Soon; Choi, Byeong-Sun

    2011-01-01

    Research highlights: → CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. → CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. → HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. → H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. → HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56 Lck , ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56 Lck , ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new antireservoir therapy.

  6. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung-Chang [National Institute of Health, Chungbuk (Korea, Republic of); School of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Hyeon Guk [National Institute of Health, Chungbuk (Korea, Republic of); Roh, Tae-Young [Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Park, Jihwan [Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Jung, Kyung-Min; Lee, Joo-Shil [National Institute of Health, Chungbuk (Korea, Republic of); Choi, Sang-Yun [School of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Sung Soon [National Institute of Health, Chungbuk (Korea, Republic of); Choi, Byeong-Sun, E-mail: byeongsun@korea.kr [National Institute of Health, Chungbuk (Korea, Republic of)

    2011-01-14

    Research highlights: {yields} CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. {yields} CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. {yields} HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. {yields} H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. {yields} HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56{sup Lck}, ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56{sup Lck}, ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new

  7. Correlation of CD4 counts with microalbuminuria in HIV patients

    Science.gov (United States)

    Bakri, S.; Haerani, R.; Hasyim, K.; Sudirman, K.; Tarukallo, N.

    2018-03-01

    One of the manifestations of kidney disease is Microalbuminuria (MA). CD4 T cells are cells that play a central role in immune protection, wherein HIV infections, they are the primary target of the virus. CD4 cells counts is an indirect reflection of the activity and viral load of HIV. This study aimed to determine the correlation of CD4 counts with MA in HIV patients. A cross-sectional with thedescriptive analytical study was in HIV patients >18 years old without a history of Diabetes Mellitus. The result of thestatistical test is significant if the value of p HIV patients.

  8. CD4 cell levels during treatment for tuberculosis (TB in Ethiopian adults and clinical markers associated with CD4 lymphocytopenia.

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    Sten Skogmar

    Full Text Available BACKGROUND: The clinical correlations and significance of subnormal CD4 levels in HIV-negative patients with TB are unclear. We have determined CD4 cell levels longitudinally during anti-tuberculosis treatment (ATT in patients, with and without HIV co-infection, and their associations with clinical variables. METHOD: Adults diagnosed with TB (maximum duration of ATT for 2 weeks, and with no history of antiretroviral therapy (ART in HIV-positive subjects were included consecutively in eight out-patient clinics in Ethiopia. Healthy individuals were recruited for comparison at one of the study health centers. Data on patient characteristics and physical findings were collected by trained nurses following a structured questionnaire at inclusion and on follow-up visits at 2 and 6 months. In parallel, peripheral blood CD4 cell levels were determined. The evolution of CD4 cell levels during ATT was assessed, and the association between clinical characteristics and low CD4 cell levels at baseline was investigated using regression analysis. RESULTS: In total, 1116 TB patients were included (307 HIV-infected. Among 809 HIV-negative patients, 200 (25% had subnormal CD4 cell counts (<500 cells/mm(3, with <350 cells/mm(3 in 82 (10% individuals. CD4 cell levels increased significantly during the course of ATT in both HIV+ and HIV- TB-patients, but did not reach the levels in healthy subjects (median 896 cells/mm(3. Sputum smear status, signs of wasting (low mid upper arm circumference (MUAC, and bedridden state were significantly associated with low CD4 cell counts. CONCLUSION: A high proportion of Ethiopian TB patients have subnormal CD4 cell counts before starting treatment. Low CD4 cell levels are associated with smear positive disease and signs of wasting. The continuous increase of CD4 cell counts during the course of ATT suggest a reversible impact of active TB on CD4 cell homeostasis, which may be considered in interpretation of CD4 cell counts in HIV

  9. Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells.

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    Joanne M Lumsden

    Full Text Available A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP-specific CD4(+ T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM and/or central memory (T(CM CD4(+ T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both T(E/EM and T(CM cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4(+ T(E/EM cells and of CD4(+ T(CM cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4(+ T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4(+ T(E/EM cells and of CD4(+ T(E/EM cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both T(E/EM and T(CM cells are major producers of IL-2.

  10. T Cell Epitope Immunotherapy Induces a CD4+ T Cell Population with Regulatory Activity

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    Verhoef Adrienne

    2005-01-01

    Full Text Available Background Synthetic peptides, representing CD4+ T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils. Methods and Findings In this study we address the mechanism of action of peptide immunotherapy (PIT in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4+ cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4neg PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4+ and CD8+ PBMCs. Conclusion This study provides evidence for the induction of a population of CD4+ T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.

  11. Role of distinct CD4(+) T helper subset in pathogenesis of oral lichen planus.

    Science.gov (United States)

    Wang, Hui; Zhang, Dunfang; Han, Qi; Zhao, Xin; Zeng, Xin; Xu, Yi; Sun, Zheng; Chen, Qianming

    2016-07-01

    Oral lichen planus (OLP) is one of the most common chronic inflammatory oral mucosal diseases with T-cell-mediated immune pathogenesis. In subepithelial and lamina propria of OLP local lesions, the presence of CD4(+) T helper (CD4(+) Th) cells appeared as the major lymphocytes. These CD4(+) T lymphocytes can differentiate into distinct Th cell types such as Th1, Th2, Treg, Th17, Th22, Th9, and Tfh within the context of certain cytokines environment. Growing evidence indicated that Th1/Th2 imbalance may greatly participate into the cytokine network of OLP immunopathology. In addition, Th1/Th2 imbalance can be regulated by the Treg subset and also greatly influenced by the emerging novel CD4(+) Th subset Th17. Furthermore, the presence of novel subsets Th22, Th9 and Tfh in OLP patients is yet to be clarified. All these Th subsets and their specific cytokines may play a critical role in determining the character, extent and duration of immune responses in OLP pathogenesis. Therefore, we review the roles of distinct CD4(+) Th subsets and their signature cytokines in determining disease severity and susceptibility of OLP and also reveal the novel therapeutic strategies based on T lymphocytes subsets in OLP treatment. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Percentages of CD4+CD161+ and CD4−CD8−CD161+ T Cells in the Synovial Fluid Are Correlated with Disease Activity in Rheumatoid Arthritis

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    Jinlin Miao

    2015-01-01

    Full Text Available Objective. CD161 has been identified as a marker of human IL-17-producing T cells that are implicated in the pathogenesis of rheumatoid arthritis (RA. This study aimed to investigate the potential link between the percentage of CD161+ T cells and disease activity in RA patients. Methods. Peripheral blood (PB from 54 RA patients and 21 healthy controls was evaluated. Paired synovial fluid (SF (n = 17 was analyzed. CD161 expression levels on CD4+, CD8+, and CD4−CD8− T cells were assessed by flow cytometry. Results. The percentage of CD4+CD161+ T cells in RA SF was higher than RA PB, and it was positively correlated with DAS28, erythrocyte sedimentation rate (ESR, and C-reactive protein (CRP. CD4−CD8−CD161+ T cell percentage was decreased in RA PB and was further reduced in RA SF, and its level in SF was inversely correlated with DAS28, ESR, and CRP. However, CD8+CD161+ T cell percentage was neither changed in RA PB and SF nor correlated with disease activity indices. Conclusion. An increased CD4+CD161+ T cell percentage and a decreased CD4−CD8−CD161+ T cell percentage are present in RA SF and are associated with disease activity, and the accumulation of CD4+CD161+ T cells in SF may contribute to the local inflammation of RA.

  13. ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis

    Science.gov (United States)

    Moguche, Albanus O.; Shafiani, Shahin; Clemons, Corey; Larson, Ryan P.; Dinh, Crystal; Higdon, Lauren E.; Cambier, C.J.; Sissons, James R.; Gallegos, Alena M.; Fink, Pamela J.

    2015-01-01

    Immune control of persistent infection with Mycobacterium tuberculosis (Mtb) requires a sustained pathogen-specific CD4 T cell response; however, the molecular pathways governing the generation and maintenance of Mtb protective CD4 T cells are poorly understood. Using MHCII tetramers, we show that Mtb-specific CD4 T cells are subject to ongoing antigenic stimulation. Despite this chronic stimulation, a subset of PD-1+ cells is maintained within the lung parenchyma during tuberculosis (TB). When transferred into uninfected animals, these cells persist, mount a robust recall response, and provide superior protection to Mtb rechallenge when compared to terminally differentiated Th1 cells that reside preferentially in the lung-associated vasculature. The PD-1+ cells share features with memory CD4 T cells in that their generation and maintenance requires intrinsic Bcl6 and intrinsic ICOS expression. Thus, the molecular pathways required to maintain Mtb-specific CD4 T cells during ongoing infection are similar to those that maintain memory CD4 T cells in scenarios of antigen deprivation. These results suggest that vaccination strategies targeting the ICOS and Bcl6 pathways in CD4 T cells may provide new avenues to prevent TB. PMID:25918344

  14. A vaccine encoding conserved promiscuous HIV CD4 epitopes induces broad T cell responses in mice transgenic to multiple common HLA class II molecules.

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    Susan Pereira Ribeiro

    Full Text Available Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, "promiscuous" (multiple HLA-DR-binding B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8. Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

  15. The early activation marker CD69 regulates the expression of chemokines and CD4 T cell accumulation in intestine.

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    Katarina Radulovic

    Full Text Available Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+ T cells and/or CD4(- cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/- CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/- CD4 T cell accumulation in colonic lamina propria (cLP was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/- mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/- CD45RB(high CD4 T cells into RAG(-/- hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.

  16. CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

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    Zhang, Xiaolong; Chang Li, Xian; Xiao, Xiang; Sun, Rui; Tian, Zhigang; Wei, Haiming

    2013-01-01

    The peripheral Foxp3+ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4+ T cells can be readily converted to Foxp3+ iTreg in vitro, and memory CD4+ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3+ T cells from various CD4+ T-cell subsets in human peripheral blood. Though naive CD4+ T cells were readily converted to Foxp3+ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3+ T cells did not express the memory marker CD45RO as do Foxp3+ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4+ T cells, defined as CD62L+ central memory T cells, could be induced by TGF-β to differentiate into Foxp3+ T cells. It is well known that Foxp3+ T cells derived from human CD4+CD25- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4+CD62L+ central memory T cell-derived Foxp3+ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4+ T cell-derived Foxp3+ T cells. But further research showed that mouse CD4+ central memory T cells also could be induced to differentiate into Foxp3+ T cells, such Foxp3+ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4+CD62L+ central memory T cells as a novel potential source of iTreg. PMID:24155942

  17. Changes and clinical significance of CD4+CD25+CD127- regulatory T cells in Graves disease

    International Nuclear Information System (INIS)

    Zou Jintao; Yu Peiling; Dong Jingwei; Liao Qihong; Liu Dongliang; Zeng Hongyi

    2012-01-01

    Objective: To investigate the mechanism of Graves disease by observing the changes of CD4 + CD25 + CD127 - regulatory T cells (Treg) population in the patients. Methods: Flow cytometry was used to detect the proportion of CD4 + CD25 + CD127 - Treg of CD4 + T cells in 90 Graves disease patients (Graves disease group) and 50 healthy adults (control group). Thyroid function and autoantibody levels were determined simultaneously. The t test was adopted for comparison between groups. The relationship between CD4 + CD25 + CD127 - Treg and thyroid function was analyzed by linear correlation analysis. Results: The percentages of CD4 + CD25 + CD127 - Treg in Graves disease group and control group were 1.39%±1.09% and 4.59%±1.14% separately. There was significant difference between the two groups (t=16.4, P<0.01). There were negative correlation between CD4 + CD25 + CD127 - Treg percentages and total triiodothyronine, total thyroxine,free triiodothyronine, free thyroxine and thyrotropin receptor antibody,thyroglobulin antibody, thyroid microsomal antibody (r=-0.62, -0.65, -0.56, -0.71, -0.50, -0.15, all P<0.01). Conclusions: The reduction of CD4 + CD25 + CD127 - Treg percentages in Graves disease group and close relations of CD4 + CD25 + CD127 - Treg with thyroid function and thyroid autoantibody levels suggest that CD4 + CD25 + CD127 - Treg decrease in the number may be associated with the onset of Graves disease. CD4 + CD25 + CD127 - may be the specific marker of Treg. (authors)

  18. Protein scissors: Photocleavage of proteins at specific locations

    Indian Academy of Sciences (India)

    Unknown

    Binding of ligands to globular proteins at hydrophobic cavities while making specific ... ched to a PTI model A1010 monochromator. UV cut-off filter ..... >1:1 stoichiometry (protein to ligand), the binding equilibrium favors the thermo- dynamically ...

  19. Reference values of CD4 T-lymphocytes in human ...

    African Journals Online (AJOL)

    exposed uninfected infants in Kano.Nigeria. ... Journal of Medicine in the Tropics ... Studies to evaluate CD4 count in vertically exposed, but human immunodeficiency virus (HIV) negative infants from this region have not been done previously.

  20. The central nervous system environment controls effector CD4+ T cell cytokine profile in experimental allergic encephalomyelitis

    DEFF Research Database (Denmark)

    Krakowski, M L; Owens, T

    1997-01-01

    In experimental allergic encephalomyelitis (EAE), CD4+ T cells infiltrate the central nervous system (CNS). We derived CD4+ T cell lines from SJL/J mice that were specific for encephalitogenic myelin basic protein (MBP) peptides and produced both Th1 and Th2 cytokines. These lines transferred EAE...... to naive mice. Peptide-specific cells re-isolated from the CNS only produced Th1 cytokines, whereas T cells in the lymph nodes produced both Th1 and Th2 cytokines. Mononuclear cells isolated from the CNS, the majority of which were microglia, presented antigen to and stimulated MBP-specific T cell lines...... in vitro. Although CNS antigen-presenting cells (APC) supported increased production of interferon (IFN)-gamma mRNA by these T cells, there was no increase in the interleukin (IL)-4 signal, whereas splenic APC induced increases in both IFN-gamma and IL-4. mRNA for IL-12 (p40 subunit) was up...

  1. A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool.

    Science.gov (United States)

    Schiller, Annemarie; Zhang, Ting; Li, Ruliang; Duechting, Andrea; Sundararaman, Srividya; Przybyla, Anna; Kuerten, Stefanie; Lehmann, Paul V

    2017-12-07

    Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus , Mycoplasma , Lactobacillus , Neisseria , Candida , Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the 'CPI pool'), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

  2. Early programming and late-acting checkpoints governing the development of CD4 T cell memory.

    Science.gov (United States)

    Dhume, Kunal; McKinstry, K Kai

    2018-04-27

    CD4 T cells contribute to protection against pathogens through numerous mechanisms. Incorporating the goal of memory CD4 T cell generation into vaccine strategies thus offers a powerful approach to improve their efficacy, especially in situations where humoral responses alone cannot confer long-term immunity. These threats include viruses such as influenza that mutate coat proteins to avoid neutralizing antibodies, but that are targeted by T cells that recognize more conserved protein epitopes shared by different strains. A major barrier in the design of such vaccines is that the mechanisms controlling the efficiency with which memory cells form remain incompletely understood. Here, we discuss recent insights into fate decisions controlling memory generation. We focus on the importance of three general cues: interleukin-2, antigen, and costimulatory interactions. It is increasingly clear that these signals have a powerful influence on the capacity of CD4 T cells to form memory during two distinct phases of the immune response. First, through 'programming' that occurs during initial priming, and second, through 'checkpoints' that operate later during the effector stage. These findings indicate that novel vaccine strategies must seek to optimize cognate interactions, during which interleukin-2-, antigen, and costimulation-dependent signals are tightly linked, well beyond initial antigen encounter to induce robust memory CD4 T cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Role of IL-4 receptor α-positive CD4(+) T cells in chronic airway hyperresponsiveness.

    Science.gov (United States)

    Kirstein, Frank; Nieuwenhuizen, Natalie E; Jayakumar, Jaisubash; Horsnell, William G C; Brombacher, Frank

    2016-06-01

    TH2 cells and their cytokines are associated with allergic asthma in human subjects and with mouse models of allergic airway disease. IL-4 signaling through the IL-4 receptor α (IL-4Rα) chain on CD4(+) T cells leads to TH2 cell differentiation in vitro, implying that IL-4Rα-responsive CD4(+) T cells are critical for the induction of allergic asthma. However, mechanisms regulating acute and chronic allergen-specific TH2 responses in vivo remain incompletely understood. This study defines the requirements for IL-4Rα-responsive CD4(+) T cells and the IL-4Rα ligands IL-4 and IL-13 in the development of allergen-specific TH2 responses during the onset and chronic phase of experimental allergic airway disease. Development of acute and chronic ovalbumin (OVA)-induced allergic asthma was assessed weekly in CD4(+) T cell-specific IL-4Rα-deficient BALB/c mice (Lck(cre)IL-4Rα(-/lox)) and respective control mice in the presence or absence of IL-4 or IL-13. During acute allergic airway disease, IL-4 deficiency did not prevent the onset of TH2 immune responses and OVA-induced airway hyperresponsiveness or goblet cell hyperplasia, irrespective of the presence or absence of IL-4Rα-responsive CD4(+) T cells. In contrast, deficiency of IL-13 prevented allergic asthma, irrespective of the presence or absence of IL-4Rα-responsive CD4(+) T cells. Importantly, chronic allergic inflammation and airway hyperresponsiveness were dependent on IL-4Rα-responsive CD4(+) T cells. Deficiency in IL-4Rα-responsive CD4(+) T cells resulted in increased numbers of IL-17-producing T cells and, consequently, increased airway neutrophilia. IL-4-responsive T helper cells are dispensable for acute OVA-induced airway disease but crucial in maintaining chronic asthmatic pathology. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  4. CD4 T cell autophagy is integral to memory maintenance.

    Science.gov (United States)

    Murera, Diane; Arbogast, Florent; Arnold, Johan; Bouis, Delphine; Muller, Sylviane; Gros, Frédéric

    2018-04-13

    Studies of mice deficient for autophagy in T cells since thymic development, concluded that autophagy is integral to mature T cell homeostasis. Basal survival and functional impairments in vivo, limited the use of these models to delineate the role of autophagy during the immune response. We generated Atg5 f/f distal Lck (dLck)-cre mice, with deletion of autophagy only at a mature stage. In this model, autophagy deficiency impacts CD8 + T cell survival but has no influence on CD4 + T cell number and short-term activation. Moreover, autophagy in T cells is dispensable during early humoral response but critical for long-term antibody production. Autophagy in CD4 + T cells is required to transfer humoral memory as shown by injection of antigen-experienced cells in naive mice. We also observed a selection of autophagy-competent cells in the CD4 + T cell memory compartment. We performed in vitro differentiation of memory CD4 + T cells, to better characterize autophagy-deficient memory cells. We identified mitochondrial and lipid load defects in differentiated memory CD4 + T cells, together with a compromised survival, without any collapse of energy production. We then propose that memory CD4 + T cells rely on autophagy for their survival to regulate toxic effects of mitochondrial activity and lipid overload.

  5. Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

    Science.gov (United States)

    Karim, Ahmad Faisal; Reba, Scott M; Li, Qing; Boom, W Henry; Rojas, Roxana E

    2017-09-01

    We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4 + T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4 + T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4 + T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4 + T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4 + T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4 + T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response.

    Science.gov (United States)

    Hus, I; Schmitt, M; Tabarkiewicz, J; Radej, S; Wojas, K; Bojarska-Junak, A; Schmitt, A; Giannopoulos, K; Dmoszyńska, A; Roliński, J

    2008-05-01

    Recently, we described that vaccination with allogeneic dendritic cells (DCs) pulsed with tumor cell lysate generated specific CD8+ T cell response in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the potential of autologous DCs pulsed ex vivo with tumor cell lysates to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Twelve patients at clinical stage 0-2 as per Rai were vaccinated intradermally up to eight times with a mean number of 7.4 x 10(6) DCs pulsed with B-CLL cell lysate. We observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. A significant increase of specific cytotoxic CD8+ T lymphocytes against the leukemia-associated antigens RHAMM or fibromodulin was detected in four patients after DC vaccination. In patients with a clinical response, an increase of interleukin 12 (IL-12) serum levels and a decrease of the frequency of CD4+CD25(+)FOXP3+ T regulatory cells were observed. Taken together, the study demonstrated that vaccination with autologous DC in CLL patients is feasible and safe. Immunological and to some extend hematological responses could be noted, justifying further investigation on this immunotherapeutical approach.

  7. How Do CD4+ T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

    Science.gov (United States)

    Haabeth, Ole Audun Werner; Tveita, Anders Aune; Fauskanger, Marte; Schjesvold, Fredrik; Lorvik, Kristina Berg; Hofgaard, Peter O.; Omholt, Hilde; Munthe, Ludvig A.; Dembic, Zlatko; Corthay, Alexandre; Bogen, Bjarne

    2014-01-01

    CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed. PMID:24782871

  8. How do CD4+ T cells detect and eliminate tumor cells that either lack or express MHC class II molecules?

    Directory of Open Access Journals (Sweden)

    Ole Audun Werner Haabeth

    2014-04-01

    Full Text Available CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor specific antigen by host antigen presenting cells (APCs appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315, where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-g stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed.

  9. Specificity of transmembrane protein palmitoylation in yeast.

    Directory of Open Access Journals (Sweden)

    Ayelén González Montoro

    Full Text Available Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs, characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD. There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as

  10. Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

    Science.gov (United States)

    Wheeler, Lee Adam; Trifonova, Radiana; Vrbanac, Vladimir; Basar, Emre; McKernan, Shannon; Xu, Zhan; Seung, Edward; Deruaz, Maud; Dudek, Tim; Einarsson, Jon Ivar; Yang, Linda; Allen, Todd M.; Luster, Andrew D.; Tager, Andrew M.; Dykxhoorn, Derek M.; Lieberman, Judy

    2011-01-01

    The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission. PMID:21576818

  11. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  12. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

    International Nuclear Information System (INIS)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru

    2013-01-01

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4 + IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4 + IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4 + IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4 + IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4 + LPLs and primed splenic CD4 + T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4 + IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

  13. Predominant CD4 T-lymphocyte tropism of human herpesvirus 6-related virus.

    OpenAIRE

    Takahashi, K; Sonoda, S; Higashi, K; Kondo, T; Takahashi, H; Takahashi, M; Yamanishi, K

    1989-01-01

    Human herpesvirus 6 (HHV-6)-related virus was isolated from CD4+ CD8- and CD3+ CD4+ mature T lymphocytes but could not be isolated from CD4- CD8+, CD4- CD8-, and CD3- T cells in the peripheral blood of exanthem subitum patients. HHV-6-related virus predominantly infected CD4+ CD8+, CD4+ CD8-, and CD3+ CD4+ cells with mature phenotypes and rarely infected CD4- CD8+ cells from cord blood mononuclear cells, which suggested predominant CD4 mature T-lymphocyte tropism of HHV-6-related virus.

  14. GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

    International Nuclear Information System (INIS)

    Igarashi, Hanna; Cao, Yujia; Iwai, Hideyuki; Piao, Jinhua; Kamimura, Yosuke; Hashiguchi, Masaaki; Amagasa, Teruo; Azuma, Miyuki

    2008-01-01

    Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25 - CD4 + effector (Teff) and CD25 + CD4 + regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4 + T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4 + T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists

  15. Colorectal cancer cells suppress CD4+ T cells immunity through canonical Wnt signaling.

    Science.gov (United States)

    Sun, Xuan; Liu, Suoning; Wang, Daguang; Zhang, Yang; Li, Wei; Guo, Yuchen; Zhang, Hua; Suo, Jian

    2017-02-28

    Understanding how colorectal cancer escapes from immunosurveillance and immune attack is important for developing novel immunotherapies for colorectal cancer. In this study we evaluated the role of canonical Wnt signaling in the regulation of T cell function in a mouse colorectal cancer model. We found that colorectal cancer cells expressed abundant Wnt ligands, and intratumoral T cells expressed various Frizzled proteins. Meanwhile, both active β-catenin and total β-catenin were elevated in intratumoral T cells. In vitro study indicated that colorectal cancer cells suppressed IFN-γ expression and increased IL-17a expression in activated CD4+ T cells. However, the cytotoxic activity of CD8+ T cells was not altered by colorectal cancer cells. To further evaluate the importance of Wnt signaling for CD4+ T cell-mediated cancer immunity, β-catenin expression was enforced in CD4+ T cells using lentiviral transduction. In an adoptive transfer model, enforced expression of β-catenin in intratumoral CD4+ T cells increased IL-17a expression, enhanced proliferation and inhibited apoptosis of colorectal cancer cells. Taken together, our study disclosed a new mechanism by which colorectal cancer impairs T cell immunity.

  16. Alternative pathway for the development of Vα14+ NKT cells directly from CD4-CD8- thymocytes that bypasses the CD4+CD8+ stage.

    Science.gov (United States)

    Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Aihara, Minako; Ozawa, Ritsuko; Kojo, Satoshi; Harada, Michishige; Endo, Takaho A; Watanabe, Takashi; Ohara, Osamu; Taniguchi, Masaru

    2017-03-01

    Although invariant V α 14 + natural killer T cells (NKT cells) are thought to be generated from CD4 + CD8 + double-positive (DP) thymocytes, the developmental origin of CD4 - CD8 - double-negative (DN) NKT cells still remains unresolved. Here we provide definitive genetic evidence obtained, through studies of mice with DP-stage-specific ablation of expression of the gene encoding the recombinase component RAG-2 (Rag2) and by a fate-mapping approach, that supports the proposal of the existence of an alternative developmental pathway through which a fraction of DN NKT cells with strong T-helper-type-1 (T H 1)-biased and cytotoxic characteristics develop from late DN-stage thymocytes, bypassing the DP stage. These findings provide new insight into understanding of the development of NKT cells and propose a role for timing of expression of the invariant T cell antigen receptor in determining the functional properties of NKT cells.

  17. Increase in IFNγ(-IL-2(+ cells in recent human CD4 T cell responses to 2009 pandemic H1N1 influenza.

    Directory of Open Access Journals (Sweden)

    Jason M Weaver

    Full Text Available Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1 induced Th1-like responses biased toward the expression of IFNγ(+TNFα(+ CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1 induced more IFNγ(-IL-2(+TNFα(+ T cells, similar to the IFNγ(-IL-2(+ non-polarized, primed precursor T cells (Thpp that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα(+, IFNγ(+, and IL-2(+ cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ(-IL-2(+TNFα(+ CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ(+TNFα(+ responses. These IFNγ(-IL-2(+TNFα(+ CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.

  18. MicroRNA-126 deficiency enhanced the activation and function of CD4+ T cells by elevating IRS-1 pathway.

    Science.gov (United States)

    Chu, F; Hu, Y; Zhou, Y; Guo, M; Lu, J; Zheng, W; Xu, H; Zhao, J; Xu, L

    2018-02-01

    Recent evidence has shown that microRNA-126 (miR-126) has been involved in the development and function of immune cells, which contributed to the pathogenesis of related clinical diseases. However, the potential role of miR-126 in the development and function of CD4 + T cells remains largely unknown. Here we first found that the activation and proliferation, as well as the expression of interferon (IFN)-γ, of CD4 + T cells from miR-126 knock-down (KD) mice using the miRNA-sponge technique were enhanced significantly in vitro, compared with those in CD4 + T cells from wild-type (WT) mice. To monitor further the possible effect of miR-126 deficiency on the function of CD4 + T cells in vivo, we used dextran sulphate sodium (DSS)-induced murine model of acute autoimmune colitis and found that miR-126 deficiency could elevate the pathology of colitis. Importantly, the proportion of CD4 + T cells in splenocytes increased significantly in miR-126KD mice. Moreover, the expression levels of CD69 and CD44 on CD4 + T cells increased significantly and the expression level of CD62L decreased significantly. Of note, adoptive cell transfer assay showed that the pathology of colitis was more serious in carboxyfluorescein succinimidyl ester (CFSE)-labelled miR-126KD CD4 + T cell-transferred group, compared with that in the CFSE-labelled WT CD4 + T cells transferred group. Consistently, the expression levels of CD69 and CD44 on CFSE + cells increased significantly. Furthermore, both the proliferation and IFN-γ secretion of CFSE + cells also increased significantly in the CFSE-labelled miR-126KD CD4 + T cell-transferred group. Mechanistic evidence showed that the expression of insulin receptor substrate 1 (IRS-1), as a functional target of miR-126, was elevated in CD4 + T cells from miR-126KD mice, accompanied by altered transduction of the extracellular regulated kinase, protein B (AKT) and nuclear factor kappa B (NF-κB) pathway. Our data revealed a novel role in which miR-126

  19. Impaired circulating CD4+ LAP+ regulatory T cells in patients with acute coronary syndrome and its mechanistic study.

    Directory of Open Access Journals (Sweden)

    Zheng-Feng Zhu

    Full Text Available OBJECTIVE: CD4(+ latency-associated peptide (LAP(+ regulatory T cells (Tregs are a newly discovered T cell subset in humans and the role of these cells in patients with acute coronary syndrome (ACS has not been explored. We designed to investigate whether circulating frequency and function of CD4(+LAP(+ Tregs are defective in ACS. METHODS: One hundred eleven ACS patients (acute myocardial infarction and unstable angina and 117 control patients were enrolled in the study. The control patients consisted of chronic stable angina (CSA and chest pain syndrome (CPS. The frequencies of circulating CD4(+LAP(+ Tregs and the expression of the transmembrane protein glycoprotein-A repetitions predominant (GARP on CD4(+ T cells were determined by flow cytometry. The function of CD4(+LAP(+ Tregs was detected using thymidine uptake. Serum interleukin-10 (IL-10 and transforming growth factor-β protein (TGF-β levels were detected using ELISA and expression of GARP mRNA in peripheral blood mononuclear cells (PBMCs was measured by real time-polymerase chain reaction. RESULTS: We found ACS patients had a significantly lower frequency of circulating CD4(+LAP(+ Tregs, and the function of these cells was reduced compared to controls. The expression of GARP in CD4(+ T cells and the serum levels of TGF-β in ACS patients were lower than those of control patients. The serum levels of IL-10 were similar between the two cohorts. CONCLUSIONS: A novel regulatory T cell subset, defined as CD4(+LAP(+ T cells is defective in ACS patients.

  20. Site-Specific PEGylation of Therapeutic Proteins

    Directory of Open Access Journals (Sweden)

    Jonathan K. Dozier

    2015-10-01

    Full Text Available The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

  1. Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells.

    Science.gov (United States)

    Singh, Yogesh; Zhou, Yuetao; Zhang, Shaqiu; Abdelazeem, Khalid N M; Elvira, Bernat; Salker, Madhuri S; Lang, Florian

    2017-01-01

    MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b. © 2017 The Author

  2. Immunity to experimental Salmonella typhimurium infections in rats. Transfer of immunity with primed CD45RC+ and CD45RC- CD4 T-cell subpopulations

    DEFF Research Database (Denmark)

    Thygesen, P; Christensen, H B; Hougen, H P

    1996-01-01

    The protective effect of primed CD4 T cells against a lethal dose of Salmonella typhimurium was studied in Lewis rats. Primed CD4 T cells were obtained by inoculating Lewis rats with a non-lethal dose of S. typhimurium. Four weeks after the infection, spleen CD4 T cells were separated by antibody......-induced increase in CD45RC+ cells is most likely due to generation of antigen-specific memory T cells....

  3. Transfer of primed CD4+OX40- T lymphocytes induces increased immunity to experimental Salmonella typhimurium infections in rats

    DEFF Research Database (Denmark)

    Thygesen, P; Christensen, H B; Hougen, H P

    1997-01-01

    The protective effect of primed CD4 T cells against a lethal dose of Salmonella typhimurium was studied in Lewis rats. Primed CD4 T cells were obtained by inoculating Lewis rats with a non-lethal dose of S. typhimurium. Four weeks after the infection, spleen non-adherent mononuclear cells were is......-specific memory T cells that have returned to a resting state....

  4. CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch.

    Science.gov (United States)

    Brown, Deborah M; Dilzer, Allison M; Meents, Dana L; Swain, Susan L

    2006-09-01

    The mechanisms whereby CD4 T cells contribute to the protective response against lethal influenza infection remain poorly characterized. To define the role of CD4 cells in protection against a highly pathogenic strain of influenza, virus-specific TCR transgenic CD4 effectors were generated in vitro and transferred into mice given lethal influenza infection. Primed CD4 effectors conferred protection against lethal infection over a broad range of viral dose. The protection mediated by CD4 effectors did not require IFN-gamma or host T cells, but did result in increased anti-influenza Ab titers compared with untreated controls. Further studies indicated that CD4-mediated protection at high doses of influenza required B cells, and that passive transfer of anti-influenza immune serum was therapeutic in B cell-deficient mice, but only when CD4 effectors were present. Primed CD4 cells also acquired perforin (Pfn)-mediated cytolytic activity during effector generation, suggesting a second mechanism used by CD4 cells to confer protection. Pfn-deficient CD4 effectors were less able to promote survival in intact BALB/c mice and were unable to provide protection in B cell-deficient mice, indicating that Ab-independent protection by CD4 effectors requires Pfn. Therefore, CD4 effectors mediate protection to lethal influenza through at least two mechanisms: Pfn-mediated cytotoxicity early in the response promoted survival independently of Ab production, whereas CD4-driven B cell responses resulted in high titer Abs that neutralized remaining virus.

  5. Utility of CD4 cell counts for early prediction of virological failure during antiretroviral therapy in a resource-limited setting

    Directory of Open Access Journals (Sweden)

    Lawn Stephen D

    2008-07-01

    Full Text Available Abstract Background Viral load monitoring is not available for the vast majority of patients receiving antiretroviral therapy in resource-limited settings. However, the practical utility of CD4 cell count measurements as an alternative monitoring strategy has not been rigorously assessed. Methods In this study, we used a novel modelling approach that accounted for all CD4 cell count and VL values measured during follow-up from the first date that VL suppression was achieved. We determined the associations between CD4 counts (absolute values and changes during ART, VL measurements and risk of virological failure (VL > 1,000 copies/ml following initial VL suppression in 330 patients in South Africa. CD4 count changes were modelled both as the difference from baseline (ΔCD4 count and the difference between consecutive values (CD4 count slope using all 3-monthly CD4 count measurements during follow-up. Results During 7093.2 patient-months of observation 3756 paired CD4 count and VL measurements were made. In patients who developed virological failure (n = 179, VL correlated significantly with absolute CD4 counts (r = - 0.08, P = 0.003, ΔCD4 counts (r = - 0.11, P P P = 0.99, P = 0.92 and P = 0.75, respectively. Moreover, in a receiver operating characteristic (ROC curve, the association between a negative CD4 count slope and virological failure was poor (area under the curve = 0.59; sensitivity = 53.0%; specificity = 63.6%; positive predictive value = 10.9%. Conclusion CD4 count changes correlated significantly with VL at group level but had very limited utility in identifying virological failure in individual patients. CD4 count is an inadequate alternative to VL measurement for early detection of virological failure.

  6. Pengaruh Suplementasi Seng terhadap CD 4+ Pengidap Human Immunodeficiency Virus

    Directory of Open Access Journals (Sweden)

    Mohammad Zen Rahfiludin

    2015-01-01

    Full Text Available ABSTRACT Optimal immune response is needed to viral elimination, but in HIV infection the immune cell is the main target. Zinc has proved to increase immune response to various infection. However, its role in HIV infection has not understood. This study aimed to analyze the effect of zinc supplementation to increase CD4+ in HIV-infected patients. This was an experimental study with pre test post test with control group design. Twenty HIV-infected persons were devided into 2 groups: control group which received Anti Retroviral Therapy (AZT and the Zn+ART group received 5 mg zinc/d orally for one month and AZT. Field workers visited patients for checking compliance. Venous blood was taken from all of the subjects, the CD4+ cell count was measured and daily nutrient intake was analyzed. CD4+ cell count was determined by flowcytometri method. Daily food intake was determined by two 24 hour recall periods during 2 non consecutive days. Differential test between the two groups was performed using independent t-test or Mann Whitney test, when the distribution was not normal. Analysis of CD4+ differences before and after treatment in each group by paired t-test. The mean of CD4+ before zinc supplementation was 371.3 ± 126.8 cell/μL and increase to 415.2 ± 194.1 cell/μL after supplementation. However the increase 43.9 ± 83.5 cell/μL was not significantly different (p= 0.131. There was not a significant change CD4+ (p= 0.112 between both groups, possibly because the dose and duration of zinc supplementation. Moreover, only one type of micronutrient given (zinc, also led to an increase CD4+ in our study did not significantly. Zinc supplementation in complement with AZT therapy was not significantly increase CD4+ in HIV patients. Keywords: zinc supplementation, CD4+, HIV, AZT.   ABSTRAK Respon imun yang optimal diperlukan untuk pemusnahan virus, namun dalam infeksi HIV justru sel sistem imun yang diserang. Seng telah terbukti dapat meningkatkan

  7. HIV-1 induces DCIR expression in CD4+ T cells.

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    Alexandra A Lambert

    2010-11-01

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  8. Clonal expansion of CD4(+) cytotoxic T lymphocytes in patients with IgG4-related disease.

    Science.gov (United States)

    Mattoo, Hamid; Mahajan, Vinay S; Maehara, Takashi; Deshpande, Vikram; Della-Torre, Emanuel; Wallace, Zachary S; Kulikova, Maria; Drijvers, Jefte M; Daccache, Joe; Carruthers, Mollie N; Castelino, Flavia V; Stone, James R; Stone, John H; Pillai, Shiv

    2016-09-01

    IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4(+) T cells constitute the major inflammatory cell population in IgG4-RD lesions. We used an unbiased approach to characterize CD4(+) T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites. We used flow cytometry to identify CD4(+) effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4(+)SLAMF7(+) cytotoxic T lymphocytes (CTLs) and CD4(+)GATA3(+) TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging. CD4(+) effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4(+) CTLs but not CD4(+)GATA3(+) memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4(+) CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4(+) CTLs. IgG4-RD is prominently linked to clonally expanded IL-1β- and TGF-β1-secreting CD4(+) CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4(+) T cells are now linked to a human disease characterized by chronic inflammation and fibrosis. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  9. CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human.

    Science.gov (United States)

    Wu, Yu-Jing; Chen, Heng-Shi; Chen, Wen-Sheng; Dong, Jin; Dong, Xiao-Jie; Dai, Xing; Huang, Qiong; Wei, Wei

    2018-01-01

    Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6'- O -benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4 + T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4 + T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4 + T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr 394 ). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4 + T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr 394 ) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4 + T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

  10. Requirement for CD40 ligand, CD4(+) T cells, and B cells in an infectious mononucleosis-like syndrome

    DEFF Research Database (Denmark)

    Brooks, J W; Hamilton-Easton, A M; Christensen, J P

    1999-01-01

    (+) CD8(+) population that is found in mice with different major histocompatibility complex (MHC) haplotypes. Aspects of the CD8(+)-T-cell response are substantially modified in mice that lack B cells, CD4(+) T cells, or the CD40 ligand (CD40L). The B-cell-deficient mice show no increase in Vbeta4(+) CD8......(+) T cells. Similar abrogation of the Vbeta4(+) CD8(+) response is seen following antibody-mediated depletion of the CD4(+) subset, through the numbers of CD8(+) CD62L(lo) cells are still significantly elevated. Virus-specific CD4(+)-T-cell frequencies are minimal in the CD40L(-/-) mice, and the Vbeta4......(+) CD8(+) population remains unexpanded. Apparently B-cell-CD4(+)-T-cell interactions play a part in the gammaHV-68 induction of both splenomegaly and non-MHC-restricted Vbeta4(+) CD8(+)-T-cell expansion....

  11. HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4(+) T Cell Responses

    DEFF Research Database (Denmark)

    Wang, M. J.; Larsen, Mette Voldby; Nielsen, Morten

    2010-01-01

    of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4(+) or CD8(+) T cells prior to the ELISPOT culture revealed...... that effectors are either CD4(+) (the majority of reactivities) or CD8(+) T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4(+) T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. Conclusions....../Significance: HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4(+) T cell responses restricted by HLA-II molecules....

  12. CD4+CD25+ T regulatory cells from FIV+ cats induce a unique anergic profile in CD8+ lymphocyte targets

    Directory of Open Access Journals (Sweden)

    Tompkins Mary B

    2010-11-01

    Full Text Available Abstract Background Using the FIV model, we reported previously that CD4+CD25+ T regulatory (Treg cells from FIV+ cats are constitutively activated and suppress CD4+CD25- and CD8+ T cell immune responses. In an effort to further explore Treg-mediated suppression, we asked whether Treg cells induce anergy through the alteration of production of cyclins, cyclin-dependent kinases and their inhibitors. Results Lymphocytes were obtained from control or FIV+ cats and sorted by FACS into CD4+CD25+ and CD8+ populations. Following co-culture with CD4+CD25+ cells, CD8+ targets were examined by Western blot for changes in cyclins D3, E and A, retinoblastoma (Rb protein, as well as the cyclin dependent kinase inhibitor p21cip1. Following co-culture with CD4+CD25+cells, we observed up-regulation of p21cip1 and cyclin E, with down-regulation of cyclin D3, in CD8+ cells from FIV+ cats. As expected, CD8+ targets from control cats were quiescent with little up-regulation of p21cip1 and cyclin E. There was also a lack of Rb phosphorylation in CD8+ targets consistent with late G1 cell cycle arrest. Further, IL-2 mRNA was down regulated in CD8+ cells after co-culture with CD4+CD25+ Treg cells. Following CD4+CD25+ co-culture, CD8+ targets from FIV+ cats also had increased Foxp3 mRNA expression; however, these CD8+Foxp3+ cells did not exhibit suppressor function. Conclusions Collectively, these data suggest that CD4+CD25+ Treg cells from FIV+ cats induce CD8+ anergy by disruption of normal G1 to S cell cycle progression.

  13. Dopamine Receptor D3 Signaling on CD4+ T Cells Favors Th1- and Th17-Mediated Immunity.

    Science.gov (United States)

    Contreras, Francisco; Prado, Carolina; González, Hugo; Franz, Dafne; Osorio-Barrios, Francisco; Osorio, Fabiola; Ugalde, Valentina; Lopez, Ernesto; Elgueta, Daniela; Figueroa, Alicia; Lladser, Alvaro; Pacheco, Rodrigo

    2016-05-15

    Dopamine receptor D3 (DRD3) expressed on CD4(+) T cells is required to promote neuroinflammation in a murine model of Parkinson's disease. However, how DRD3 signaling affects T cell-mediated immunity remains unknown. In this study, we report that TCR stimulation on mouse CD4(+) T cells induces DRD3 expression, regardless of the lineage specification. Importantly, functional analyses performed in vivo using adoptive transfer of OVA-specific OT-II cells into wild-type recipients show that DRD3 deficiency in CD4(+) T cells results in attenuated differentiation of naive CD4(+) T cells toward the Th1 phenotype, exacerbated generation of Th2 cells, and unaltered Th17 differentiation. The reciprocal regulatory effect of DRD3 signaling in CD4(+) T cells favoring Th1 generation and impairing the acquisition of Th2 phenotype was also reproduced using in vitro approaches. Mechanistic analysis indicates that DRD3 signaling evokes suppressor of cytokine signaling 5 expression, a negative regulator of Th2 development, which indirectly favors acquisition of Th1 phenotype. Accordingly, DRD3 deficiency results in exacerbated eosinophil infiltration into the airways of mice undergoing house dust mite-induced allergic response. Interestingly, our results show that, upon chronic inflammatory colitis induced by transfer of naive CD4(+) T cells into lymphopenic recipients, DRD3 deficiency not only affects Th1 response, but also the frequency of Th17 cells, suggesting that DRD3 signaling also contributes to Th17 expansion under chronic inflammatory conditions. In conclusion, our findings indicate that DRD3-mediated signaling in CD4(+) T cells plays a crucial role in the balance of effector lineages, favoring the inflammatory potential of CD4(+) T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

  14. Naturally occurring, tumor-specific, therapeutic proteins.

    Science.gov (United States)

    Argiris, Konstantinos; Panethymitaki, Chrysoula; Tavassoli, Mahvash

    2011-05-01

    The emerging approach to cancer treatment known as targeted therapies offers hope in improving the treatment of therapy-resistant cancers. Recent understanding of the molecular pathogenesis of cancer has led to the development of targeted novel drugs such as monoclonal antibodies, small molecule inhibitors, mimetics, antisense and small interference RNA-based strategies, among others. These compounds act on specific targets that are believed to contribute to the development and progression of cancers and resistance of tumors to conventional therapies. Delivered individually or combined with chemo- and/or radiotherapy, such novel drugs have produced significant responses in certain types of cancer. Among the most successful novel compounds are those which target tyrosine kinases (imatinib, trastuzumab, sinutinib, cetuximab). However, these compounds can cause severe side-effects as they inhibit pathways such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor, which are also important for normal functions in non-transformed cells. Recently, a number of proteins have been identified which show a remarkable tumor-specific cytotoxic activity. This toxicity is independent of tumor type or specific genetic changes such as p53, pRB or EGFR aberrations. These tumor-specific killer proteins are either derived from common human and animal viruses such as E1A, E4ORF4 and VP3 (apoptin) or of cellular origin, such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and MDA-7 (melanoma differentiation associated-7). This review aims to present a current overview of a selection of these proteins with preferential toxicity among cancer cells and will provide an insight into the possible mechanism of action, tumor specificity and their potential as novel tumor-specific cancer therapeutics.

  15. Specific detection of proteins using Nanomechanical resonators

    DEFF Research Database (Denmark)

    Fischer, Lee MacKenzie; Wright, V.A.; Guthy, C.

    2008-01-01

    of probes onto their surfaces in order to enable the specificity of the detection. Such nanoresonator-based specific detection of proteins is here reported using streptavidin as target system, and immobilized biotin as probe. Nanomechanical resonators resistant to stiction were first realized from silicon...... carbonitride using a novel fabrication method. Vapor-phase deposition of mercaptopropyl trimethoxysilane was performed, and an added mass of 2.22 +/- 0.07 fg/mu m(2) was measured. This linker molecule was used to attach biotin onto the devices, enabling the specific detection of streptavidin. A mass of 3.6 fg....../mu m(2) was attributed to the added streptavidin, corresponding to one molecule per 27 nm(2). The specificity of this recognition was confirmed by exposing the devices to a solution of streptavidin that was already saturated with biotin. An additional negative control was also performed by also...

  16. Site-Specific Infrared Probes of Proteins

    Science.gov (United States)

    Ma, Jianqiang; Pazos, Ileana M.; Zhang, Wenkai; Culik, Robert M.; Gai, Feng

    2015-01-01

    Infrared spectroscopy has played an instrumental role in studying a wide variety of biological questions. However, in many cases it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and/or environmental information in a site-specific manner. To overcome this limitation, many recent efforts have been dedicated to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural and/or environmental properties. In this Review, we highlight some recent advancements of this rapidly growing research area. PMID:25580624

  17. Immunophenotypic enumeration of CD4 + T-lymphocyte values in ...

    African Journals Online (AJOL)

    Background: The enumeration of CD4+ T-lymphocytes in Human Immunodeficiency Virus (HIV)-infected individuals is an essential tool for staging HIV disease, to make decisions for initiation of anti-retroviral therapy (ART), for monitoring response to ART and to initiate chemoprophylaxis against opportunistic infections.

  18. Autoimmune gastritis mediated by CD4+ T cells promotes the development of gastric cancer.

    Science.gov (United States)

    Nguyen, Thanh-Long M; Khurana, Shradha S; Bellone, Clifford J; Capoccia, Benjamin J; Sagartz, John E; Kesman, Russell A; Mills, Jason C; DiPaolo, Richard J

    2013-04-01

    Chronic inflammation is a major risk factor for cancer, including gastric cancers and other gastrointestinal cancers. For example, chronic inflammation caused by autoimmune gastritis (AIG) is associated with an increased risk of gastric polyps, gastric carcinoid tumors, and possibly adenocarcinomas. In this study, we characterized the progression of gastric cancer in a novel mouse model of AIG. In this model, disease was caused by CD4(+) T cells expressing a transgenic T-cell receptor specific for a peptide from the H(+)/K(+) ATPase proton pump, a protein expressed by parietal cells in the stomach. AIG caused epithelial cell aberrations that mimicked most of those seen in progression of human gastric cancers, including chronic gastritis followed by oxyntic atrophy, mucous neck cell hyperplasia, spasmolytic polypeptide-expressing metaplasia, dysplasia, and ultimately gastric intraepithelial neoplasias. Our work provides the first direct evidence that AIG supports the development of gastric neoplasia and provides a useful model to study how inflammation drives gastric cancer. ©2013 AACR.

  19. Calpain 4 is not necessary for LFA-1-mediated function in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Sarah A Wernimont

    2010-05-01

    Full Text Available T cell activation and immune synapse formation require the appropriate activation and clustering of the integrin, LFA-1. Previous work has reported that the calpain family of calcium-dependent proteases are important regulators of integrin activation and modulate T cell adhesion and migration. However, these studies have been limited by the use of calpain inhibitors, which have known off-target effects.Here, we used a LoxP/CRE system to specifically deplete calpain 4, a small regulatory calpain subunit required for expression and activity of ubiquitously expressed calpains 1 and 2, in CD4+ T cells. CD4+ and CD8+ T cells developed normally in Capn4(F/F:CD4-CRE mice and had severely diminished expression of Calpain 1 and 2, diminished talin proteolysis and impaired casein degradation. Calpain 4-deficient T cells showed no difference in adhesion or migration on the LFA-1 ligand ICAM-1 compared to control T cells. Moreover, there was no impairment in conjugation between Capn4(F/F:CD4-CRE T cells and antigen presenting cells, and the conjugates were still capable of polarizing LFA-1, PKC-theta and actin to the immune synapse. Furthermore, T cells from Capn4(F/F:CD4-CRE mice showed normal proliferation in response to either anti-CD3/CD28 coated beads or cognate antigen-loaded splenocytes. Finally, there were no differences in the rates of apoptosis following extrinsic and intrinsic apoptotic stimuli.Our findings demonstrate that calpain 4 is not necessary for LFA-1-mediated adhesion, conjugation or migration. These results challenge previous reports that implicate a central role for calpains in the regulation of T cell LFA-1 function.

  20. Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells.

    Science.gov (United States)

    Wang, Xiaomei; Su, Jin; Sherman, Alexandra; Rogers, Geoffrey L; Liao, Gongxian; Hoffman, Brad E; Leong, Kam W; Terhorst, Cox; Daniell, Henry; Herzog, Roland W

    2015-04-09

    Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-β (TGF-β) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses. © 2015 by The American Society of Hematology.

  1. CD4+ T cell autoimmunity to hypocretin/orexin and cross-reactivity to a 2009 H1N1 influenza A epitope in narcolepsy

    DEFF Research Database (Denmark)

    De la Herrán-Arita, Alberto K; Kornum, Birgitte Rahbek; Mahlios, Josh

    2013-01-01

    the wake-promoting neuropeptide hypocretin (HCRT) (orexin). We identified two DQ0602-binding HCRT epitopes, HCRT56-68 and HCRT87-99, that activated a subpopulation of CD4(+) T cells in narcolepsy patients but not in DQ0602-positive healthy control subjects. Because of the established association...... to the 2009 H1N1 strain, pHA1275-287, with homology to HCRT56-68 and HCRT87-99. In vitro stimulation of narcolepsy CD4(+) T cells with pH1N1 proteins or pHA1275-287 increased the frequency of HCRT56-68- and HCRT87-99-reactive T cells. Our data indicate the presence of CD4(+) T cells that are reactive to HCRT...... of narcolepsy with the 2009 H1N1 influenza A strain (pH1N1), we administered a seasonal influenza vaccine (containing pH1N1) to patients with narcolepsy and found an increased frequency of circulating HCRT56-68- and HCRT87-99-reactive T cells. We also identified a hemagglutinin (HA) pHA1 epitope specific...

  2. A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

    Directory of Open Access Journals (Sweden)

    Aniuska Becerra-Artiles

    Full Text Available Most of humanity is chronically infected with human herpesvirus 6 (HHV-6, with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48 and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune

  3. The value of point-of-care CD4+ and laboratory viral load in tailoring antiretroviral therapy monitoring strategies to resource limitations.

    Science.gov (United States)

    Hyle, Emily P; Jani, Ilesh V; Rosettie, Katherine L; Wood, Robin; Osher, Benjamin; Resch, Stephen; Pei, Pamela P; Maggiore, Paolo; Freedberg, Kenneth A; Peter, Trevor; Parker, Robert A; Walensky, Rochelle P

    2017-09-24

    To examine the clinical and economic value of point-of-care CD4 (POC-CD4) or viral load monitoring compared with current practices in Mozambique, a country representative of the diverse resource limitations encountered by HIV treatment programs in sub-Saharan Africa. We use the Cost-Effectiveness of Preventing AIDS Complications-International model to examine the clinical impact, cost (2014 US$), and incremental cost-effectiveness ratio [$/year of life saved (YLS)] of ART monitoring strategies in Mozambique. We compare: monitoring for clinical disease progression [clinical ART monitoring strategy (CLIN)] vs. annual POC-CD4 in rural settings without laboratory services and biannual laboratory CD4 (LAB-CD4), biannual POC-CD4, and annual viral load in urban settings with laboratory services. We examine the impact of a range of values in sensitivity analyses, using Mozambique's 2014 per capita gross domestic product ($620) as a benchmark cost-effectiveness threshold. In rural settings, annual POC-CD4 compared to CLIN improves life expectancy by 2.8 years, reduces time on failed ART by 0.6 years, and yields an incremental cost-effectiveness ratio of $480/YLS. In urban settings, biannual POC-CD4 is more expensive and less effective than viral load. Compared to biannual LAB-CD4, viral load improves life expectancy by 0.6 years, reduces time on failed ART by 1.0 year, and is cost-effective ($440/YLS). In rural settings, annual POC-CD4 improves clinical outcomes and is cost-effective compared to CLIN. In urban settings, viral load has the greatest clinical benefit and is cost-effective compared to biannual POC-CD4 or LAB-CD4. Tailoring ART monitoring strategies to specific settings with different available resources can improve clinical outcomes while remaining economically efficient.

  4. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    International Nuclear Information System (INIS)

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-01-01

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  5. [Effect of total glucosides of peony on expression and DNA methylation status of ITGAL gene in CD4(+) T cells of systemic lupus erythematosus].

    Science.gov (United States)

    Zhao, Ming; Liang, Gongping; Luo, Shuangyan; Lu, Qianjin

    2012-05-01

    To investigate the effect of total glucosides of peony (TGP) on expression and DNA methylation status of ITGAL gene (CD11a) in CD4(+) T cells from patients with systemic lupus erythematosus (SLE). CD4(+) T cells were isolated by positive selection using CD4 beads. CD4(+) T cells were treated by TGP at 0, 62.5, 312.5 and 1562.5 mg/L for 48 h. The MTT method was used to assess cell viability; mRNA expression level was measured by realtime-PCR; protein level of CD11a was measured by flow cytometric analysis; DNA methylation status was assayed by bisulfite sequencing. No significant change in cell viability was found in CD4(+) T cells among the different concentration groups (P>0.05). Compared with control, the mRNA and protein levels of ITGAL were down-regulated significantly in SLE CD4(+) T cells treated with TGP (1562.5 mg/L) (PTGP (1562.5 mg/L) treated CD4(+) T cells compared with control group (PTGP can repress CD11a gene expression through enhancing DNA methylation of ITGAL promoter in CD4(+) T cells from patients with SLE. This observation represents a preliminary step in understanding the mechanism of TGP in SLE therapy.

  6. Increased numbers of CD4+ and CD8+ T cells in lesional skin of cats with allergic dermatitis.

    Science.gov (United States)

    Roosje, P J; van Kooten, P J; Thepen, T; Bihari, I C; Rutten, V P; Koeman, J P; Willemse, T

    1998-07-01

    The aim of this study was to characterize T cells in the skin of cats with an allergic dermatitis histologically compatible with atopic dermatitis, since T cells play an important role in the pathogenesis of atopic dermatitis in humans. We observed a significantly greater number of T cells in lesional skin of domestic short-haired cats with allergic dermatitis (n = 10; median age 5.8 years) than in the skin of healthy control animals (n = 10; median age 5.0 years). In the skin of the healthy control animals, one or two CD4+ cells and no CD8+ cells were found. A predominant increase of CD4+ T cells and a CD4+/CD8+ ratio (mean +/- SD: 3.9 +/- 2.0) was found in the lesional skin of 10 cats with allergic dermatitis. The CD4+/CD8+ cell ratio in the skin of healthy control animals could not be determined because of the absence of CD8+ cells. The CD4+/CD8+ cell ratio in the peripheral blood of 10 cats with allergic dermatitis (mean +/- SD: 1.9 +/- 0.4) did not differ significantly from that in 10 healthy control animals (2.2 +/- 0.4). The CD4+/CD8+ cell ratio and predominance of CD4+ T cells in the lesional skin of cats with allergic dermatitis is comparable to that found in atopic dermatitis in humans. In addition, the observed increase of CD4+ T cells in the nonlesional skin of cats with allergic dermatitis compared to the skin of healthy cats is similar to what is seen in humans. Cytokines produced by T cells and antigen-specific T cells are important mediators in the inflammatory cascade resulting in atopic dermatitis in humans. This study is a first step to investigate their role in feline allergic dermatitis.

  7. Selective Loss of Early Differentiated, Highly Functional PD1high CD4 T Cells with HIV Progression.

    Directory of Open Access Journals (Sweden)

    Robert M Paris

    Full Text Available The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127high CD4 T cells within the early/intermediate differentiated (EI (CD27highCD45RAlow T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load. A significant loss of circulating PD-1highCTLA-4low CD4 T cells was found specifically in the CD127highCD27highCD45RAlow compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1high compared to PD-1low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1low EI CD4 T cells. In line with our previous findings, PD-1high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.

  8. Ubiquitination of specific mitochondrial matrix proteins

    International Nuclear Information System (INIS)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

    2016-01-01

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  9. Ubiquitination of specific mitochondrial matrix proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  10. Functional and phenotypical analysis of IL-6-secreting CD4+ T cells in human adipose tissue.

    Science.gov (United States)

    de Jong, Anja J; Pollastro, Sabrina; Kwekkeboom, Joanneke C; Andersen, Stefan N; Dorjée, Annemarie L; Bakker, Aleida M; Alzaid, Fawaz; Soprani, Antoine; Nelissen, Rob G H H; Mullers, Jan B; Venteclef, Nicolas; de Vries, Niek; Kloppenburg, Margreet; Toes, René E M; Ioan-Facsinay, Andreea

    2018-03-01

    Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL-6-secretion by CD4 + T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL-6 + CD4 + T cells from SVF display a more activated phenotype than the IL-6 - T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL-6-secreting CD4 + T cells cannot be assigned to a conventional Th subset. TCRβ gene analysis revealed that IL-6 + and IL-6 - CD4 + T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL-6 production by CD4 + T cells. Thus, IL-6 + CD4 + T cells are TCRαβ T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

    Science.gov (United States)

    Ramberger, Melanie; Högl, Birgit; Stefani, Ambra; Mitterling, Thomas; Reindl, Markus; Lutterotti, Andreas

    2017-03-01

    Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease. We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools. Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope. A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients. © Sleep Research Society 2016. Published by Oxford University Press [on behalf of the Sleep Research Society].

  12. Histone modification profiles are predictive for tissue/cell-type specific expression of both protein-coding and microRNA genes

    Directory of Open Access Journals (Sweden)

    Zhang Michael Q

    2011-05-01

    Full Text Available Abstract Background Gene expression is regulated at both the DNA sequence level and through modification of chromatin. However, the effect of chromatin on tissue/cell-type specific gene regulation (TCSR is largely unknown. In this paper, we present a method to elucidate the relationship between histone modification/variation (HMV and TCSR. Results A classifier for differentiating CD4+ T cell-specific genes from housekeeping genes using HMV data was built. We found HMV in both promoter and gene body regions to be predictive of genes which are targets of TCSR. For example, the histone modification types H3K4me3 and H3K27ac were identified as the most predictive for CpG-related promoters, whereas H3K4me3 and H3K79me3 were the most predictive for nonCpG-related promoters. However, genes targeted by TCSR can be predicted using other type of HMVs as well. Such redundancy implies that multiple type of underlying regulatory elements, such as enhancers or intragenic alternative promoters, which can regulate gene expression in a tissue/cell-type specific fashion, may be marked by the HMVs. Finally, we show that the predictive power of HMV for TCSR is not limited to protein-coding genes in CD4+ T cells, as we successfully predicted TCSR targeted genes in muscle cells, as well as microRNA genes with expression specific to CD4+ T cells, by the same classifier which was trained on HMV data of protein-coding genes in CD4+ T cells. Conclusion We have begun to understand the HMV patterns that guide gene expression in both tissue/cell-type specific and ubiquitous manner.

  13. Delineating CD4 dependency of HIV-1: Adaptation to infect low level CD4 expressing target cells widens cellular tropism but severely impacts on envelope functionality.

    Directory of Open Access Journals (Sweden)

    David Beauparlant

    2017-03-01

    Full Text Available A hallmark of HIV-1 infection is the continuously declining number of the virus' predominant target cells, activated CD4+ T cells. With diminishing CD4+ T cell levels, the capacity to utilize alternate cell types and receptors, including cells that express low CD4 receptor levels such as macrophages, thus becomes crucial. To explore evolutionary paths that allow HIV-1 to acquire a wider host cell range by infecting cells with lower CD4 levels, we dissected the evolution of the envelope-CD4 interaction under in vitro culture conditions that mimicked the decline of CD4high target cells, using a prototypic subtype B, R5-tropic strain. Adaptation to CD4low targets proved to severely alter envelope functions including trimer opening as indicated by a higher affinity to CD4 and loss in shielding against neutralizing antibodies. We observed a strikingly decreased infectivity on CD4high target cells, but sustained infectivity on CD4low targets, including macrophages. Intriguingly, the adaptation to CD4low targets altered the kinetic of the entry process, leading to rapid CD4 engagement and an extended transition time between CD4 and CCR5 binding during entry. This phenotype was also observed for certain central nervous system (CNS derived macrophage-tropic viruses, highlighting that the functional perturbation we defined upon in vitro adaptation to CD4low targets occurs in vivo. Collectively, our findings suggest that CD4low adapted envelopes may exhibit severe deficiencies in entry fitness and shielding early in their evolution. Considering this, adaptation to CD4low targets may preferentially occur in a sheltered and immune-privileged environment such as the CNS to allow fitness restoring compensatory mutations to occur.

  14. Regulation of allergic airway inflammation by adoptive transfer of CD4+ T cells preferentially producing IL-10.

    Science.gov (United States)

    Matsuda, Masaya; Doi, Kana; Tsutsumi, Tatsuya; Fujii, Shinya; Kishima, Maki; Nishimura, Kazuma; Kuroda, Ikue; Tanahashi, Yu; Yuasa, Rino; Kinjo, Toshihiko; Kuramoto, Nobuyuki; Mizutani, Nobuaki; Nabe, Takeshi

    2017-10-05

    Anti-inflammatory pharmacotherapy for asthma has mainly depended on the inhalation of glucocorticoids, which non-specifically suppress immune responses. If the anti-inflammatory cytokine interleukin (IL)-10 can be induced by a specific antigen, asthmatic airway inflammation could be suppressed when individuals are exposed to the antigen. The purpose of this study was to develop cellular immunotherapeutics for atopic diseases using IL-10-producing CD4 + T cells. Spleen cells isolated from ovalbumin (OVA)-sensitized mice were cultured with the antigen, OVA and growth factors, IL-21, IL-27 and TGF-β for 7 days. After the 7-day culture, the CD4 + T cells were purified using a murine CD4 magnetic beads system. When the induced CD4 + T cells were stimulated by OVA in the presence of antigen-presenting cells, IL-10 was preferentially produced in vitro. When CD4 + T cells were adoptively transferred to OVA-sensitized mice followed by intratracheal OVA challenges, IL-10 was preferentially produced in the serum and bronchoalveolar lavage fluid in vivo. IL-10 production coincided with the inhibition of eosinophilic airway inflammation and epithelial mucus plugging. Most of the IL-10-producing CD4 + T cells were negative for Foxp3 and GATA-3, transcription factors of naturally occurring regulatory T cells and Th2 cells, respectively, but double positive for LAG-3 and CD49b, surface markers of inducible regulatory T cells, Tr1 cells. Collectively, most of the induced IL-10-producing CD4 + T cells could be Tr1 cells, which respond to the antigen to produce IL-10, and effectively suppressed allergic airway inflammation. The induced Tr1 cells may be useful for antigen-specific cellular immunotherapy for atopic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. cd4 t-lymphocyte subsets in women with invasive cervical cancer

    African Journals Online (AJOL)

    2013-10-01

    Oct 1, 2013 ... Only nine (20%) of the 45 HIV+ women had CD4 cell count of 0-200. HIV+ women had lower CD4 ... that may have biological influence on the dependent variable. A P value ... Married. 206. 60. Widowed. 86. 25. Divorced/separated. 24. 7. Polygamous (344) ..... Guidelines for the performance of CD4+CD4.

  16. TNF-α blockade induces IL-10 expression in human CD4+ T cells

    NARCIS (Netherlands)

    Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

    2014-01-01

    IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is

  17. Blood feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Wikel Stephen K

    2009-10-01

    Full Text Available Abstract Background Tick modulation of host defenses facilitates both blood feeding and pathogen transmission. Several tick species deviate host T cell responses toward a Th2 cytokine profile. The majority of studies of modulation of T cell cytokine expression by ticks were performed with lymphocytes from infested mice stimulated in vitro with polyclonal T cell activators. Those reports did not examine tick modulation of antigen specific responses. We report use of a transgenic T cell receptor (TCR adoptive transfer model reactive with influenza hemagglutinin peptide (110-120 to examine CD4+ T cell intracellular cytokine responses during infestation with the metastriate tick, Dermacentor andersoni, or exposure to salivary gland extracts. Results Infestation with pathogen-free D. andersoni nymphs or administration of an intradermal injection of female or male tick salivary gland extract induced significant increases of IL-4 transcripts in skin and draining lymph nodes of BALB/c mice as measured by quantitative real-time RT-PCR. Furthermore, IL-10 transcripts were significantly increased in skin while IL-2 and IFN-γ transcripts were not significantly changed by tick feeding or intradermal injection of salivary gland proteins, suggesting a superimposed Th2 response. Infestation induced TCR transgenic CD4+ T cells to divide more frequently as measured by CFSE dilution, but more notably these CD4+ T cells also gained the capacity to express IL-4. Intracellular levels of IL-4 were significantly increased. A second infestation administered 14 days after a primary exposure to ticks resulted in partially reduced CFSE dilution with no change in IL-4 expression when compared to one exposure to ticks. Intradermal inoculation of salivary gland extracts from both male and female ticks also induced IL-4 expression. Conclusion This is the first report of the influence of a metastriate tick on the cytokine profile of antigen specific CD4+ T cells. Blood feeding

  18. Genome-wide expression profiling analysis to identify key genes in the anti-HIV mechanism of CD4+ and CD8+ T cells.

    Science.gov (United States)

    Gao, Lijie; Wang, Yunqi; Li, Yi; Dong, Ya; Yang, Aimin; Zhang, Jie; Li, Fengying; Zhang, Rongqiang

    2018-07-01

    Comprehensive bioinformatics analyses were performed to explore the key biomarkers in response to HIV infection of CD4 + and CD8 + T cells. The numbers of CD4 + and CD8 + T cells of HIV infected individuals were analyzed and the GEO database (GSE6740) was screened for differentially expressed genes (DEGs) in HIV infected CD4 + and CD8 + T cells. Gene Ontology enrichment, KEGG pathway analyses, and protein-protein interaction (PPI) network were performed to identify the key pathway and core proteins in anti-HIV virus process of CD4 + and CD8 + T cells. Finally, we analyzed the expressions of key proteins in HIV-infected T cells (GSE6740 dataset) and peripheral blood mononuclear cells(PBMCs) (GSE511 dataset). 1) CD4 + T cells counts and ratio of CD4 + /CD8 + T cells decreased while CD8 + T cells counts increased in HIV positive individuals; 2) 517 DEGs were found in HIV infected CD4 + and CD8 + T cells at acute and chronic stage with the criterial of P-value T cells. The main biological processes of the DEGs were response to virus and defense response to virus. At chronic stage, ISG15 protein, in conjunction with IFN-1 pathway might play key roles in anti-HIV responses of CD4 + T cells; and 4) The expression of ISG15 increased in both T cells and PBMCs after HIV infection. Gene expression profile of CD4 + and CD8 + T cells changed significantly in HIV infection, in which ISG15 gene may play a central role in activating the natural antiviral process of immune cells. © 2018 Wiley Periodicals, Inc.

  19. Requirement for CD4 T Cell Help in Generating Functional CD8 T Cell Memory

    Science.gov (United States)

    Shedlock, Devon J.; Shen, Hao

    2003-04-01

    Although primary CD8 responses to acute infections are independent of CD4 help, it is unknown whether a similar situation applies to secondary responses. We show that depletion of CD4 cells during the recall response has minimal effect, whereas depletion during the priming phase leads to reduced responses by memory CD8 cells to reinfection. Memory CD8 cells generated in CD4+/+ mice responded normally when transferred into CD4-/- hosts, whereas memory CD8 cells generated in CD4-/- mice mounted defective recall responses in CD4+/+ adoptive hosts. These results demonstrate a previously undescribed role for CD4 help in the development of functional CD8 memory.

  20. Reduced Memory CD4+ T-Cell Generation in the Circulation of Young Children May Contribute to the Otitis-Prone Condition

    Science.gov (United States)

    Sharma, Sharad K.; Casey, Janet R.

    2011-01-01

    Background. An explanation for the immunologic dysfunction that causes children to be prone to repeated episodes of acute otitis media (AOM) has long been sought. Poor antibody response has been associated with the otitis-prone condition; however, there is no precise mechanistic explanation for this condition. Methods. Non–otitis-prone and otitis-prone children with AOM or nasopharyngeal (NP) colonization caused by either Streptococcus pneumoniae or Haemophilus influenzae were compared for pathogen-specific CD4+ T-helper memory responses by stimulating peripheral blood mononuclear cells using 6 vaccine candidate S. pneumoniae and 3 H. influenzae protein antigens. Samples were analyzed by multi-parameter flow cytometry. Results. Significantly reduced percentages of functional CD45RALow memory CD4+ T cells producing specific cytokines (interferon γ, interleukin [IL]–2, IL-4 and IL-17a) were observed in otitis-prone children following AOM and NP colonization with either S. pneumoniae or H. influenzae. Immunoglobulin (Ig) G responses to the studied protein antigens were reduced, which suggests that antigen-specific B-cell function may be compromised as a result of poor T-cell help. Staphylococcal enterotoxin B stimulated similar cytokine patterns in memory CD4+T cells in both groups of children. Conclusions. Otitis-prone children have suboptimal circulating functional T-helper memory and reduced IgG responses to S. pneumoniae or H. influenzae after colonization and after AOM; this immune dysfunction causes susceptibility to recurrent AOM infections. PMID:21791667

  1. Identification of a type 1 diabetes-associated CD4 promoter haplotype with high constitutive activity

    DEFF Research Database (Denmark)

    Kristiansen, O P; Karlsen, A E; Larsen, Z M

    2004-01-01

    screened the human CD4 promoter for mutations and identified three frequent single nucleotide polymorphisms (SNPs): CD4-181C/G, CD4-521C/G and CD4-1050T/C. The SNPs are in strong linkage disequilibrium (LD) and association with the CD4-1188(TTTTC)(5-14) alleles, and we observed nine CD4 promoter haplotypes...... promoter activity and (2) the CD4-181G variant encodes higher stimulated promoter activity than the CD4-181C variant. This difference is in part neutralized in the frequently occurring CD4 promoter haplotypes by the more upstream genetic variants. Thus, we report functional impact of a novel CD4-181C/G SNP...

  2. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

    Directory of Open Access Journals (Sweden)

    Mattias N E Forsell

    2008-10-01

    Full Text Available The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3. Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4 rabbits with envelope glycoprotein (Env trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

  3. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

    Science.gov (United States)

    Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

    2008-10-03

    The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

  4. Oct1 and OCA-B are selectively required for CD4 memory T cell function.

    Science.gov (United States)

    Shakya, Arvind; Goren, Alon; Shalek, Alex; German, Cody N; Snook, Jeremy; Kuchroo, Vijay K; Yosef, Nir; Chan, Raymond C; Regev, Aviv; Williams, Matthew A; Tantin, Dean

    2015-11-16

    Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4(+) memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4(+) T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4(+) T cell memory. © 2015 Shakya et al.

  5. Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells.

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    Xiaosheng Wu

    Full Text Available Greater than 75% of all hematologic malignancies derive from germinal center (GC or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID, GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR. Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells.

  6. Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

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    Patsharaporn Techasintana

    Full Text Available Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs and microRNAs (miRNAs. RNA immunoprecipitation (RIP methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1 and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

  7. Human CD4+ T cells lyse target cells via granzyme/perforin upon circumvention of MHC class II restriction by an antibody-like immunoreceptor.

    Science.gov (United States)

    Hombach, Andreas; Köhler, Heike; Rappl, Gunter; Abken, Hinrich

    2006-10-15

    Immune elimination of tumor cells requires the close cooperation between CD8+ CTL and CD4+ Th cells. We circumvent MHC class II-restriction of CD4+ T cells by expression of a recombinant immunoreceptor with an Ab-derived binding domain redirecting specificity. Human CD4+ T cells grafted with an immunoreceptor specific for carcinoembryonic Ag (CEA) are activated to proliferate and secrete cytokines upon binding to CEA+ target cells. Notably, redirected CD4+ T cells mediate cytolysis of CEA+ tumor cells with high efficiencies. Lysis by redirected CD4+ T cells is independent of death receptor signaling via TNF-alpha or Fas, but mediated by perforin and granzyme because cytolysis is inhibited by blocking the release of cytotoxic granules, but not by blocking of Fas ligand or TNF-alpha. CD4+ T cells redirected by Ab-derived immunoreceptors in a MHC class II-independent fashion substantially extend the power of an adoptive, Ag-triggered immunotherapy not only by CD4+ T cell help, but also by cytolytic effector functions. Because cytolysis is predominantly mediated via granzyme/perforin, target cells that are resistant to death receptor signaling become sensitive to a cytolytic attack by engineered CD4+ T cells.

  8. Pre-existing malignancy results in increased prevalence of distinct populations of CD4+ T cells during sepsis.

    Science.gov (United States)

    Xie, Jianfeng; Robertson, Jennifer M; Chen, Ching-Wen; Zhang, Wenxiao; Coopersmith, Craig M; Ford, Mandy L

    2018-01-01

    The presence of pre-existing malignancy in murine hosts results in increased immune dysregulation and risk of mortality following a septic insult. Based on the known systemic immunologic changes that occur in cancer hosts, we hypothesized that the presence of pre-existing malignancy would result in phenotypic and functional changes in CD4+ T cell responses following sepsis. In order to conduct a non-biased, unsupervised analysis of phenotypic differences between CD4+ T cell compartments, cohorts of mice were injected with LLC1 tumor cells and tumors were allowed to grow for 3 weeks. These cancer hosts and age-matched non-cancer controls were then subjected to CLP. Splenocytes were harvested at 24h post CLP and flow cytometry and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) were used to analyze populations of CD4+ cells most different between the two groups. Results indicated that relative to non-cancer controls, cancer mice contained more resting memory CD4+ T cells, more activated CD4+ effectors, and fewer naïve CD4+ T cells during sepsis, suggesting that the CD4+ T cell compartment in cancer septic hosts is one of increased activation and differentiation. Moreover, cancer septic animals exhibited expansion of two distinct subsets of CD4+ T cells relative to previously healthy septic controls. Specifically, we identified increases in both a PD-1hi population and a distinct 2B4hi BTLAhi LAG-3hi population in cancer septic animals. By combining phenotypic analysis of exhaustion markers with functional analysis of cytokine production, we found that PD-1+ CD4+ cells in cancer hosts failed to make any cytokines following CLP, while the 2B4+ PD-1lo cells in cancer mice secreted increased TNF during sepsis. In sum, the immunophenotypic landscape of cancer septic animals is characterized by both increased CD4+ T cell activation and exhaustion, findings that may underlie the observed increased mortality in mice with pre-existing malignancy

  9. Pre-existing malignancy results in increased prevalence of distinct populations of CD4+ T cells during sepsis.

    Directory of Open Access Journals (Sweden)

    Jianfeng Xie

    Full Text Available The presence of pre-existing malignancy in murine hosts results in increased immune dysregulation and risk of mortality following a septic insult. Based on the known systemic immunologic changes that occur in cancer hosts, we hypothesized that the presence of pre-existing malignancy would result in phenotypic and functional changes in CD4+ T cell responses following sepsis. In order to conduct a non-biased, unsupervised analysis of phenotypic differences between CD4+ T cell compartments, cohorts of mice were injected with LLC1 tumor cells and tumors were allowed to grow for 3 weeks. These cancer hosts and age-matched non-cancer controls were then subjected to CLP. Splenocytes were harvested at 24h post CLP and flow cytometry and SPADE (Spanning-tree Progression Analysis of Density-normalized Events were used to analyze populations of CD4+ cells most different between the two groups. Results indicated that relative to non-cancer controls, cancer mice contained more resting memory CD4+ T cells, more activated CD4+ effectors, and fewer naïve CD4+ T cells during sepsis, suggesting that the CD4+ T cell compartment in cancer septic hosts is one of increased activation and differentiation. Moreover, cancer septic animals exhibited expansion of two distinct subsets of CD4+ T cells relative to previously healthy septic controls. Specifically, we identified increases in both a PD-1hi population and a distinct 2B4hi BTLAhi LAG-3hi population in cancer septic animals. By combining phenotypic analysis of exhaustion markers with functional analysis of cytokine production, we found that PD-1+ CD4+ cells in cancer hosts failed to make any cytokines following CLP, while the 2B4+ PD-1lo cells in cancer mice secreted increased TNF during sepsis. In sum, the immunophenotypic landscape of cancer septic animals is characterized by both increased CD4+ T cell activation and exhaustion, findings that may underlie the observed increased mortality in mice with pre

  10. CD4+ and CD8+ T cell activation are associated with HIV DNA in resting CD4+ T cells.

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    Leslie R Cockerham

    Full Text Available The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1 in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.

  11. Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4+ T-Cell Responses-Brief Report

    NARCIS (Netherlands)

    Meiler, Svenja; Smeets, Esther; Winkels, Holger; Shami, Annelie; Pascutti, Maria Fernanda; Nolte, Martijn A.; Beckers, Linda; Weber, Christian; Gerdes, Norbert; Lutgens, Esther

    2016-01-01

    Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4(+) effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood. Low-density lipoprotein

  12. Circumvention of regulatory CD4(+) T cell activity during cross-priming strongly enhances T cell-mediated immunity.

    Science.gov (United States)

    Heit, Antje; Gebhardt, Friedemann; Lahl, Katharina; Neuenhahn, Michael; Schmitz, Frank; Anderl, Florian; Wagner, Hermann; Sparwasser, Tim; Busch, Dirk H; Kastenmüller, Kathrin

    2008-06-01

    Immunization with purified antigens is a safe and practical vaccination strategy but is generally unable to induce sustained CD8(+) T cell-mediated protection against intracellular pathogens. Most efforts to improve the CD8(+) T cell immunogenicity of these vaccines have focused on co-administration of adjuvant to support cross-presentation and dendritic cell maturation. In addition, it has been shown that CD4(+) T cell help during the priming phase contributes to the generation of protective CD8(+) memory T cells. In this report we demonstrate that the depletion of CD4(+) T cells paradoxically enhances long-lasting CD8-mediated protective immunity upon protein vaccination. Functional and genetic in vivo inactivation experiments attribute this enhancement primarily to MHC class II-restricted CD4(+) regulatory T cells (Treg), which appear to physiologically suppress the differentiation process towards long-living effector memory T cells. Since, in functional terms, this suppression by Treg largely exceeds the positive effects of conventional CD4(+) T cell help, even the absence of all CD4(+) T cells or lack of MHC class II-mediated interactions on priming dendritic cells result in enhanced CD8(+) T cell immunogenicity. These findings have important implications for the improvement of vaccines against intracellular pathogens or tumors, especially in patients with highly active Treg.

  13. CD4+RORγt++ and Tregs in a Mouse Model of Diet-Induced Nonalcoholic Steatohepatitis

    Directory of Open Access Journals (Sweden)

    Luisa Vonghia

    2015-01-01

    Full Text Available Background and Aims. Inflammatory mediators that cross-talk in different metabolically active organs are thought to play a crucial role in the pathogenesis of Nonalcoholic Steatohepatitis (NASH. This study was aimed at investigating the CD4+RORγt+ T-helper cells and their counterpart, the CD4+CD25+FOXP3+ regulatory T cells in the liver, subcutaneous adipose tissue (SAT, and abdominal adipose tissue (AAT in a high fat diet (HFD mouse model. Methods. C57BL6 mice were fed a HFD or a normal diet (ND. Liver enzymes, metabolic parameters, and liver histology were assessed. The expression of CD4+RORγt+ cells and regulatory T cells in different organs (blood, liver, AAT, and SAT were analyzed by flow cytometry. Cytokine and adipokine tissue expression were studied by RT-PCR. Results. Mice fed a HFD developed NASH and metabolic alterations compared to normal diet. CD4+RORγt++ cells were significantly increased in the liver and the AAT while an increase of regulatory T cells was observed in the SAT of mice fed HFD compared to ND. Inflammatory cytokines were also upregulated. Conclusions. CD4+RORγt++ cells and regulatory T cells are altered in NASH with a site-specific pattern and correlate with the severity of the disease. These site-specific differences are associated with increased cytokine expression.

  14. Growth arrest specific protein (GAS) 6

    DEFF Research Database (Denmark)

    Haase, T N; Rasmussen, Morten; Jaksch, C A M

    2013-01-01

    using RNA microarray and quantitative PCR. The role of a differentially expressed gene, growth arrest specific protein 6 (GAS6), was evaluated in vitro using neonatal rat islets. Results The mRNA level of Gas6, known to be mitogenic in other tissues, was reduced in LP offspring. The mRNA content of Mafa...... was increased in LP offspring suggesting an early maturation of beta cells. When applied in vitro, GAS6 increased proliferation of neonatal pancreatic beta cells, while reducing glucose-stimulated insulin secretion without changing the total insulin content of the islets. In addition, GAS6 decreased the m......RNA content of Mafa. Conclusions/interpretation We propose a role for GAS6 in the regulation of pancreatic beta cells in the critical period around the time of birth. Our results support the hypothesis that the reduced beta cell mass seen in LP offspring is caused by a change in the intra-uterine environment...

  15. Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7 Integrin CD4+ T Cells: Implications for HIV Microbicides.

    Science.gov (United States)

    Mukhopadhya, Indrani; Murray, Graeme I; Duncan, Linda; Yuecel, Raif; Shattock, Robin; Kelly, Charles; Iannelli, Francesco; Pozzi, Gianni; El-Omar, Emad M; Hold, Georgina L; Hijazi, Karolin

    2016-09-06

    CD4+ T lymphocytes in the colorectal mucosa are key in HIV-1 transmission and dissemination. As such they are also the primary target for antiretroviral (ARV)-based rectal microbicides for pre-exposure prophylaxis. Drug transporters expressed in mucosal CD4+ T cells determine ARV distribution across the cell membrane and, most likely, efficacy of microbicides. We describe transporters for antiretroviral drugs in colorectal mucosal CD4+ T lymphocytes and compare gene expression with circulating α4β7+CD4+ T cells, which traffic to the intestine and have been shown to be preferentially infected by HIV-1. Purified total CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic separation. CD4+ T cells expressing α4β7 integrin were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells of healthy volunteers. Expressions of 15 efflux and uptake drug transporter genes were quantified using Taqman qPCR assays. Expression of efflux transporters MRP3, MRP5, and BCRP and uptake transporter CNT2 were significantly higher in colorectal CD4+ T cells compared to circulating CD4+ T cells (p = 0.01-0.03). Conversely, circulating α4β7+CD4+ T cells demonstrated significantly higher expression of OATPD compared to colorectal CD4+ T cells (p = 0.001). To the best of our knowledge this is the first report of drug transporter gene expression in colorectal CD4+ and peripheral α4β7+CD4+ T cells. The qualitative and quantitative differences in drug transporter gene expression profiles between α4β7+CD4+ T cells and total mucosal CD4+ T cells may have significant implications for the efficacy of rectally delivered ARV-microbicides. Most notably, we have identified efflux drug transporters that could be targeted by selective inhibitors or beneficial drug-drug interactions to enhance intracellular accumulation of antiretroviral drugs.

  16. Neuropilin-1highCD4+CD25+ Regulatory T Cells Exhibit Primary Negative Immunoregulation in Sepsis

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    Yu-Lei Gao

    2016-01-01

    Full Text Available Regulatory T cells (Tregs appear to be involved in sepsis-induced immune dysfunction; neuropilin-1 (Nrp-1 was identified as a surface marker for CD4+CD25+Tregs. In the current study, we investigated the negative immunoregulation of Nrp-1highCD4+CD25+Tregs and the potential therapeutic value of Nrp-1 in sepsis. Splenic CD4+CD25+Tregs from cecal ligation and puncture (CLP mouse models were further segregated into Nrp-1highTregs and Nrp-1lowTregs; they were cocultured with CD4+CD25−  T cells. The expression of forkhead/winged helix transcription factor-3 (Foxp-3, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4, membrane associated transforming growth factor-β (TGF-βm+, apoptotic rate, and secretive ability [including TGF-β and interleukin-10 (IL-10] for various types of Tregs, as well as the immunosuppressive ability of Tregs on CD4+CD25−  T cells, were determined. Meanwhile, the impact of recombinant Nrp-1 polyclonal antibody on the demethylation of Foxp-3-TSDR (Treg-specific demethylated region was measured in in vitro study. Sepsis per se markedly promoted the expression of Nrp-1 of CD4+CD25+Tregs. Foxp-3/CTLA-4/TGF-βm+ of Nrp-1highTregs were upregulated by septic challenge. Nrp-1highTregs showed strong resilience to apoptosis and secretive ability and the strongest immunosuppressive ability on CD4+CD25−  T cells. In the presence of lipopolysaccharide (LPS, the recombinant Nrp-1 polyclonal antibody reduced the demethylation of Foxp-3-TSDR. Nrp-1highTregs might reveal primary negative immunoregulation in sepsis; Nrp-1 could represent a new potential therapeutic target for the study of immune regulation in sepsis.

  17. Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease

    Science.gov (United States)

    McLane, Laura M.; Steblyanko, Maria; Anikeeva, Nadia; Ablanedo-Terrazas, Yuria; Demers, Korey; Eller, Michael A.; Streeck, Hendrik; Jansson, Marianne; Sönnerborg, Anders; Canaday, David H.; Naji, Ali; Wherry, E. John; Robb, Merlin L.; Reyes-Teran, Gustavo; Sykulev, Yuri; Betts, Michael R.

    2018-01-01

    CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues. PMID:29652923

  18. Conglutinin binds the HIV-1 envelope glycoprotein gp 160 and inhibits its interaction with cell membrane CD4

    DEFF Research Database (Denmark)

    Andersen, Ove; Sørensen, A M; Svehag, S E

    1991-01-01

    The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose...... residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160...... of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein....

  19. Assessment of metabolic and mitochondrial dynamics in CD4+ and CD8+ T cells in virologically suppressed HIV-positive individuals on combination antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Jesse J R Masson

    Full Text Available Metabolism plays a fundamental role in supporting the growth, proliferation and effector functions of T cells. We investigated the impact of HIV infection on key processes that regulate glucose uptake and mitochondrial biogenesis in subpopulations of CD4+ and CD8+ T cells from 18 virologically-suppressed HIV-positive individuals on combination antiretroviral therapy (cART; median CD4+ cell count: 728 cells/μl and 13 HIV seronegative controls. Mitochondrial membrane potential (MMP and reactive oxygen species (ROS production were also analysed in total CD4+ and CD8+ T cells. Among HIV+/cART individuals, expression of glucose transporter (Glut1 and mitochondrial density were highest within central memory and naïve CD4+ T cells, and lowest among effector memory and transitional memory T cells, with similar trends in HIV-negative controls. Compared to HIV-negative controls, there was a trend towards higher percentage of circulating CD4+Glut1+ T cells in HIV+/cART participants. There were no significant differences in mitochondrial dynamics between subject groups. Glut1 expression was positively correlated with mitochondrial density and MMP in total CD4+ T cells, while MMP was also positively correlated with ROS production in both CD4+ and CD8+ T cells. Our study characterizes specific metabolic features of CD4+ and CD8+ T cells in HIV-negative and HIV+/cART individuals and will invite future studies to explore the immunometabolic consequences of HIV infection.

  20. Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

    Science.gov (United States)

    Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; Klein, Kerenaftali; McCarthy, James S.; Doolan, Denise L.

    2016-01-01

    Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752 PMID:27662621

  1. Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1

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    Maia Kavanagh Williamson

    2018-03-01

    Full Text Available Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out.

  2. Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Brittany A Goods

    Full Text Available Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1 have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM, the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25-CD127+Foxp3-effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1-CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1-CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial.

  3. CD4+CD25hiFOXP3+ cells in cord blood of neonates born from filaria infected mother are negatively associated with CD4+Tbet+ and CD4+RORγt+ T cells.

    Science.gov (United States)

    Ateba-Ngoa, Ulysse; Mombo-Ngoma, Ghyslain; Zettlmeissl, Eva; van der Vlugt, Luciën E P M; de Jong, Sanne E; de Jong, Sanne; Matsiegui, Pierre-Blaise; Ramharter, Michael; Kremsner, Peter G; Yazdanbakhsh, Maria; Adegnika, Ayola Akim

    2014-01-01

    Children who have been exposed in utero to maternal filarial infection are immunologically less responsive to filarial antigens, have less pathology, and are more susceptible to acquire infection than offspring of uninfected mothers. Moreover children from filaria infected mothers have been shown to be less responsive to vaccination as a consequence of an impairment of their immune response. However, it is not well known how in utero exposure to parasite antigens affects cellular immune responses. Here, 30 pregnant women were examined for the presence of microfilaria of Loa loa and Mansonella perstans in peripheral blood. At delivery, cord blood mononuclear cells (CBMC) were obtained and the CD4+T cells were phenotyped by expression of the transcription factors Tbet, RORγt, and FOXP3. No significant difference was observed between newborns from infected versus uninfected mothers in the frequencies of total CD4+T cells and CD4+T cells subsets including CD4+Tbet+, CD4+RORγt+ T and CD4+CD25hiFOXP3+ T cells. However, there was a negative association between CD4+CD25hiFOXP3+T cells and CD4+Tbet+ as well as CD4+RORγt+ T cells in the infected group only (B = -0.242, P = 0.002; B = -0.178, P = 0.013 respectively). Our results suggest that filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep TH1 and TH17 in check.

  4. Transplantation of mouse HSCs genetically modified to express a CD4-restricted TCR results in long-term immunity that destroys tumors and initiates spontaneous autoimmunity.

    Science.gov (United States)

    Ha, Sung P; Klemen, Nicholas D; Kinnebrew, Garrett H; Brandmaier, Andrew G; Marsh, Jon; Hangoc, Giao; Palmer, Douglas C; Restifo, Nicholas P; Cornetta, Kenneth; Broxmeyer, Hal E; Touloukian, Christopher E

    2010-12-01

    The development of effective cancer immunotherapies has been consistently hampered by several factors, including an inability to instigate long-term effective functional antitumor immunity. This is particularly true for immunotherapies that focus on the adoptive transfer of activated or genetically modified mature CD8+ T cells. In this study, we sought to alter and enhance long-term host immunity by genetically modifying, then transplanting, mouse HSCs. We first cloned a previously identified tumor-reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen tyrosinase-related protein 1 (Tyrp1), then constructed both a high-expression lentivirus vector and a TCR-transgenic mouse expressing the genes encoding this TCR. Using these tools, we demonstrated that both mouse and human HSCs established durable, high-efficiency TCR gene transfer following long-term transplantation into lethally irradiated mice transgenic for HLA-DR4. Recipients of genetically modified mouse HSCs developed spontaneous autoimmune vitiligo that was associated with the presence of a Th1-polarized memory effector CD4+ T cell population that expressed the Tyrp1-specific TCR. Most importantly, large numbers of CD4+ T cells expressing the Tyrp1-specific TCR were detected in secondary HLA-DR4-transgenic transplant recipients, and these mice were able to destroy subcutaneously administered melanoma cells without the aid of vaccination, immune modulation, or cytokine administration. These results demonstrate the creation of what we believe to be a novel translational model of durable lentiviral gene transfer that results in long-term effective immunity.

  5. Transcriptional Reprogramming during Effector-to-Memory Transition Renders CD4+ T Cells Permissive for Latent HIV-1 Infection.

    Science.gov (United States)

    Shan, Liang; Deng, Kai; Gao, Hongbo; Xing, Sifei; Capoferri, Adam A; Durand, Christine M; Rabi, S Alireza; Laird, Gregory M; Kim, Michelle; Hosmane, Nina N; Yang, Hung-Chih; Zhang, Hao; Margolick, Joseph B; Li, Linghua; Cai, Weiping; Ke, Ruian; Flavell, Richard A; Siliciano, Janet D; Siliciano, Robert F

    2017-10-17

    The latent reservoir for HIV-1 in resting memory CD4 + T cells is the major barrier to curing HIV-1 infection. Studies of HIV-1 latency have focused on regulation of viral gene expression in cells in which latent infection is established. However, it remains unclear how infection initially becomes latent. Here we described a unique set of properties of CD4 + T cells undergoing effector-to-memory transition including temporary upregulation of CCR5 expression and rapid downregulation of cellular gene transcription. These cells allowed completion of steps in the HIV-1 life cycle through integration but suppressed HIV-1 gene transcription, thus allowing the establishment of latency. CD4 + T cells in this stage were substantially more permissive for HIV-1 latent infection than other CD4 + T cells. Establishment of latent HIV-1 infection in CD4 + T could be inhibited by viral-specific CD8 + T cells, a result with implications for elimination of latent HIV-1 infection by T cell-based vaccines. Copyright © 2017. Published by Elsevier Inc.

  6. Utility of the point of care CD4 analyzer, PIMA, to enumerate CD4 counts in the field settings in India

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    Thakar Madhuri

    2012-09-01

    Full Text Available Abstract Background In resource limited settings non-availability of CD4 count facility at the site could adversely affect the ART roll out programme. Point of care CD4 enumerating equipments can make the CD4 count available at the site of care and improve the patients’ management considerably. This study is aimed at determining the utility of a Point of Care PIMA CD4 analyzer (Alere, Germany in the field settings in India. Method The blood samples were collected from 1790 participants at 21 ART centers from different parts of the country and tested using PIMA and the reference methods (FACSCalibur, FACSCount and CyFlow SL3. The paired finger prick and venous blood samples from 175 participants were tested by the PIMA CD4 Analyzer and then by FACSCalibur. Result The CD4 counts obtained by PIMA CD4 analyzer showed excellent correlation with the counts obtained by the reference methods; for venous blood the Pearson’s r was 0.921, p 500 cells/mm3, the differences in the median CD4 counts obtained by the reference method and the PIMA analyzer were not significant (P > 0.05 and the relative bias were low (−7 to 5.1%. The Intermachine comparison showed variation within the acceptable limit of%CV of 10%. Conclusion In the field settings, the POC PIMA CD4 analyzer gave CD4 counts comparable to the reference methods for all CD4 ranges. The POC equipment could identify the patients eligible for ART in 91% cases. Adequate training is necessary for finger prick sample collection for optimum results. Decentralization of CD4 testing by making the CD4 counts available at primary health centers, especially in remote areas with minimum or no infrastructure would reduce the missed visits and improve adherence of the patients.

  7. Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis ‬

    KAUST Repository

    Zeitelhofer, Manuel

    2017-02-15

    Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.

  8. Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis ‬

    KAUST Repository

    Zeitelhofer, Manuel; Adzemovic, Milena Z.; Gomez-Cabrero, David; Bergman, Petra; Hochmeister, Sonja; N'diaye, Marie; Paulson, Atul; Ruhrmann, Sabrina; Almgren, Malin; Tegner, Jesper; Ekströ m, Tomas J.; Guerreiro-Cacais, André Ortlieb; Jagodic, Maja

    2017-01-01

    Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.

  9. Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Magdalena Radwanska

    2007-05-01

    Full Text Available Effector responses induced by polarized CD4+ T helper 2 (Th2 cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha. IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(creIL-4Ralpha(-/lox deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(creIL-4Ralpha(-/lox mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(creIL-4Ralpha(-/lox mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L

  10. Multifunctional Analysis of CD4+ T-Cell Response as Immune-Based Model for Tuberculosis Detection

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    Miriam Lichtner

    2015-01-01

    Full Text Available Mono- and multifunctional specific CD4+ and CD8+ T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB and latent infection (LTBI. We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2 of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI and 10 uninfected controls. Cytokines production by CD4+ T cells at single-cell levels was higher in TB-infected subjects than uninfected controls P0.45%, it was possible to differentiate TB-infected (>0.45% by uninfected subjects (0.182%. The magnitude of CD8+ T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4+ T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.

  11. No Neurocognitive Advantage for Immediate Antiretroviral Treatment in adults with greater than 500 CD4+ T Cell Counts

    DEFF Research Database (Denmark)

    Wright, Edwina J; Grund, Birgit; Robertson, Kevin R

    2018-01-01

    OBJECTIVE: To compare the effect of immediate versus deferred antiretroviral treatment (ART) on neuropsychological test performance in treatment-naive HIV-positive adults with >500 CD4+ cells/μL. DESIGN: Randomized trial. METHODS: The START parent study randomized participants to commence immediate...... versus deferred ART until CD4+ cells/μL. The START Neurology substudy used 8 neuropsychological tests, at baseline, months 4, 8, 12 and annually, to compare groups for changes in test performance. Test results were internally standardized to z-scores. The primary outcome was the average of the eight...... test z-scores (QNPZ-8). Mean changes in QNPZ-8 from baseline were compared by intent-to-treat using longitudinal mixed models. Changes from baseline to specific time points were compared using ANCOVA models. RESULTS: 592 participants had a median age of 34 years; median baseline CD4+ count of 629 cells...

  12. Increased frequency of CD8+ and CD4+ regulatory T cells in chronic lymphocytic leukemia: association with disease progression.

    Science.gov (United States)

    Jadidi-Niaragh, Farhad; Yousefi, Mehdi; Memarian, Ali; Hojjat-Farsangi, Mohammad; Khoshnoodi, Jalal; Razavi, Seyed Mohsen; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2013-02-01

    Little is known regarding the immunobiology of regulatory T (Treg) cells in hematopoietic malignancies, particularly in chronic lymphocytic leukemia (CLL). In the present study, we showed that the frequencies of CD8(+) and CD4(+) Treg cells were significantly increased in progressive as compared with indolent CLL patients and normal subjects. Enriched CD4(+) Treg cells induced a similar level of inhibition in polyclonally activated B cells and effector T cells from CLL patients and normal subjects. Our results suggest that the increase in circulating Treg cells may result in downregulation of tumor-specific immune response, leading to tumor expansion and disease progression.

  13. Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase

    DEFF Research Database (Denmark)

    Pedersen, B K; Kharazmi, A; Theander, T G

    1987-01-01

    The binding of monoclonal antibodies against CD4 was specifically inhibited by treatment of human CD4+ cells with either alkaline protease (AP) or elastase (Ela), purified from Pseudomonas aeruginosa. Binding of antibodies against CD3 (pan T), CD5 (pan T), CD8 (T suppressor/cytotoxic), HLA-ABC, HLA......-DR, HLA-DQ, HLA-DP/DR, and beta 2 microglobulin was not inhibited by AP or Ela. Heat-inactivation of the proteases at 65 degrees C for 20 min or treatment with the metal chelator EDTA abolished the inhibitory activity of both proteases. These findings may serve to develop novel immunological methods...

  14. Mass Cytometry Analysis Reveals the Landscape and Dynamics of CD32a+ CD4+ T Cells From Early HIV Infection to Effective cART.

    Science.gov (United States)

    Coindre, Sixtine; Tchitchek, Nicolas; Alaoui, Lamine; Vaslin, Bruno; Bourgeois, Christine; Goujard, Cecile; Avettand-Fenoel, Veronique; Lecuroux, Camille; Bruhns, Pierre; Le Grand, Roger; Beignon, Anne-Sophie; Lambotte, Olivier; Favier, Benoit

    2018-01-01

    CD32a has been proposed as a specific marker of latently HIV-infected CD4 + T cells. However, CD32a was recently found to be expressed on CD4 + T cells of healthy donors, leading to controversy on the relevance of this marker in HIV persistence. Here, we used mass cytometry to characterize the landscape and variation in the abundance of CD32a + CD4 + T cells during HIV infection. To this end, we analyzed CD32a + CD4 + T cells in primary HIV infection before and after effective combination antiretroviral therapy (cART) and in healthy donors. We found that CD32a + CD4 + T cells include heterogeneous subsets that are differentially affected by HIV infection. Our analysis revealed that naive ( N ), central memory ( CM ), and effector/memory ( Eff/Mem ) CD32a + CD4 + T-cell clusters that co-express LILRA2- and CD64-activating receptors were more abundant in primary HIV infection and cART stages. Conversely, LILRA2 - CD32a + CD4 + T-cell clusters of either the T N , T CM , or T Eff/Mem phenotype were more abundant in healthy individuals. Finally, an activated CD32a + CD4 + T Eff/Mem cell cluster co-expressing LILRA2, CD57, and NKG2C was more abundant in all HIV stages, particularly during primary HIV infection. Overall, our data show that multiple abundance modifications of CD32a + CD4 + T-cell subsets occur in the early phase of HIV infection, and some of which are conserved after effective cART. Our study brings a better comprehension of the relationship between CD32a expression and CD4 + T cells during HIV infection.

  15. Mass Cytometry Analysis Reveals the Landscape and Dynamics of CD32a+ CD4+ T Cells From Early HIV Infection to Effective cART

    Directory of Open Access Journals (Sweden)

    Sixtine Coindre

    2018-06-01

    Full Text Available CD32a has been proposed as a specific marker of latently HIV-infected CD4+ T cells. However, CD32a was recently found to be expressed on CD4+ T cells of healthy donors, leading to controversy on the relevance of this marker in HIV persistence. Here, we used mass cytometry to characterize the landscape and variation in the abundance of CD32a+ CD4+ T cells during HIV infection. To this end, we analyzed CD32a+ CD4+ T cells in primary HIV infection before and after effective combination antiretroviral therapy (cART and in healthy donors. We found that CD32a+ CD4+ T cells include heterogeneous subsets that are differentially affected by HIV infection. Our analysis revealed that naive (N, central memory (CM, and effector/memory (Eff/Mem CD32a+ CD4+ T-cell clusters that co-express LILRA2- and CD64-activating receptors were more abundant in primary HIV infection and cART stages. Conversely, LILRA2− CD32a+ CD4+ T-cell clusters of either the TN, TCM, or TEff/Mem phenotype were more abundant in healthy individuals. Finally, an activated CD32a+ CD4+ TEff/Mem cell cluster co-expressing LILRA2, CD57, and NKG2C was more abundant in all HIV stages, particularly during primary HIV infection. Overall, our data show that multiple abundance modifications of CD32a+ CD4+ T-cell subsets occur in the early phase of HIV infection, and some of which are conserved after effective cART. Our study brings a better comprehension of the relationship between CD32a expression and CD4+ T cells during HIV infection.

  16. Percentage and function of CD4+CD25+ regulatory T cells in patients with hyperthyroidism

    Science.gov (United States)

    Jiang, Ting-Jun; Cao, Xue-Liang; Luan, Sha; Cui, Wan-Hui; Qiu, Si-Huang; Wang, Yi-Chao; Zhao, Chang-Jiu; Fu, Peng

    2018-01-01

    The current study observed the percentage of peripheral blood (PB) CD4+CD25+ regulatory T cells (Tregs) and the influence of CD4+CD25+ Tregs on the proliferation of naïve CD4 T cells in patients with hyperthyroidism. Furthermore, preliminary discussions are presented on the action mechanism of CD4+CD25+ Tregs on hyperthyroidism attacks. The present study identified that compared with the percentage of PB CD4+CD25+ Tregs in healthy control subjects, no significant changes were observed in the percentage of PB CD4+CD25+ Tregs in patients with hyperthyroidism (P>0.05). For patients with hyperthyroidism, CD4+CD25+ Tregs exhibited significantly reduced inhibition of the proliferation of naïve CD4 T cells and decreased secretion capacity on the cytokines of CD4 T cells, compared with those of healthy control subjects (Phyperthyroidism was significantly improved (Phyperthyroidism before treatment, no significant changes were observed in the percentage of PB CD4+CD25+ Tregs in hyperthyroidism patients following treatment (P>0.05). In the patients with hyperthyroidism, following treatment, CD4+CD25+ Tregs exhibited significantly increased inhibition of the proliferation of naïve CD4 T cells and increased secretion capacity of CD4 T cell cytokines, compared with those of the patients with hyperthyroidism prior to treatment (Phyperthyroidism, and its non-proportional decrease may be closely associated with the occurrence and progression of hyperthyroidism. PMID:29207121

  17. Inhibitory effects of Trypanosoma cruzi sialoglycoproteins on CD4+ T cells are associated with increased susceptibility to infection.

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    Marise Pinheiro Nunes

    Full Text Available BACKGROUND: The Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens characterized by decreased IL-2 synthesis. Trypanosoma cruzi mucin (Tc Muc has been implicated in this phenomenom. These molecules contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the ability of Tc Muc to modulate the activation of CD4(+ T cells. Our data show that cross-linking of CD3 on naïve CD4(+ T cells in the presence of Tc Muc resulted in the inhibition of both cytokine secretion and proliferation. We further show that the sialylated O-Linked Glycan residues from tc mucin potentiate the suppression of T cell response by inducing G1-phase cell cycle arrest associated with upregulation of mitogen inhibitor p27(kip1. These inhibitory effects cannot be reversed by the addition of exogenous IL-2, rendering CD4(+ T cells anergic when activated by TCR triggering. Additionally, in vivo administration of Tc Muc during T. cruzi infection enhanced parasitemia and aggravated heart damage. Analysis of recall responses during infection showed lower frequencies of IFN-γ producing CD4(+ T cells in the spleen of Tc Muc treated mice, compared to untreated controls. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Tc Muc mediates inhibitory efects on CD4(+ T expansion and cytokine production, by blocking cell cycle progression in the G1 phase. We propose that the sialyl motif of Tc Muc is able to interact with sialic acid-binding Ig-like lectins (Siglecs on CD4(+ T cells, which may allow the parasite to modulate the immune system.

  18. EOMES-positive CD4+ T cells are increased in PTPN22 (1858T) risk allele carriers.

    Science.gov (United States)

    Chemin, Karine; Ramsköld, Daniel; Diaz-Gallo, Lina-Marcela; Herrath, Jessica; Houtman, Miranda; Tandre, Karolina; Rönnblom, Lars; Catrina, Anca; Malmström, Vivianne

    2018-04-01

    The presence of the PTPN22 risk allele (1858T) is associated with several autoimmune diseases including rheumatoid arthritis (RA). Despite a number of studies exploring the function of PTPN22 in T cells, the exact impact of the PTPN22 risk allele on T-cell function in humans is still unclear. In this study, using RNA sequencing, we show that, upon TCR-activation, naïve human CD4 + T cells homozygous for the PTPN22 risk allele overexpress a set of genes including CFLAR and 4-1BB, which are important for cytotoxic T-cell differentiation. Moreover, the protein expression of the T-box transcription factor Eomesodermin (EOMES) was increased in T cells from healthy donors homozygous for the PTPN22 risk allele and correlated with a decreased number of naïve CD4 + T cells. There was no difference in the frequency of other CD4 + T-cell subsets (Th1, Th17, Tfh, Treg). Finally, an accumulation of EOMES + CD4 + T cells was observed in synovial fluid of RA patients with a more pronounced production of Perforin-1 in PTPN22 risk allele carriers. Altogether, we propose a novel mechanism of action of PTPN22 risk allele through the generation of cytotoxic CD4 + T cells and identify EOMES + CD4 + T cells as a relevant T-cell subset in RA pathogenesis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. [Changes of CD(4)(+) Foxp3+ regulatory T cells and CD(4)(+)IL-17+T cells in acrolein exposure rats].

    Science.gov (United States)

    Wei, Ming; Tu, Ling; Liang, Yinghong; Li, Jia; Gong, Yanjie; Zhang, Yihua; Yang, Lu

    2015-09-01

    To evaluate the changes of CD(4)(+) IL-17+T (Th17) and CD(4)(+)Foxp3+regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF) , and therefore to explore the role of Th17 and Treg in acrolein exposure airway inflammation in rats. Forty male Wistar rats were randomly divided into 4 groups: a 2 wk acrolein exposure group, a 4 wk acrolein exposure group, a 2 wk control group and a 4 wk control group (n=10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts.IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17+T and CD(4)(+) Foxp3+Treg in peripheral blood and BALF were determined by flow cytometry.The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups. Levels of IL-17 were remarkable increased in the 2 wk acrolein exposure group and the 4 wk acrolein exposure group in serum [(52.64 ± 1.89) ng/L, (76.73 ± 5.57) ng/L], and BALF [(79.07 ± 5.67) ng/L, (96.61 ± 6.44) ng/L] compared with the 2 wk control group [(40.05 ± 3.12) ng/L, (56.75 ± 4.37) ng/L] and the 4 wk control group [(38.75 ± 3.23) ng/L, (53.27 ± 4.48) ng/L], all Pcells and macrophages (r=0.5126, 0.5437, all Pcells and an vary of inflammatory cytokines were evident in airway inflammation of acrolein exposed rats, suggesting that Treg was involved in the immunological regulation and Th17 was associated with the persistent inflammation in acrolein induced airway inflammation in rats.

  20. Mucosal immunization in macaques upregulates the innate APOBEC 3G anti-viral factor in CD4(+) memory T cells.

    Science.gov (United States)

    Wang, Yufei; Bergmeier, Lesley A; Stebbings, Richard; Seidl, Thomas; Whittall, Trevor; Singh, Mahavir; Berry, Neil; Almond, Neil; Lehner, Thomas

    2009-02-05

    APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p<0.0002 to p< or =0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4(+)CCR5(+) memory T cells in circulating (p=0.0001), splenic (p=0.0001), iliac lymph nodes (p=0.002) and rectal (p=0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4(+)CCR5(+) circulating cells (p<0.01) and the draining iliac lymph node cells (p<0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5(+)CD4(+) memory and CD4(+)CD95(+)CCR7(-) effector memory T cells.

  1. CD4+CD28null T Cells are related to previous cytomegalovirus infection but not to accelerated atherosclerosis in ANCA-associated vasculitis.

    Science.gov (United States)

    Slot, Marjan C; Kroon, Abraham A; Damoiseaux, Jan G M C; Theunissen, Ruud; Houben, Alfons J H M; de Leeuw, Peter W; Tervaert, Jan Willem Cohen

    2017-05-01

    Previous studies have suggested an increased risk for cardiovascular events in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). We analyzed the presence of atherosclerotic damage in patients with AAV in relation to the presence of CD4 + CD28 null T cells and antibodies against cytomegalovirus (CMV) and human Heat-Shock Protein 60 (hHSP60). In this cross-sectional study, patients with inactive AAV were compared with healthy controls (HC). Carotid intima-media thickness (IMT) and aortic pulse-wave velocity (PWV) were measured. In addition, CD4 + CD28 null T cells, anti-CMV, and anti-hHSP60 levels were determined. Forty patients with AAV were included. Patients' spouses were recruited as HC (N = 38). CD4 + CD28 null T cells are present in patients with AAV in a higher percentage (median 3.1, range 0.01-85) than in HC (0.28, 0-36, P CD4 + CD28 null T cells (0.33 vs 13.8, P CD4 + CD28 null T cells and/or a previous CMV infection and IMT or PWV. There was no relation between anti-hHSP60 and CD4 + CD28 null T cells. Increased PWV values suggest atherosclerotic damage in patients with AAV. Plaque size, as determined by IMT, did not differ. CD4 + CD28 null T cells are increased in AAV and related to the previous CMV infection.

  2. What happens in the thymus does not stay in the thymus: how T cells recycle the CD4+-CD8+ lineage commitment transcriptional circuitry to control their function

    Science.gov (United States)

    Vacchio, Melanie S.; Bosselut, Rémy

    2016-01-01

    MHC-restricted CD4+ and CD8+ T cell are at the core of most adaptive immune responses. Although these cells carry distinct functions, they arise from a common precursor during thymic differentiation, in a developmental sequence that matches CD4 and CD8 expression and functional potential with MHC restriction. While the transcriptional control of CD4+-CD8+ lineage choice in the thymus is now better understood, less was known about what maintains the CD4+- and CD8+-lineage integrity of mature T cells. In this review, we discuss the mechanisms that establish in the thymus, and maintain in post-thymic cells, the separation of these lineages. We focus on recent studies that address the mechanisms of epigenetic control of Cd4 expression and emphasize how maintaining a transcriptional circuitry nucleated around Thpok and Runx proteins, the key architects of CD4+-CD8+ lineage commitment in the thymus, is critical for CD4+ T cell helper functions. PMID:27260768

  3. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

  4. Targeting CD4(+) T-Helper Cells Improves the Induction of Antitumor Responses in Dendritic Cell-Based Vaccination

    NARCIS (Netherlands)

    Aarntzen, Erik H. J. G.; de Vries, I. Jolanda M.; Lesterhuis, W. Joost; Schuurhuis, Danita; Jacobs, Joannes F. M.; Bol, Kalijn; Schreibelt, Gerty; Mus, Roel; de Wilt, Johannes H. W.; Haanen, John B. A. G.; Schadendorf, Dirk; Croockewit, Alexandra; Blokx, Willeke A. M.; van Rossum, Michelle M.; Kwok, William W.; Adema, Gosse J.; Punt, Cornelis J. A.; Figdor, Carl G.

    2013-01-01

    To evaluate the relevance of directing antigen-specific CD4(+) T helper cells as part of effective anticancer immunotherapy, we investigated the immunologic and clinical responses to vaccination with dendritic cells (DC) pulsed with either MHC class I (MHC-I)-restricted epitopes alone or both MHC

  5. Radiographic manifestations of Tuberculosis in HIV positive patients: Correlation with CD4+ T-cell count

    Directory of Open Access Journals (Sweden)

    Mehrdad Bakhshayesh-Karam

    2016-01-01

    Conclusion: In CD4+ cell count <500, the dominant radiographic pattern of Tuberculosis is atypical presentation. At this level of immunity, CD4+ T cell dysfunction may play a deterministic role in TB radiographic manifestation.

  6. Synthetic TLR4 agonists enhance functional antibodies and CD4+ T-cell responses against the Plasmodium falciparum GMZ2.6C multi-stage vaccine antigen

    DEFF Research Database (Denmark)

    Baldwin, Susan L; Roeffen, Will; Singh, Susheel K

    2016-01-01

    A subunit vaccine targeting both transmission and pathogenic asexual blood stages of Plasmodium falciparum, i.e., a multi-stage vaccine, could be a powerful tool to combat malaria. Here, we report production and characterization of the recombinant protein GMZ2.6C, which contains a fragment of the......-γ and TNF in response to GMZ2.6C. Both of these agonists have good safety records in humans....... of the sexual-stage protein Pfs48/45-6C genetically fused to GMZ2, an asexual vaccine antigen in advanced clinical development. To select the most suitable vaccine formulation for downstream clinical studies, GMZ2.6C was tested with various immune modulators in different adjuvant formulations (stable emulsions......, liposomes, and alum) in C57BL/6 mice. Some, but not all, formulations containing either the synthetic TLR4 agonist GLA or SLA elicited the highest parasite-specific antibody titers, the greatest IFN-γ responses in CD4+ TH1 cells, and the highest percentage of multifunctional CD4+ T cells expressing IFN...

  7. Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki; Yoichiro; Tanabe, Kazumi; Umeki, Shigeko; Nakamura, Nori; Yamakido, Michio; Hamamoto, Kazuko.

    1990-04-01

    The T-cell receptor CD3 (TCR/CD3) complex plays a central role in antigen recognition and activation of mature T cells, and therefore abnormalities in the expression of the complex should induce unresponsiveness of T cells to antigen stimulus. Using flow cytometry, we detected and enumerated variant cells with loss or alteration of surface TCR/CD3 expression among human mature CD4 + T cells. The presence of variant CD4 + T cells was demonstrated by isolating and cloning them from peripheral blood, and their abnormalities can be accounted for by alterations in TCR expression such as defects of protein expression and partial protein deletion. The variant frequency in peripheral blood increased with aging in normal donors and was highly elevated in patients with ataxia telangiectasia, an autosomal recessive inherited disease with defective DNA repair and variable T-cell immunodeficiency. These findings suggest that such alterations in TCR expression are induced by somatic mutagenesis of TCR genes and can be important factors related to age-dependent and genetic disease-associated T-cell dysfunction. (author)

  8. Evaluation of CD4+/CD8+ status and urinary tract infections ...

    African Journals Online (AJOL)

    Evaluation of CD4+/CD8+ status and urinary tract infections associated with urinary schistosomiasis ... African Health Sciences ... by <50 ova /10ml of urine had a mean CD4+:CD8+ ratio of 1.57 while those with heavy infections as ... Key words: CD4+, CD8+, urinary tract infections, urinary schistosomiasis, rural Nigerians

  9. Nutritional status, quality of life and CD4 cell count of adults living ...

    African Journals Online (AJOL)

    Keywords: CD4 cell count; Quality of Life; adults; nutritional status; nutritional intake. Nutritional status .... used to calculate the Body Mass Index (BMI). BMI was ... fat consumption as a percentage of the total food energy intake, and component ..... except when the CD4 counts fall very low.5,27 The CD4 cell count may offer a ...

  10. Inter- and intra-laboratory variability of CD4 cell counts in Swaziland

    African Journals Online (AJOL)

    2012-06-01

    Jun 1, 2012 ... The gold standard technique for CD4 enumeration is flow cytometry.3,4 Biological and analytical (laboratory) variations are known to affect CD4 enumeration; biological factors can that influence CD4 results include haemodilution in pregnancy, seasonal and diurnal variations (lowest at approximately 12: ...

  11. Induction of CD4 suppressor T cells with anti-Leu-8 antibody

    International Nuclear Information System (INIS)

    Kanof, M.E.; Strober, W.; James, S.P.

    1987-01-01

    To characterize the conditions under which CD