Neuronal surface antigen antibodies in limbic encephalitis

Science.gov (United States)

Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J.; Ribalta, T; Dalmau, J

2008-01-01

Objective: To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Methods: Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. Results: NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). Conclusions: In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies. GLOSSARY GAD = glutamic acid decarboxylase; LE = limbic encephalitis; NMDAR = N-methyl-D-aspartate receptor; NSA = neuronal surface antigens; nNSA = novel NSA; SCLC = small-cell lung cancer; VGKC = voltage-gated potassium channels

Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

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M Jedi-Tehrani

2005-10-01

Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

DEFF Research Database (Denmark)

Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

2010-01-01

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Science.gov (United States)

Akl, Ahmed; Lepetit, Maud; Crochette, Romain; Giral, Magali; Lepourry, Julie; Pallier, Annaick; Castagnet, Stéphanie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean-Paul

2013-01-01

In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells. PMID:24386360

Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner.

Science.gov (United States)

Riedl, Petra; Reiser, Michael; Stifter, Katja; Krieger, Jana; Schirmbeck, Reinhold

2014-07-01

Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

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Blaise Ndjamen

2014-03-01

Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

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Smith, Mark L; Mason, Hugh S; Shuler, Michael L

2002-12-30

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed. Copyright 2002 Wiley Periodicals, Inc.

Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

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Serkan Yazıcı

2015-01-01

Full Text Available We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF. 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+, B cells (HLA-DR+, CD19+, and HLA-DR+CD19+, NKT cells (CD3+CD16+CD56+, and NK cells (CD3−CD16+CD56+. The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato

1988-01-01

We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-γ. In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G 2 /M phases of the cell cycle than in G 0 /G 1 . The expression of YH206 antigen was enhanced in the S and G 2 /M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens. (author)

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

Science.gov (United States)

Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

2017-01-15

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this

A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells.

Science.gov (United States)

Takamatsu, H-H; Denyer, M S; Wileman, T E

2002-09-10

A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.

Chimeric antigen receptor T cells: a novel therapy for solid tumors

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Shengnan Yu

2017-03-01

Full Text Available Abstract The chimeric antigen receptor T (CAR-T cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2, and mesothelin (MSLN, as well as the challenges for CAR-T cell therapy.

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

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Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

1984-01-01

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

Analysis of antigen-specific B-cell memory directly ex vivo.

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McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Glycoprotein on cell surfaces

International Nuclear Information System (INIS)

Muramatsu, T.

1975-01-01

There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

Chimeric antigen receptor T-cell therapy for solid tumors

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Kheng Newick

2016-01-01

Full Text Available Chimeric antigen receptor (CAR T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias. This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

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Michael C. Gong

1999-06-01

Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

21 CFR 660.40 - Hepatitis B Surface Antigen.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral-Antigen Pathway.

Science.gov (United States)

Hewitt, Rachel E; Robertson, Jack; Haas, Carolin T; Pele, Laetitia C; Powell, Jonathan J

2017-01-01

Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer's patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4 + T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4 + T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen-PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer's patch T cell responses.

Shedding of the immunodominant P20 surface antigen of Eimeria bovis sporozoites.

OpenAIRE

Speer, C A; Whitmire, W M

1989-01-01

P20 is an immunodominant surface antigen of Eimeria bovis sporozoites. As parasites underwent merogony within cultured bovine monocytes and Madin-Darby bovine kidney (MDBK) cells, P20 appeared to be shed gradually by meronts and was absent in type 1 and 2 first-generation merozoites. Meronts of E. bovis appeared to shed P20 into the parasitophorous vacuole of bovine monocytes, whereas MDBK cells evidently released P20 into the culture medium or destroyed its antigenic determinant.

Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity

International Nuclear Information System (INIS)

Dyer, M.J.; Hunt, S.V.

1981-01-01

The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes

The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

DEFF Research Database (Denmark)

Kemp, M; Handman, E; Kemp, K

1998-01-01

The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune...... to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes....... Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor...

Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia

International Nuclear Information System (INIS)

Epstein, L.M.; Forney, J.D.

1984-01-01

A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei

Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

International Nuclear Information System (INIS)

Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

1988-01-01

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes

Effective antigen presentation to helper T cells by human eosinophils.

Science.gov (United States)

Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

2016-12-01

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral–Antigen Pathway

Science.gov (United States)

Hewitt, Rachel E.; Robertson, Jack; Haas, Carolin T.; Pele, Laetitia C.; Powell, Jonathan J.

2017-01-01

Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer’s patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4+ T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4+ T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen–PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer’s patch T cell responses. PMID:28367148

The Antigen Presenting Cells Instruct Plasma Cell Differentiation

Directory of Open Access Journals (Sweden)

Wei eXu

2014-01-01

Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

The antigen presenting cells instruct plasma cell differentiation.

Science.gov (United States)

Xu, Wei; Banchereau, Jacques

2014-01-06

The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

Science.gov (United States)

2012-01-01

Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

Binding of hydrophobic antigens to surfaces

DEFF Research Database (Denmark)

2017-01-01

A first aspect of the present invention is a method of detecting antibodies comprising the steps of: i) providing a first group of beads comprising a surface modified with C1-C10 alkyl groups comprising amine, ammonium, ether and/or hydroxyl groups, ii) contacting said first group of beads......-antigen-antibody conjugates, and v) detecting said bead-antigen-antibody conjugates. Further aspects include an antibody detection kit, a bead-antigen conjugate and a composition comprising at least two different groups of bead-antigen-conjugates....

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

International Nuclear Information System (INIS)

Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J.

1990-01-01

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins

Can resting B cells present antigen to T cells

International Nuclear Information System (INIS)

Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

1985-01-01

Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

The Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation

Science.gov (United States)

Mou, Yongbin; Xing, Yun; Ren, Hongyan; Cui, Zhihua; Zhang, Yu; Yu, Guangjie; Urba, Walter J.; Hu, Qingang; Hu, Hongming

2017-01-01

Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells. Immunological study using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Hareyama, Masato

1988-12-01

We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-..gamma... In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G/sub 2//M phases of the cell cycle than in G/sub 0//G/sub 1/. The expression of YH206 antigen was enhanced in the S and G/sub 2//M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens.

Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

Energy Technology Data Exchange (ETDEWEB)

Michelin, S.; Gallegos, C.E.; Dubner, D. [Radiopathology Laboratory, Nuclear Regulatory Authority, Buenos Aires (Argentina); Favier, B.; Carosella, E.D. [CEA, I2BM, Hopital Saint-Louis, IUH, Service de Recherches en Hemato-Immunologie, Paris (France)

2009-07-01

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of {gamma}-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that {gamma}-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

International Nuclear Information System (INIS)

Michelin, S.; Gallegos, C.E.; Dubner, D.; Favier, B.; Carosella, E.D.

2009-01-01

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of γ-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that γ-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

DEFF Research Database (Denmark)

Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

2006-01-01

Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

Science.gov (United States)

Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

2012-04-01

Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma

Directory of Open Access Journals (Sweden)

Long Zheng

2017-12-01

Full Text Available T cells expressing chimeric antigen receptors (CARs recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

Science.gov (United States)

Lai, Zengzu; Schreiber, John R

2009-05-21

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

Science.gov (United States)

Oka, Tatsuya; Rios, Eon J; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J

2013-10-01

Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Science.gov (United States)

Williams, L A; McLellan, A D; Summers, K L; Sorg, R V; Fearnley, D B; Hart, D N

1996-01-01

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro. PMID:8911149

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

Energy Technology Data Exchange (ETDEWEB)

Kleinhenz, M.E.; Ellner, J.J.

1985-07-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

International Nuclear Information System (INIS)

Kleinhenz, M.E.; Ellner, J.J.

1985-01-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

Heterologous expression of carcinoembryonic antigen in Lactococcus lactis via LcsB-mediated surface displaying system for oral vaccine development.

Science.gov (United States)

Zhang, Xiaowei; Hu, Shumin; Du, Xue; Li, Tiejun; Han, Lanlan; Kong, Jian

2016-12-01

Carcinoembryonic antigen (CEA) is an attractive target for immunotherapy because it is expressed minimally in normal tissue, but is overexpressed in a wide variety of malignant epithelial tissues. Lactic acid bacteria (LABs), widely used in food processes, are attractive candidates for oral vaccination. Thus, we examined whether LABs could be used as a live vaccine vector to deliver CEA antigen. CEA was cloned into an Escherichia coli/Lactococcus lactis shuttle vector pSEC:LEISS under the control of a nisin promoter. For displaying the CEA on the cell surface of the L. lactis strain, the anchor motif LcsB from the S-layer protein of Lactobacillus crispatus was fused with CEA. Intracellular and cell surface expression of the CEA-LcsB fusion was confirmed by western blot analysis. Significantly higher levels of CEA-specific secretory immunoglobulin A in the sera of mice were observed upon oral administration of strain cultures containing the CEA-LcsB fused protein. In addition, the CEA-LcsB antigen group showed a higher spleen index compared to the CEA antigen alone or negative control, demonstrating that surface-displayed CEA antigen could induce a higher immune response. These results provided the first evidence for displaying CEA antigen on the cell surfaces of LABs as oral vaccines against cancer or infectious diseases. Copyright © 2014. Published by Elsevier B.V.

Leukemia-associated antigens in man.

Science.gov (United States)

Brown, G; Capellaro, D; Greaves, M

1975-12-01

Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

Antigen presentation by resting B cells. Radiosensitivity of the antigen-presentation function and two distinct pathways of T cell activation

International Nuclear Information System (INIS)

Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

1984-01-01

In this report we have examined the ability of small resting B cells to act as antigen-presenting cells (APC) to antigen-specific MHC-restricted T cells as assessed by either T cell proliferation or T cell-dependent B cell stimulation. We found that 10 of 14 in vitro antigen-specific MHC-restricted T cell clones and lines and three of four T cell hybridomas could be induced to either proliferate or secrete IL-2 in the presence of lightly irradiated (1,000 rads) purified B cells and the appropriate foreign antigen. All T cell lines and hybridomas were stimulated to proliferate or make IL-2 by macrophage- and dendritic cell-enriched populations and all T cells tested except one hybridoma caused B cell activation when stimulated with B cells as APC. Furthermore, lightly irradiated, highly purified syngeneic B cells were as potent a source of APC for inducing B cell activation as were low density dendritic and macrophage-enriched cells. Lymph node T cells freshly taken from antigen-primed animals were also found to proliferate when cultured with purified B cells and the appropriate antigen. This APC function was easily measured when the cells were irradiated with 1,000 rads, but was greatly diminished or absent when they were irradiated with 3,300 rads. In addition, this radiosensitivity allowed us to easily distinguish B cell antigen presentation from presentation by the dendritic cell and macrophage, as the latter was resistant to 3,300 rads. Finally, one T cell clone that failed to proliferate when B cells were used as APC was able to recruit allogeneic B cells to proliferate in the presence of syngeneic B cells and the appropriate antigen. This result suggests that there are at least two distinct pathways of activation in T cells, one that leads to T cell proliferation and one that leads to the secretion of B cell recruitment factor(s)

Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences.

Science.gov (United States)

Howe, Daniel K; Gaji, Rajshekhar Y; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-02-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).

Sarcocystis neurona Merozoites Express a Family of Immunogenic Surface Antigens That Are Orthologues of the Toxoplasma gondii Surface Antigens (SAGs) and SAG-Related Sequences†

Science.gov (United States)

Howe, Daniel K.; Gaji, Rajshekhar Y.; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-01-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s). PMID:15664946

Protein-scaffold Directed Nanoscale Assembly of T Cell Ligands: Artificial Antigen Presentation with Defined Valency, Density and Ratio.

Science.gov (United States)

Smith, Mason R; Tolbert, Stephanie V; Wen, Fei

2018-05-07

Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein-scaffold directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast-cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency, but instead determined by the overall pMHC surface density. When intercellular adhesion molecule 1 (ICAM-1) was co-assembled with pMHC, it enhanced antigen recognition sensitivity by 6-fold. Further, T cells responded with different magnitudes to varying ratios of pMHC and ICAM-1 and exhibited a maximum response at a ratio of 15% pMHC and 85% ICAM-1, introducing an additional parameter for tuning T cell activation. This protein-scaffold directed assembly technology is readily transferrable to acellular surfaces for translational research as well as large-scale T-cell manufacturing.

Presentation of lipid antigens to T cells.

Science.gov (United States)

Mori, Lucia; De Libero, Gennaro

2008-04-15

T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

Viral sequestration of antigen subverts cross presentation to CD8(+ T cells.

Directory of Open Access Journals (Sweden)

Eric F Tewalt

2009-05-01

Full Text Available Virus-specific CD8(+ T cells (T(CD8+ are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC. Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T(CD8+. Direct presentation of vaccinia virus (VACV antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T(CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T(CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

DEFF Research Database (Denmark)

Kemp, M; Hey, A S; Kurtzhals, J A

1994-01-01

of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after...... the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL...... stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from...

Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

Directory of Open Access Journals (Sweden)

Marc Cartellieri

2010-01-01

Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Science.gov (United States)

Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

2016-01-01

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

Redirecting T cells to eradicate B-cell acute lymphoblastic leukemia: bispecific T-cell engagers and chimeric antigen receptors.

Science.gov (United States)

Aldoss, I; Bargou, R C; Nagorsen, D; Friberg, G R; Baeuerle, P A; Forman, S J

2017-04-01

Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

Science.gov (United States)

Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

2017-07-28

-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4 + and CD8 + T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and characterization of surface antigens in parasites, using radiolabelling techniques

International Nuclear Information System (INIS)

Ramasamy, R.

1982-04-01

Surface proteins of Schistosoma sp and Leishmania sp were studied using 125-Iodine as tracer. The surface proteins were labelled by the Lactoperoxidase method and the proteins then separated using SDS PAG electrophoresis and autoradiography. The possible immunogens were then separated using immunoprecipitation and Fluorescent Antibody techniques using sera from patients or from artificially immunized rabbits. Four common antigens were identified from the surfaces of male and female adult worms, cercariae and schistosomulae of S.mansoni. These antigens, which had molecular weights of 150,000, 78,000, 45,000, and 22,000 were also isolated from the surfaces of S.haematobium adults. The surface antigens on promastigotes of a Kenyan strain of Leishmania donovani were separated into three protein antigens with molecular weights of 66,000, 59,000 and 43,000 respectively. The 59,000 molecular weight antigen was a glycoprotein and was common to promastigotes of an American and Indian strain of L.donovani and to L.braziliensis mexicana. None of the isolated antigens have been shown to have a protective effect when vaccinated into mice, but the study illustrates the value of radionuclide tracers in the unravelling of the mosaic of antigens which parasites possess

Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

Directory of Open Access Journals (Sweden)

Hinrich eAbken

2013-11-01

Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2

DEFF Research Database (Denmark)

Faissner, A; Kruse, J; Goridis, C

1984-01-01

The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal m...

Harnessing Dendritic Cells for Tumor Antigen Presentation

Energy Technology Data Exchange (ETDEWEB)

Nierkens, Stefan [Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, Nijmegen 6525 GA (Netherlands); Janssen, Edith M., E-mail: edith.janssen@cchmc.org [Division of Molecular Immunology, Cincinnati Children' s Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229 (United States)

2011-04-26

Dendritic cells (DC) are professional antigen presenting cells that are crucial for the induction of anti-tumor T cell responses. As a consequence, research has focused on the harnessing of DCs for therapeutic interventions. Although current strategies employing ex vivo-generated and tumor-antigen loaded DCs have been proven feasible, there are still many obstacles to overcome in order to improve clinical trial successes and offset the cost and complexity of customized cell therapy. This review focuses on one of these obstacles and a pivotal step for the priming of tumor-specific CD8{sup +} and CD4{sup +} T cells; the in vitro loading of DCs with tumor antigens.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

Science.gov (United States)

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

Directory of Open Access Journals (Sweden)

Ewoud Bernardus Compeer

2012-03-01

Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

The E7-associated cell-surface antigen: a marker for the 11p13 chromosomal deletion associated with aniridia-Wilms tumor.

OpenAIRE

Scoggin, C H; Fisher, J H; Shoemaker, S A; Morse, H; Leigh, T; Riccardi, V M

1985-01-01

Unbalanced interstitial deletions of the p13 region of human chromosome 11 have been associated with congenital hypoplasia or aplasia of the iris, mental retardation, ambiguous genitalia, and predisposition to Wilms tumor of the kidney. Utilizing somatic cell hybrids containing either the normal or abnormal chromosome 11 from a child with Wilms tumor and aniridia, we previously mapped the E7 cell-surface antigen to the 11p1300-to-11p15.1 region. To localize even further the site of this antig...

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma

DEFF Research Database (Denmark)

Xu-Monette, Zijun Y; Li, Ling; Byrd, John C

2016-01-01

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its...

The value of serum Hepatitis B surface antigen quantification in ...

African Journals Online (AJOL)

The value of serum Hepatitis B surface antigen quantification in determining viralactivity in chronic Hepatitis B virus infection. ... ofCHB andalso higher in hepatitis e antigen positive patients compared to hepatitis e antigen negative patients.

Granulocytes: New Members of the Antigen-Presenting Cell Family

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Ang Lin

2017-12-01

Full Text Available Granulocytes, the most abundant types of leukocytes, are the first line of defense against pathogen invasion. However, the plasticity and diversity of granulocytes have been increasingly revealed, especially with regard to their versatile functions in orchestrating adaptive immune responses. A substantial body of recent evidence demonstrates that granulocytes can acquire the function as antigen-presenting cells under pathological or inflammatory conditions. In addition, they can acquire surface expression of MHC class II and costimulatory molecules as well as T cell stimulatory behavior when cultured with selected cytokines. The classic view of granulocytes as terminally differentiated, short-lived phagocytes is therefore changing to phenotypically and functionally heterogeneous cells that are engaged in cross-talk with other leukocyte populations and provide an additional link between innate and adaptive immunity. In this brief review, we summarize the current knowledge on the antigen-presenting capacity of granulocyte subsets (neutrophils, eosinophils, and basophils. Underlying mechanisms, relevant physiological significance and potential controversies are also discussed.

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

Science.gov (United States)

Schmid-Siegert, Emanuel; Richard, Sophie; Luraschi, Amanda; Mühlethaler, Konrad; Pagni, Marco; Hauser, Philippe M

2017-11-07

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Science.gov (United States)

Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

Science.gov (United States)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Identification of Surface Exposed Elementary Body Antigens of ...

African Journals Online (AJOL)

This study sought to identify the surface exposed antigenic components of Cowdria ruminantium elementary body (EB) by biotin labeling, determine effect of reducing and non-reducing conditions and heat on the mobility of these antigens and their reactivity to antibodies from immunized animals by Western blotting.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

Science.gov (United States)

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

DEFF Research Database (Denmark)

Mustafa, A S; Amoudy, H A; Wiker, H G

1998-01-01

GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity.

Science.gov (United States)

van Montfoort, Nadine; Camps, Marcel G; Khan, Selina; Filippov, Dmitri V; Weterings, Jimmy J; Griffith, Janice M; Geuze, Hans J; van Hall, Thorbald; Verbeek, J Sjef; Melief, Cornelis J; Ossendorp, Ferry

2009-04-21

Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.

Concurrence of hepatitis B surface antibodies and surface antigen: implications for postvaccination control of health care workers

NARCIS (Netherlands)

Zaaijer, Hans L.; Lelie, P. N.; Vandenbroucke-Grauls, C. M. J. E.; Koot, M.

2002-01-01

Among 1081 persons testing positive for hepatitis B surface antigen, 106 (10%) tested positive for antibodies to surface antigen (anti-HBs) in the same blood sample. Thirty of these persons were studied in detail: seven tested positive for hepatitis B e-antigen, nine were apparently healthy blood

Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

Science.gov (United States)

Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

2016-01-01

Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.

Science.gov (United States)

Tsaltas, G; Ford, C H

1993-02-01

Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

Science.gov (United States)

Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

2008-05-01

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages

B cell antigen receptor signaling and internalization are mutually exclusive events.

Directory of Open Access Journals (Sweden)

Ping Hou

2006-07-01

Full Text Available Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

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Kumar Sukhdeo

Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

Cell-free soluble-phase radioimmunoassay for Thy-1 antigen

Energy Technology Data Exchange (ETDEWEB)

Shalev, A.; Zuckerman, F. (Ben-Gurion Univ. of the Negev, Beersheba (Israel))

1983-12-01

A cell-free, soluble-phase, radioimmunoassay has been developed for Thy-1 antigen. The method is based on immunoprecipitation of radiolabelled Thy-1 molecules with specific antibodies, antiimmunoglobulin serum and polyethyleneglycol (PEG). The method can be used with convenience to screen for the presence of Thy-1 in various fluids as well as on cell surfaces for qualitative or quantitative purposes. Presence of antibodies or autoantibodies against Thy-1 can also be detected specifically. Evidence that the dog, carp, hamster and goldfish carry Thy-1-like molecules on neuronal (brain) cells is demonstrated by this method.

Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Hareyama, M.; Imai, K.; Kubo, K.; Takahashi, H.; Koshiba, H.; Hinoda, Y.; Shidou, M.; Oouchi, A.; Yachi, A.; Morita, K. (Sapporo Medical College (Japan))

1991-05-01

The changes of antigenic expression of cultured human gastric adenocarcinoma MKN45 cells caused by irradiation were investigated to elucidate the immune responses to localized irradiation. The expression of carcinoembryonic antigen (CEA) showed remarkable increases in the culture supernatant and on the surface of the membrane of irradiated cells. The expression of major histocompatibility complex Class I antigen on the membrane also was enhanced by irradiation. In addition, the irradiated cell groups, when analyzed using a CEA-specific probe, showed remarkable increases in the CEA mRNA. These enhancements increased in the 10-Gy and 15-Gy irradiated populations compared with the 5-Gy irradiated population. These results suggest that the enhancement of expression of CEA by radiation takes place at the CEA gene expression (mRNA) level but not at the protein level.

Some problems associated with radiolabeling surface antigens on helminth parasites: a brief review

Energy Technology Data Exchange (ETDEWEB)

Hayunga, E.G. (Division of Tropical Public Health, Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD (USA)); Murrell, K.D. (Agricultural Research Service, Beltsville, MD (USA))

1982-06-01

Recent developments in technology have facilitated substantial advances in the characterization of surface antigens from a wide variety of both normal and neoplastic cells. However, the immunochemistry of parasites has lagged behind. Efforts to apply conventional radiolabeling methods to helminths have not always been successful. Experimental work with Schistosoma mansoni is reviewed to illustrate common problems encountered in surface labeling studies. These findings should provide insight for the future investigation of other helminth species.

Some problems associated with radiolabeling surface antigens on helminth parasites: a brief review

International Nuclear Information System (INIS)

Hayunga, E.G.; Murrell, K.D.

1982-01-01

Recent developments in technology have facilitated substantial advances in the characterization of surface antigens from a wide variety of both normal and neoplastic cells. However, the immunochemistry of parasites has lagged behind. Efforts to apply conventional radiolabeling methods to helminths have not always been successful. Experimental work with Schistosoma mansoni is reviewed to illustrate common problems encountered in surface labeling studies. These findings should provide insight for the future investigation of other helminth species. (Auth.)

Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma

International Nuclear Information System (INIS)

Kato, H.; Torigoe, T.

1977-01-01

A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with other carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All patients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care

Cell-surface antigens associated with dualtropic and thymotropic murine leukemia viruses inducing thymic and nonthymic lymphomas

International Nuclear Information System (INIS)

Haas, M.; Patch, V.

1980-01-01

Unique type-specific antigens were detected on cells infected with dualtropic and thymotropic viruses and x-ray-induced T cell- and B cell-malignant lymphomas of C57BL/6 mice. These findings support the contention that T cell lymphoma (TCL)-inducing and B cell lymphoma (BCL)-indcing viruses isolated from x-irradiated C57BL/6 mice are env gene recombinants in which ecotropic gene sequences have been substituted by xenotropic sequences. We found that unique antigenicities are associated with each TCL-inducing and BCL-inducing dualtropic virus, and that the thymotropic TCL-inducing virus isolates represent a separate serologic group. These virus mapping experiments indicated that many serologically different recombinant viruses can be isolated from C57BL/6 mice. It is suggested that many distinct recombinant viruses may exist in lymphomagenic C57BL/6 mice, some of which are associated with specific lyphoma induction

Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

Science.gov (United States)

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

2017-01-01

Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method. PMID:28761820

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

Science.gov (United States)

Chapanian, Rafi; Constantinescu, Iren; Rossi, Nicholas A A; Medvedev, Nadia; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

2012-11-01

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic

Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

International Nuclear Information System (INIS)

Sadeghi, H.; Da Silva, A.M.; Klein, C.

1988-01-01

The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [ 3 H]myristate, [ 3 H]palmitate, and [ 14 C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [ 14 C]ethanolamine but not with [ 3 H]myristate of [ 3 H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

Science.gov (United States)

Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

2011-02-01

Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

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Arnot David E

2008-06-01

Full Text Available Abstract Background The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development. Methods A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy Results Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1. Conclusion A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.

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.

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

Science.gov (United States)

Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

2016-06-01

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Virosomes for antigen and DNA delivery

NARCIS (Netherlands)

Daemen, T; de Mare, A; Bungener, L; de Jonge, J; Huckriede, A; Wilschut, J

2005-01-01

Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cell-mediated immunity. Prophylactic vaccination

A Molecular-Level Account of the Antigenic Hantaviral Surface

Directory of Open Access Journals (Sweden)

Sai Li

2016-05-01

Full Text Available Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV, a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells

DEFF Research Database (Denmark)

Kirkin, Alexei F.; Dzhandzhugazyan, Karine N.; Guldberg, Per

2018-01-01

In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4+ T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT...... antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25...... patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can...

Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

DEFF Research Database (Denmark)

Mustafa, A S; Amoudy, H A; Wiker, H G

1998-01-01

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r......GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

A portion of the Pf155/RESA antigen of Plasmodium falciparum is accessible on the surface of infected erythrocytes

International Nuclear Information System (INIS)

Saul, A.; Maloy, W.L.; Howard, R.J.; Rock, E.P.

1988-01-01

An investigation of antigens accessible to lactoperoxidase-catalysed cell surface iodination on intact Plasmodium falciparum-infected red blood cells (RBC) has identified a 125 I-labelled antigen with an apparent size of about 155 kD. This labelled protein was specifically immunoprecipitated by the following antibodies: a rabbit antiserum and a mouse monoclonal antibody raised against a synthetic peptide comprising the 3',8-mer repeat EENVEHDA of the Pf155/RESA protein; a rabbit antiserum raised against a synthetic octapeptide comprising two copies of the 3',4-mer repeat EENV of the Pf155/RESA protein; and rabbit antisera against another synthetic peptide C(MYSNNNVED) 2 . The last antibody shows a strong reaction in asexual blood state parasites with the Pf155/RESA antigen. While this antigen has been described previously as a submembrane component of the outer membrane of infected RBC, this report shows that at least part of it is accessible to the surface of both ring and late trophozoite-infected erythrocytes. 21 refs., 4 figs

The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells.

Science.gov (United States)

den Haan, Joke M M; Arens, Ramon; van Zelm, Menno C

2014-12-01

The adaptive immune system consists of T and B cells that express clonally distributed antigen receptors. To achieve functional adaptive immune responses, antigen-specific T cell populations are stimulated by professional antigen-presenting cells like dendritic cells (DCs), which provide crucial stimulatory signals for efficient expansion and development of effector functions. Antigen-specific B cells receive costimulatory signals from helper T cells to stimulate affinity maturation and isotype switching. Here we elaborate on the interactions between DCs, T cells and B cells, and on the important signals for efficient induction of adaptive immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

Induction of anti-HBs in HB vaccine nonresponders in vivo by hepatitis B surface antigen-pulsed blood dendritic cells.

Science.gov (United States)

Fazle Akbar, Sk Md; Furukawa, Shinya; Yoshida, Osamu; Hiasa, Yoichi; Horiike, Norio; Onji, Morikazu

2007-07-01

Antigen-pulsed dendritic cells (DCs) are now used for treatment of patients with cancers, however, the efficacy of these DCs has never been evaluated for prophylactic purposes. The aim of this study was (1) to prepare hepatitis B surface antigen (HBsAg)-pulsed human blood DCs, (2) to assess immunogenicity of HBsAg-pulsed DCs in vitro and (3) to evaluate the efficacy of HBsAg-pulsed DCs in hepatitis B (HB) vaccine nonresponders. Human peripheral blood DCs were cultured with HBsAg to prepare HBsAg-pulsed DCs. The expression of immunogenic epitopes of HBsAg on HBsAg-pulsed DCs was assessed in vitro. Finally, HBsAg-pulsed DCs were administered, intradermally to six HB vaccine nonresponders and the levels of antibody to HBsAg (anti-HBs) in the sera were assessed. HB vaccine nonresponders did not exhibit features of immediate, early or delayed adverse reactions due to administration of HBsAg-pulsed DCs. Anti-HBs were detected in the sera of all HB vaccine nonresponders within 28 days after administration of HBsAg-pulsed DCs. This study opens a new field of application of antigen-pulsed DCs for prophylactic purposes when adequate levels of protective antibody cannot be induced by traditional vaccination approaches.

Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Borodina, Irina; Jensen, B. M.; Søndergaard, Ib

2010-01-01

Background: Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast...... were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS) after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained...... their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating...

Specificity of antigens on UV radiation-induced antigenic tumor cell variants measured in vitro and in vivo

International Nuclear Information System (INIS)

Hostetler, L.W.; Romerdahl, C.A.; Kripke, M.L.

1989-01-01

The purpose of this study was to determine whether antigenic variants cross-react immunologically with the parental tumor and whether the UVR-associated antigen unique to UVR-induced tumors is also present on the variants. Antigenic (regressor) variants and nonimmunogenic (progressor) clones derived from UV-irradiated cultures of the C3H K1735 melanoma and SF19 spontaneous fibrosarcoma cell lines were used to address these questions. In an in vivo immunization and challenge assay, the antigenic variants did not induce cross-protection among themselves, but each induced immunity against the immunizing variant, the parent tumor cells, and nonimmunogenic clones derived from UV-irradiated parent cultures. Therefore, the variants can be used to induce in mice a protective immunity that prevents the growth of the parent tumor and nonimmunogenic clones, but not other antigenic variants. In contrast, immunization with cells of the parental tumor or the nonimmunogenic clones induced no protective immunity against challenge with any of the cell lines. Utilizing the K1735 melanoma-derived cell lines in vitro, T-helper (Th) cells isolated from tumor-immunized mice were tested for cross-reactivity by their ability to collaborate with trinitrophenyl-primed B-cells in the presence of trinitrophenyl-conjugated tumor cells. Also, the cross-reactivity of cytotoxic T-lymphocytes from tumor-immunized mice was assessed by a 4-h 51Cr-release assay. Antigenic variants induced cytotoxic T-lymphocytes and Th activity that was higher than that induced by the parent tumor and nonimmunogenic clones from the UVR-exposed parent tumor and cross-reacted with the parental tumor cells and nonimmunogenic clones, but not with other antigenic variants

Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

International Nuclear Information System (INIS)

Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

1987-01-01

Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

International Nuclear Information System (INIS)

Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

2011-01-01

Highlights: → Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. → An ideal artificial APCs system was successfully prepared in vivo. → Controlled release of IL-2 leads to much more T-cell expansion. → This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

Energy Technology Data Exchange (ETDEWEB)

Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

2011-08-05

Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis

Directory of Open Access Journals (Sweden)

Patrycja Anna Kobierecka

2016-02-01

Full Text Available Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type Campylobacter jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analysed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ LAB (Lactic Acid Bacteria strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered

Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

International Nuclear Information System (INIS)

Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

2010-01-01

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

Science.gov (United States)

Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

Comparison of microglia and infiltrating CD11c+ cells as antigen presenting cells for T cell proliferation and cytokine response

DEFF Research Database (Denmark)

Wlodarczyk, Agnieszka; Løbner, Morten; Cédile, Oriane

2014-01-01

BACKGROUND: Tissue-resident antigen-presenting cells (APC) exert a major influence on the local immune environment. Microglia are resident myeloid cells in the central nervous system (CNS), deriving from early post-embryonic precursors, distinct from adult hematopoietic lineages. Dendritic cells...... (DC) and macrophages infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE). Microglia are not considered to be as effective APC as DC or macrophages. METHODS: In this work we compared the antigen presenting capacity of CD11c+ and CD11c- microglia subsets with infiltrating CD11c......+ APC, which include DC. The microglial subpopulations (CD11c- CD45dim CD11b+ and CD11c+ CD45dim CD11b+) as well as infiltrating CD11c+ CD45high cells were sorted from CNS of C57BL/6 mice with EAE. Sorted cells were characterised by flow cytometry for surface phenotype and by quantitative real-time PCR...

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

Science.gov (United States)

Moguche, Albanus O; Musvosvi, Munyaradzi; Penn-Nicholson, Adam; Plumlee, Courtney R; Mearns, Helen; Geldenhuys, Hennie; Smit, Erica; Abrahams, Deborah; Rozot, Virginie; Dintwe, One; Hoff, Søren T; Kromann, Ingrid; Ruhwald, Morten; Bang, Peter; Larson, Ryan P; Shafiani, Shahin; Ma, Shuyi; Sherman, David R; Sette, Alessandro; Lindestam Arlehamn, Cecilia S; McKinney, Denise M; Maecker, Holden; Hanekom, Willem A; Hatherill, Mark; Andersen, Peter; Scriba, Thomas J; Urdahl, Kevin B

2017-06-14

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

DEFF Research Database (Denmark)

Ditzel, Henrik

2009-01-01

A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Antigen Loss Variants: Catching Hold of Escaping Foes.

Science.gov (United States)

Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

2017-01-01

Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse. CD19 and CD20 antigen loss in acute lymphocytic leukemia and chronic lymphocytic leukemia, respectively, and lineage switching in leukemia associated with mixed lineage leukemia (MLL) gene rearrangements are well-documented evidences in this regard. Although increasing number of novel immunotherapies are being developed, majority of these do not address the control of antigen loss variants. Here, we review the occurrence of antigen loss variants in leukemia and discuss the therapeutic strategies to tackle the same. We also present an approach of dual-targeting immunoligand effectively retargeting NK cells against antigen loss variants in MLL-associated leukemia. Novel immunotherapies simultaneously targeting more than one tumor antigen certainly hold promise to completely eradicate tumor and prevent therapy-resistant relapses.

A molecular approach to immunoscintigraphy: A study of the T-antigen conformation on the surface of tumors

International Nuclear Information System (INIS)

Noujaim, A.; Selvaraj, S.; Suresh, M.R.; Turner, C.; McLean, G.; Willans, D.; Longenecker, B.M.; Haines, D.M.

1987-01-01

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both α and β configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models. (orig.) [de

Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

Science.gov (United States)

Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

2015-05-01

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

International Nuclear Information System (INIS)

Abbas, A.K.; Haber, S.; Rock, K.L.

1985-01-01

Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

Chloroquine inhibits accessory cell presentation of soluble natural and synthetic protein antigens

DEFF Research Database (Denmark)

Buus, S; Werdelin, O

1984-01-01

We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...... was time- and dose-dependent. A brief treatment solely of the accessory cells with the drug compromised their ability to stimulate primed T cells in a subsequent culture provided the accessory cells were treated with chloroquine before their exposure to the antigen. These results suggest that chloroquine...... acts on an early event in the antigen handling by accessory cells. Chloroquine is a well known inhibitor of lysosomal proteolysis, and it is likely that its effect on antigen presentation is caused by an inhibition of antigen degradation....

Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Directory of Open Access Journals (Sweden)

Ana Teresa B.F. Antonangelo

2012-09-01

Full Text Available Babesiosis is one of the most important diseases affecting livestock agriculture worldwide. Animals from the subspecies Bos taurus indicus are more resistant to babesiosis than those from Bos taurus taurus. The genera Babesia and Plasmodium are Apicomplexa hemoparasites and share features such as invasion of red blood cells (RBC. The glycoprotein Duffy is the only human erythrocyte receptor for Pasmodium vivax and a mutation which abolishes expression of this glycoprotein on erythrocyte surfaces is responsible for making the majority of people originating from the indigenous populations of West Africa resistant to P. vivax. The current work detected and quantified the Duffy antigen on Bos taurus indicus and Bos taurus taurus erythrocyte surfaces using a polyclonal antibody in order to investigate if differences in susceptibility to Babesia are due to different levels of Duffy antigen expression on the RBCs of these animals, as is known to be the case in human beings for interactions of Plasmodium vivax-Duffy antigen. ELISA tests showed that the antibody that was raised against Duffy antigens detected the presence of Duffy antigen in both subspecies and that the amount of this antigen on those erythrocyte membranes was similar. These results indicate that the greater resistance of B. taurus indicus to babesiosis cannot be explained by the absence or lower expression of Duffy antigen on RBC surfaces.

Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

International Nuclear Information System (INIS)

Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

1987-01-01

The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

Energy Technology Data Exchange (ETDEWEB)

Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

1987-11-01

The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

Science.gov (United States)

Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

2018-05-11

Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

Science.gov (United States)

Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

Identification of a peptide binding protein that plays a role in antigen presentation

International Nuclear Information System (INIS)

Lakey, E.K.; Margoliash, E.; Pierce, S.K.

1987-01-01

The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 4 /PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation

Kefiran suppresses antigen-induced mast cell activation.

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Furuno, Tadahide; Nakanishi, Mamoru

2012-01-01

Kefir is a traditional fermented milk beverage produced by kefir grains in the Caucasian countries. Kefiran produced by Lactobacillus kefiranofaciens in kefir grains is an exopolysaccharide having a repeating structure with glucose and galactose residues in the chain sequence and has been suggested to exert many health-promoting effects such as immunomodulatory, hypotensive, hypocholesterolemic activities. Here we investigated the effects of kefiran on mast cell activation induced by antigen. Pretreatment with kefiran significantly inhibited antigen-induced Ca(2+) mobilization, degranulation, and tumor necrosis factor-α production in bone marrow-derived mast cells (BMMCs) in a dose-dependent manner. The phosphorylation of Akt, glycogen synthase kinase 3β, and extracellular signal-regulated kinases (ERKs) after antigen stimulation was also suppressed by pretreatment of BMMCs with kefiran. These findings indicate that kefiran suppresses mast cell degranulation and cytokine production by inhibiting the Akt and ERKs pathways, suggesting an anti-inflammatory effect for kefiran.

Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

DEFF Research Database (Denmark)

Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

2016-01-01

Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...

Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

Science.gov (United States)

Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

2013-04-18

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

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de Jong, R. N.; Beurskens, F. J.; Verploegen, S.

2016-01-01

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology......) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce...... conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD...

Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

International Nuclear Information System (INIS)

Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F.

1989-01-01

The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3 H-fatty acids, [ 3 H]ethanolamine, and [ 3 H]carbohydrates. Treatment of 3 H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment

Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

Energy Technology Data Exchange (ETDEWEB)

Elliott, E V; Pindar, A; Stevenson, F K; Stevenson, G T [Southampton General Hospital (UK). Tenovus Research Lab.

1978-03-01

Three antibody populations were raised in rabbits against surface antigens on guinea-pig L/sub 2/C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L/sub 2/C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.

Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

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Wu, Julie H; Simonette, Rebecca A; Nguyen, Harrison P; Rady, Peter L; Tyring, Stephen K

2016-06-01

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

Carbohydrates as T-cell antigens with implications in health and disease.

Science.gov (United States)

Sun, Lina; Middleton, Dustin R; Wantuch, Paeton L; Ozdilek, Ahmet; Avci, Fikri Y

2016-10-01

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antigen dynamics of follicular dendritic cells

NARCIS (Netherlands)

Heesters, B.A.

2015-01-01

Stromal-derived follicular dendritic cells (FDCs) are a major depot for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate and high-affinity antibody production takes place. Historically, FDCs have been characterized as ‘accessory’

Rh antigen expression during erythropoeisis: Comparison of cord and adult derived CD34 + cells

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Gupta Namita

2008-01-01

Full Text Available Objectives: Concentrations of O 2 and CO 2 in the fetal circulation differ to that in maternal blood. Previous studies done in algae demonstrate the functional role of Rh antigen as CO 2 transporter. As a preliminary study, it was the aim of this project to compare the expression of Rh polypeptides on cord and adult red blood cell progenitors during ex vivo proliferation and differentiation of CD34 + cells during erythropoeisis. Materials and Methods: CD34 positive hematopoeitic progenitor cells were isolated from umbilical cord blood and adult peripheral blood using an immunomagnetic system and cultured in serum free medium containing erythropoietin in order to compel them along the erythroid lineage. Cultured cells were analyzed for cell surface marker expression by flow cytometry, using monoclonal antibodies to RhAG, Glycophorin A, Rh polypeptides, CD47 and Band 3. Cytospin analysis was also done to study the morphology of cultured cells. Results: The appearance of cell surface markers analyzed on different days of culture varied slightly between samples. There was no evidence to suggest that RhAG, GPA, CD47 and Band 3 expression was any different between adult and cord derived cells. Nevertheless, the results of Rh antigenic expression suggest a reasonable difference between the two groups with adult sample derived cells showing higher and earlier expression than cord blood derived cells. These preliminary findings require further investigation. Conclusion: Comparing the expression of cell surface markers especially Rh polypeptides between adult and cord blood derived erythroid progenitors might assist in discerning their functions and could be valuable in the study of erythropoeisis.

Clonal analysis of T-cell responses to herpes simplex virus: isolation, characterization and antiviral properties of an antigen-specific helper T-cell clone.

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Leung, K N; Nash, A A; Sia, D Y; Wildy, P

1984-12-01

A herpes simplex virus (HSV)-specific long-term T-cell clone has been established from the draining lymph node cells of BALB/c mice; the cells required repeated in vitro restimulation with UV-irradiated virus. The established T-cell clone expresses the Thy-1 and Lyt-1+2,3- surface antigens. For optimal proliferation of the cloned cells, both the presence of specific antigen and an exogenous source of T-cell growth factor are required. The proliferative response of the cloned T cells was found to be virus-specific but it did not distinguish between HSV-1 and HSV-2. Adoptive cell transfer of the cloned T cells helped primed B cells to produce anti-herpes antibodies: the response was antigen-specific and cell dose-dependent. The clone failed to produce a significant DTH reaction in vivo, but did produce high levels of macrophage-activating factor. Furthermore, the T-cell clone could protect from HSV infection, as measured by a reduction in local virus growth, and by enhanced survival following the challenge of mice with a lethal dose of virus. The mechanism(s) whereby this clone protects in vivo is discussed.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

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Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

DEFF Research Database (Denmark)

Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

2004-01-01

's thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture......B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto...

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

DEFF Research Database (Denmark)

Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

2004-01-01

B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto......'s thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture...

Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

Science.gov (United States)

Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

2017-06-01

We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

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Robert Moot

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs. VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

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Cecilia González

Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

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Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Prostate Stem Cell Antigen: A Prospective Therapeutic and Diagnostic Target

Science.gov (United States)

Raff, Adam B.; Gray, Andrew; Kast, W. Martin

2009-01-01

The development of novel clinical tools to combat cancer is an intense field of research and recent efforts have been directed at the identification of proteins that may provide diagnostic, prognostic and/or therapeutic applications due to their restricted expression. To date, a number of protein candidates have emerged as potential clinical tools in the treatment of prostate cancer. Discovered over ten year ago, prostate stem cell antigen (PSCA) is a cell surface antigen that belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol-anchored proteins. PSCA is highly overexpressed in human prostate cancer, with limited expression in normal tissues, making it an ideal target for both diagnosis and therapy. Several studies have now clearly correlated the expression of PSCA with relevant clinical benchmarks, such as Gleason score and metastasis, while others have demonstrated the efficacy of PSCA targeting in treatment through various modalities. The purpose of this review is to present the current body of knowledge about PSCA and its potential role in the treatment of human prostate cancer. PMID:18838214

Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

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Warren L Denning

2011-02-01

Full Text Available Harnessing the ability of cytotoxic T lymphocytes (CTLs to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

Variability in surface antigen expression on neuroblastoma cells as revealed by monoclonal antibodies

International Nuclear Information System (INIS)

Malpas, J.S.; Kemshead, J.T.; Pritchard, J.; Greaves, M.F.

1982-01-01

In treatment programmes for neuroblastoma involving autologous bone marrow transplantation, a problem exists in the identification of small numbers of metastatic tumour cells present in the marrow aspirates. Reinfusion of tumour cells along with normal bone marrow may reseed the tumour within a patient who has received high dose chemotherapy. Formalin-induced fluorescence in neuroblastoma is a possible diagnostic aid, but this method has no therapeutic potential. Other methods of detecting tumour relying on gross physiological changes in the patient are not suitable for diagnosis of minimal metastatic disease. As an immunological approach to the problem, rabbit antisera to neuroblastoma have been raised but these reagents suffer from low titre after absorption to make them specific. The authors have used the technique of somatic cell hybridisation to raise monoclonal antibodies which bind to neuroblastoma cells and not to normal haemopoietic progenitors. A panel of such reagents to demonstrate heterogeneity in antigen expression amongst metastatic neuroblastoma cells was employed in a radioimmunoassay as diagnostic aid for this problem. (Auth.)

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)

Effect of antigen on localization of immunologically specific B cells

International Nuclear Information System (INIS)

Ponzio, N.M.; Chapman, J.M.; Thorbecke, G.J.

1976-01-01

Studies were conducted to demonstrate homing of memory B cells to sites of antigen localization in lymph nodes, using functional criteria to detect local presence of memory cells at varying intervals after intravenous injection. Cell suspensions were prepared from spleens of donor mice injected with complete Freund's adjuvant. Recipient mice were injected with Escherichia coli endotoxin and immune or normal spleen cells and were gamma-irradiated. Results indicated that passively transferred unilateral B cell memory was established. The development over a period of several days of this difference between left and right lymph nodes suggests that recirculating memory B cells are being progressively selected by antigen in the lymph node, rather than that this difference is due to a specific exit of cells from the circulation towards the antigen

Shedding of leukemia-associated P24 antigen by lymphoblastoid cell lines.

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Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

1987-12-01

We report the development of a unique enzyme-linked immunosorbent assay (ELISA) which makes possible the detection of leukemia-associated P24 antigen, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4 simultaneously. Using the RCA1/SJ-9A4-ELISA, P24 antigen, as few as 50 X 10(3) cells from a common acute lymphoblastic leukemia (C-ALL) cell line could be detected. The presence of D-galactose gave complete and specific inhibition of P24 antigen binding to RCA1. Matched concentrations of D-glucose and D-sucrose had no effect on binding. The release of the P24 antigen into the culture medium by a C-ALL cell line maintained at 37 degrees C could be detected; however, no P24 antigen was present in the culture medium when the cells were maintained at 4 degrees C. Sequential analysis of the culture medium for soluble P24 antigen revealed that release of the P24 antigen associated with cell growth. Molecular sieve chromatography of concentrated culture medium indicated that shed P24 antigen was eluted in the macromolecule fraction. P24 antigen was detected in the cerebrospinal fluid (CSF) of four patients with P24 positive ALL at the time of relapse of the central nervous system (CNS) and was undetectable while in complete remission. The CSF from three patients with P24 negative ALL and three patients with aseptic meningitis had no detectable activity.

Impact of aging on antigen presentation cell function of dendritic cells.

Science.gov (United States)

Wong, Christine; Goldstein, Daniel R

2013-08-01

Older people exhibit increased mortality to infections and cancer as compared to younger people, indicating that aging impairs immunity. Dendritic cells (DCs) are key for bridging the innate and adaptive arms of the immune system by priming antigen specific T cells. Discerning how aging impacts DC function to initiate adaptive immune responses is of great biomedical importance as this could lead to the development of novel therapeutics to enhance immunity with aging. This review details reports indicating that aging impairs the antigen presenting function of DCs but highlights other studies indicating preserved DC function with aging. How aging impacts antigen presentation by DCs is complex and without a clear unifying biological underpinning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

Science.gov (United States)

Boas, Ulrik; Lind, Peter; Riber, Ulla

2014-11-15

A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use. Copyright © 2014 Elsevier Inc. All rights reserved.

Tumor cell surface proteins

International Nuclear Information System (INIS)

Kennel, S.J.; Braslawsky, G.R.; Flynn, K.; Foote, L.J.; Friedman, E.; Hotchkiss, J.A.; Huang, A.H.L.; Lankford, P.K.

1982-01-01

Cell surface proteins mediate interaction between cells and their environment. Unique tumor cell surface proteins are being identified and quantified in several tumor systems to address the following questions: (i) how do tumor-specific proteins arise during cell transformation; (ii) can these proteins be used as markers of tumor cell distribution in vivo; (iii) can cytotoxic drugs be targeted specifically to tumor cells using antibody; and (iv) can solid state radioimmunoassay of these proteins provide a means to quantify transformation frequencies. A tumor surface protein of 180,000 M/sub r/ (TSP-180) has been identified on cells of several lung carcinomas of BALB/c mice. TSP-180 was not detected on normal lung tissue, embryonic tissue, or other epithelial or sarcoma tumors, but it was found on lung carcinomas of other strains of mice. Considerable amino acid sequence homology exists among TSP-180's from several cell sources, indicating that TSP-180 synthesis is directed by normal cellular genes although it is not expressed in normal cells. The regulation of synthesis of TSP-180 and its relationship to normal cell surface proteins are being studied. Monoclonal antibodies (MoAb) to TSP-180 have been developed. The antibodies have been used in immunoaffinity chromatography to isolate TSP-180 from tumor cell sources. This purified tumor antigen was used to immunize rats. Antibody produced by these animals reacted at different sites (epitopes) on the TSP-180 molecule than did the original MoAb. These sera and MoAb from these animals are being used to identify normal cell components related to the TSP-180 molecule

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Directory of Open Access Journals (Sweden)

Lynette Beattie

2010-03-01

Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

Science.gov (United States)

Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

1993-01-01

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

A radioimmunoassay for human antibody specific for microbial antigens

International Nuclear Information System (INIS)

Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

1977-01-01

A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

EVIR: chimeric receptors that enhance dendritic cell cross-dressing with tumor antigens.

Science.gov (United States)

Squadrito, Mario Leonardo; Cianciaruso, Chiara; Hansen, Sarah K; De Palma, Michele

2018-03-01

We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8 + T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.

Prediction of antigenic epitopes on protein surfaces by consensus scoring

Directory of Open Access Journals (Sweden)

Zhang Chi

2009-09-01

Full Text Available Abstract Background Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance. Results We present a new antigen Epitope Prediction method, which uses ConsEnsus Scoring (EPCES from six different scoring functions - residue epitope propensity, conservation score, side-chain energy score, contact number, surface planarity score, and secondary structure composition. Applied to unbounded antigen structures from an independent test set, EPCES was able to predict antigenic eptitopes with 47.8% sensitivity, 69.5% specificity and an AUC value of 0.632. The performance of the method is statistically similar to other published methods. The AUC value of EPCES is slightly higher compared to the best results of existing algorithms by about 0.034. Conclusion Our work shows consensus scoring of multiple features has a better performance than any single term. The successful prediction is also due to the new score of residue epitope propensity based on atomic solvent accessibility.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

Science.gov (United States)

Urbanova, Linda; Hradilova, Nada; Moserova, Irena; Vosahlikova, Sarka; Sadilkova, Lenka; Hensler, Michal; Spisek, Radek; Adkins, Irena

2017-07-01

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8 + T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa). Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Pattern of distribution of blood group antigens on human epidermal cells during maturation

DEFF Research Database (Denmark)

Dabelsteen, Erik; Buschard, Karsten; Hakomori, Sen-Itiroh

1984-01-01

The distribution in human epidermis of A, B, and H blood group antigens and of a precursor carbohydrate chain, N-acetyl-lactosamine, was examined using immunofluorescence staining techniques. The material included tissue from 10 blood group A, 4 blood group B, and 9 blood group O persons. Murine...... on the lower spinous cells whereas H antigen was seen predominantly on upper spinous cells or on the granular cells. Epithelia from blood group A or B persons demonstrated A or B antigens, respectively, but only if the tissue sections were trypsinized before staining. In such cases A or B antigens were found...... monoclonal antibodies were used to identify H antigen (type 2 chain) and N-acetyl-lactosamine. Human antisera were used to identify A and B antigens. In all groups N-acetyl-lactosamine and H antigen were found on the cell membranes of the spinous cell layer. N-acetyl-lactosamine was present mainly...

Engineering tolerance using biomaterials to target and control antigen presenting cells.

Science.gov (United States)

Tostanoski, Lisa H; Gosselin, Emily A; Jewell, Christopher M

2016-05-01

Autoimmune diseases occur when cells of the adaptive immune system incorrectly recognize and attack "self" tissues. Importantly, the proliferation and differentiation of these cells is triggered and controlled by interactions with antigen presenting cells (APCs), such as dendritic cells. Thus, modulating the signals transduced by APCs (e.g., cytokines, costimulatory surface proteins) has emerged as a promising strategy to promote tolerance for diseases such as multiple sclerosis, type 1 diabetes, and lupus. However, many approaches have been hindered by non-specific activity of immunosuppressive or immunoregulatory cues, following systemic administration of soluble factors via traditional injections routes (e.g., subcutaneous, intravenous). Biomaterials offer a unique opportunity to control the delivery of tolerogenic signals in vivo via properties such as controlled particle size, tunable release kinetics, and co-delivery of multiple classes of cargo. In this review, we highlight recent reports that exploit these properties of biomaterials to target APCs and promote tolerance via three strategies, i) passive or active targeting of particulate carriers to APCs, ii) biomaterial-mediated control over antigen localization and processing, and iii) targeted delivery of encapsulated or adsorbed immunomodulatory signals. These reports represent exciting advances toward the goal of more effective therapies for autoimmune diseases, without the broad suppressive effects associated with current clinically-approved therapies.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

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Martin Kreutz

Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Novel Pneumocystis Antigen Discovery Using Fungal Surface Proteomics

OpenAIRE

Zheng, Mingquan; Cai, Yang; Eddens, Taylor; Ricks, David M.; Kolls, Jay K.

2014-01-01

Pneumonia due to the fungus Pneumocystis jirovecii is a life-threatening infection that occurs in immunocompromised patients. The inability to culture the organism as well as the lack of an annotated genome has hindered antigen discovery that could be useful in developing novel vaccine- or antibody-based therapies as well as diagnostics for this infection. Here we report a novel method of surface proteomics analysis of Pneumocystis murina that reliably detected putative surface proteins that ...

Kinetics of human T-cell expression of LFA-1, IL-2 receptor, and ICAM-1 following antigenic stimulation in vitro

DEFF Research Database (Denmark)

Hviid, L; Felsing, A; Theander, T G

1993-01-01

-specific stimulation is available. In the present study we have examined phenotypic T-cell changes after in vitro stimulation by the antigens purified derivative of tuberculin (PPD) and tetanus toxoid (TT). We show that the well-established differences in kinetics of mitogen- and antigen-induced T-cell proliferation...... of all 3 surface antigens showed similar kinetics, and correlated with the magnitude of the lymphoproliferative response. By day 8 (PHA-stimulation) or day 12 (PPD or TT stimulation), the lymphoproliferative response was essentially completed, the expression of CD11a and CD54 had approached...

Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

Science.gov (United States)

Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

2012-01-01

Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

DEFF Research Database (Denmark)

Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

2015-01-01

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses....... Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

Prevalence of Hepatitis B surface antigen among pregnant women ...

African Journals Online (AJOL)

Prevalence of Hepatitis B surface antigen among pregnant women attending antenatal ... Majigo Mtebe, Nyambura Moremi, Jeremiah Seni, Stephen E. Mshana. Abstract. In developing countries there is no routine screening of hepatitis B virus ...

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

Science.gov (United States)

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

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Gomez Bianca P

2012-10-01

Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

A population dynamics analysis of the interaction between adaptive regulatory T cells and antigen presenting cells.

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David Fouchet

Full Text Available BACKGROUND: Regulatory T cells are central actors in the maintenance of tolerance of self-antigens or allergens and in the regulation of the intensity of the immune response during infections by pathogens. An understanding of the network of the interaction between regulatory T cells, antigen presenting cells and effector T cells is starting to emerge. Dynamical systems analysis can help to understand the dynamical properties of an interaction network and can shed light on the different tasks that can be accomplished by a network. METHODOLOGY AND PRINCIPAL FINDINGS: We used a mathematical model to describe a interaction network of adaptive regulatory T cells, in which mature precursor T cells may differentiate into either adaptive regulatory T cells or effector T cells, depending on the activation state of the cell by which the antigen was presented. Using an equilibrium analysis of the mathematical model we show that, for some parameters, the network has two stable equilibrium states: one in which effector T cells are strongly regulated by regulatory T cells and another in which effector T cells are not regulated because the regulatory T cell population is vanishingly small. We then simulate different types of perturbations, such as the introduction of an antigen into a virgin system, and look at the state into which the system falls. We find that whether or not the interaction network switches from the regulated (tolerant state to the unregulated state depends on the strength of the antigenic stimulus and the state from which the network has been perturbed. CONCLUSION/SIGNIFICANCE: Our findings suggest that the interaction network studied in this paper plays an essential part in generating and maintaining tolerance against allergens and self-antigens.

Immune responses of dendritic cells after acquiring antigen from apoptotic hepatocholangioma cells caused by γ-ray

International Nuclear Information System (INIS)

Wu Gang; Gu Hongguang; Han Benli; Pei Xuetao

2002-01-01

Objective: To investigate the induction of cytotoxic T lymphocytes (CTLs) in antitumor responsiveness and therapeutic effects after dendritic cells (DCs) acquired antigen from apoptotic hepatocholangioma cells. Methods: DCs from blood mononuclear cells that maintain the characteristics of immaturity-anti-gen-capturing and-processing capacity were established in vitro by using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Then, apoptosis in hepatocholangioma cells was induced with γ-radiation. The experimental groups included (1) co-culture of DCs, and apoptotic cancer cells and T cells; (2) co-culture of DCs necrotic cancer cells and T cells; (3) co-culture of DCs-cultured cancer cell and T cells. These cells were co-cultured for 7 days. DCs and T cell were enriched separately. Finally, antitumor response test was carried out. Results: These cells had typical dendritic morphology, expressed high levels of CD1a, B7 and acquired antigen from apoptotic cells caused by γ-rays and induced an increased T cell-stimulatory capacity in MLR. Conclusions: DCs obtained from blood mononuclear cells using GM-CSF and IL-4 and DCs can efficiently present antigen driven from apoptotic cells caused by γ-rays and induce T cells increasing obviously. It can probably become an effective approach of DC transduction with antigen

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

International Nuclear Information System (INIS)

Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

1986-01-01

In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

International Nuclear Information System (INIS)

Rivera, Julie A.; McGuire, Travis C.

2005-01-01

To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV WSU5 infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with 51 Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection

Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

Science.gov (United States)

Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

2017-11-01

Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Simultaneous detection of Hepatitis B surface antigen and its antibody by radioimmunoassay

International Nuclear Information System (INIS)

Crouzat-Reynes, Gerard; Perigois, Francois; Lecureuil, Michel; Lejeune, Bernard

1981-01-01

The authors describe an original radioimmunoassay which allows the simultaneous detection of hepatitis B surface antigen and its antibody in a biological sample. Antigen and antibody are indiscriminately detected in a first step and then distinguished in a second step using the same reagents [fr

A novel method to generate T-cell receptor-deficient chimeric antigen receptor T cells.

Science.gov (United States)

Kamiya, Takahiro; Wong, Desmond; Png, Yi Tian; Campana, Dario

2018-03-13

Practical methods are needed to increase the applicability and efficacy of chimeric antigen receptor (CAR) T-cell therapies. Using donor-derived CAR-T cells is attractive, but expression of endogenous T-cell receptors (TCRs) carries the risk for graft-versus-host-disease (GVHD). To remove surface TCRαβ, we combined an antibody-derived single-chain variable fragment specific for CD3ε with 21 different amino acid sequences predicted to retain it intracellularly. After transduction in T cells, several of these protein expression blockers (PEBLs) colocalized intracellularly with CD3ε, blocking surface CD3 and TCRαβ expression. In 25 experiments, median TCRαβ expression in T lymphocytes was reduced from 95.7% to 25.0%; CD3/TCRαβ cell depletion yielded virtually pure TCRαβ-negative T cells. Anti-CD3ε PEBLs abrogated TCRαβ-mediated signaling, without affecting immunophenotype or proliferation. In anti-CD3ε PEBL-T cells, expression of an anti-CD19-41BB-CD3ζ CAR induced cytokine secretion, long-term proliferation, and CD19 + leukemia cell killing, at rates meeting or exceeding those of CAR-T cells with normal CD3/TCRαβ expression. In immunodeficient mice, anti-CD3ε PEBL-T cells had markedly reduced GVHD potential; when transduced with anti-CD19 CAR, these T cells killed engrafted leukemic cells. PEBL blockade of surface CD3/TCRαβ expression is an effective tool to prepare allogeneic CAR-T cells. Combined PEBL and CAR expression can be achieved in a single-step procedure, is easily adaptable to current cell manufacturing protocols, and can be used to target other T-cell molecules to further enhance CAR-T-cell therapies. © 2018 by The American Society of Hematology.

Chimeric Antigen Receptor-Engineered T Cells in Tumor Immunotherapy: From Bench to Beside

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Peng WANG

2017-06-01

Full Text Available Chimeric antigen receptor-engineered T cells (CAR-T cells, a classification of cultured T cells after modification of gene engineering technology, can recognize specific tumor antigens in a major histocompatibility complex (MHC-independent manner, consequently leading to the activation of antitumor function. The recent studies have confirmed that a variety of tumor-associated antigens (TAAs can act as target antigens for CAR-T cells. Nowadays, CAR T-cell therapy, one of the most potential tumor immunotherapies, has made great breakthroughs in hematological malignancies and promising outcomes in solid tumors. In this article, the biological characteristics and antitumor mechanism of CAR-T cells, and their application in tumor treatment were mainly reviewed.

Antigen-Specific Polyclonal Cytotoxic T Lymphocytes Induced by Fusions of Dendritic Cells and Tumor Cells

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Shigeo Koido

2010-01-01

Full Text Available The aim of cancer vaccines is induction of tumor-specific cytotoxic T lymphocytes (CTLs that can reduce the tumor mass. Dendritic cells (DCs are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Thus, DCs-based vaccination represents a potentially powerful strategy for induction of antigen-specific CTLs. Fusions of DCs and whole tumor cells represent an alternative approach to deliver, process, and subsequently present a broad spectrum of antigens, including those known and unidentified, in the context of costimulatory molecules. Once DCs/tumor fusions have been infused back into patient, they migrate to secondary lymphoid organs, where the generation of antigen-specific polyclonal CTL responses occurs. We will discuss perspectives for future development of DCs/tumor fusions for CTL induction.

Goblet cells contribute to ocular surface immune tolerance—implications for dry eye disease

NARCIS (Netherlands)

Barbosa, Flavia L.; Xiao, Yangyan; Bian, Fang; Coursey, Terry G.; Ko, Byung Yi; Clevers, Hans; de Paiva, Cintia S.; Pflugfelder, Stephen C.

2017-01-01

Conjunctival goblet cell (GC) loss in dry eye is associated with ocular surface inflammation. This study investigated if conjunctival GCs contribute to ocular surface immune tolerance. Antigens applied to the ocular surface, imaged by confocal microscopy, passed into the conjunctival stroma through

Goblet Cells Contribute to Ocular Surface Immune Tolerance-Implications for Dry Eye Disease

NARCIS (Netherlands)

Barbosa, Flavia L; Xiao, Yangyan; Bian, Fang; Coursey, Terry G; Ko, Byung Yi; Clevers, Hans; de Paiva, Cintia S; Pflugfelder, Stephen C

2017-01-01

Conjunctival goblet cell (GC) loss in dry eye is associated with ocular surface inflammation. This study investigated if conjunctival GCs contribute to ocular surface immune tolerance. Antigens applied to the ocular surface, imaged by confocal microscopy, passed into the conjunctival stroma through

Interaction of Cowpea Mosaic Virus (CPMV) Nanoparticles with Antigen Presenting Cells In Vitro and In Vivo

Science.gov (United States)

Rae, Chris S.; Manchester, Marianne

2009-01-01

Background Plant viruses such as Cowpea mosaic virus (CPMV) are increasingly being developed for applications in nanobiotechnology including vaccine development because of their potential for producing large quantities of antigenic material in plant hosts. In order to improve efficacy of viral nanoparticles in these types of roles, an investigation of the individual cell types that interact with the particles is critical. In particular, it is important to understand the interactions of a potential vaccine with antigen presenting cells (APCs) of the immune system. CPMV was previously shown to interact with vimentin displayed on cell surfaces to mediate cell entry, but the expression of surface vimentin on APCs has not been characterized. Methodology The binding and internalization of CPMV by several populations of APCs was investigated both in vitro and in vivo by flow cytometry and fluorescence confocal microscopy. The association of the particles with mouse gastrointestinal epithelium and Peyer's patches was also examined by confocal microscopy. The expression of surface vimentin on APCs was also measured. Conclusions We found that CPMV is bound and internalized by subsets of several populations of APCs both in vitro and in vivo following intravenous, intraperitoneal, and oral administration, and also by cells isolated from the Peyer's patch following gastrointestinal delivery. Surface vimentin was also expressed on APC populations that could internalize CPMV. These experiments demonstrate that APCs capture CPMV particles in vivo, and that further tuning the interaction with surface vimentin may facilitate increased uptake by APCs and priming of antibody responses. These studies also indicate that CPMV particles likely access the systemic circulation following oral delivery via the Peyer's patch. PMID:19956734

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

International Nuclear Information System (INIS)

Noda, T.; Satake, M.; Robins, T.; Ito, Y.

1986-01-01

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10 6 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Science.gov (United States)

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Natural Killer Cell Characteristics in Patients With Chronic Hepatitis B Virus (HBV) Infection Are Associated With HBV Surface Antigen Clearance After Combination Treatment With Pegylated Interferon Alfa-2a and Adefovir

NARCIS (Netherlands)

Stelma, Femke; de Niet, Annikki; Tempelmans Plat-Sinnige, Marjan J.; Jansen, Louis; Takkenberg, R. Bart; Reesink, Hendrik W.; Kootstra, Neeltje A.; van Leeuwen, Ester M. M.

2015-01-01

The role of natural killer (NK) cells in the process of hepatitis B virus (HBV) surface antigen (HBsAg) clearance and whether their phenotype is related to treatment outcome in patients with chronic hepatitis B are currently unknown. Patients with chronic hepatitis B (HBV DNA load, >17 000 IU/mL)

Cell-surface associated with transformation of human hepatocytes to the malignant phenotype

International Nuclear Information System (INIS)

Wilson, B.; Ozturk, M.; Takahashi, H.; Motte, P.; Kew, M.; Isselbacher, K.J.; Wands, J.R.

1988-01-01

Hepatocellular carcinoma is one of the leading causes of cancer death in the world. To understand the cellular changes associated with transformation of hepatocytes to the malignant state, the authors have made several libraries of monoclonal antibodies against the hepatocellular carcinoma cell line FOCUS and have found six antibodies (AF-20, SF-25, SF-31, SF-90, XF-4, and XF-8) that recognize antigens expressed at consistently higher levels on hepatoma cells. They have studied malignant and nontransformed liver tissue from the same individual by using direct 125 I-labeled antibody binding and immunoperoxidase staining techniques. For each of these antibodies, they found striking increases in antigen expression on the transformed tissues. These antigens were found to be expressed throughout the tumor and on distant metastases, with little, if any, expression on the nontransformed adjacent liver. These antibodies demonstrate that hepatic transformation may be accompanied by stereotyped and predictable antigenic changes. The uniformity of such antigenic changes suggests an association between these cell-surface alterations and the malignant transformation process

Invariant NKT cells are required for airway inflammation induced by environmental antigens.

Science.gov (United States)

Wingender, Gerhard; Rogers, Paul; Batzer, Glenda; Lee, Myung Steve; Bai, Dong; Pei, Bo; Khurana, Archana; Kronenberg, Mitchell; Horner, Anthony A

2011-06-06

Invariant NKT cells (iNKT cells) are a unique subset of T lymphocytes that rapidly carry out effector functions. In this study, we report that a majority of sterile house dust extracts (HDEs) tested contained antigens capable of activating mouse and human iNKT cells. HDEs had adjuvant-like properties in an ovalbumin (OVA)-induced asthma model, which were dependent on Vα14i NKT cells, as vaccinated animals deficient for iNKT cells displayed significantly attenuated immune responses and airway inflammation. Furthermore, the administration of HDEs together with OVA mutually augmented the synthesis of cytokines by Vα14i NKT cells and by conventional CD4(+) T cells in the lung, demonstrating a profound immune response synergy for both Th2 cytokines and IL-17A. These data demonstrate that iNKT cell antigens are far more widely dispersed in the environment than previously anticipated. Furthermore, as the antigenic activity in different houses varied greatly, they further suggest that iNKT cell responses to ambient antigens, particular to certain environments, might promote sensitization to conventional respiratory allergens.

A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

Science.gov (United States)

Miller, A

1977-01-01

The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

Axotomy induces MHC class I antigen expression on rat nerve cells

DEFF Research Database (Denmark)

Maehlen, J; Schröder, H D; Klareskog, L

1988-01-01

Immunomorphological staining demonstrates that class I major histocompatibility complex (MHC)-coded antigen expression can be selectively induced on otherwise class I-negative rat nerve cells by peripheral axotomy. Induction of class I as well as class II antigen expression was simultaneously seen...... on non-neural cells in the immediate vicinity of the injured nerve cells. As nerve regeneration after axotomy includes growth of new nerve cell processes and formation of new nerve cell contacts, the present findings raise the question of a role for MHC-coded molecules in cell-cell interactions during...... nerve cell growth....

Ultraviolet radiation (UVR) induces cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: a possible mechanism for the UVR induction of cutaneous lupus lesions

International Nuclear Information System (INIS)

Jones, S.K.

1992-01-01

Antinuclear antibodies are useful markers of connective tissue disease. In this study, UVB but not UVA induced the expression of Ro/SSA antigen on keratinocyte surfaces in vitro. This expression was also found with the extractable nuclear antigens RnP and Sm, but not with single or double-stranded DNA. The expression was prevented by blocking protein synthesis, suggesting that it was an active process. The results suggest that UVB exposure may result in the expression of Ro/SSA antigen on the surfaces of basal keratinocytes in vivo. This antigen could then bind circulating antibody leading to the cutaneous lesions in neonatal and subacute cutaneous lupus erythematosus. (Author)

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

Science.gov (United States)

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

Chimeric antigen receptors for adoptive T cell therapy in acute myeloid leukemia

Directory of Open Access Journals (Sweden)

Mingxue Fan

2017-08-01

Full Text Available Abstract Currently, conventional therapies for acute myeloid leukemia (AML have high failure and relapse rates. Thus, developing new strategies is crucial for improving the treatment of AML. With the clinical success of anti-CD19 chimeric antigen receptor (CAR T cell therapies against B-lineage malignancies, many studies have attempted to translate the success of CAR T cell therapy to other malignancies, including AML. This review summarizes the current advances in CAR T cell therapy against AML, including preclinical studies and clinical trials, and discusses the potential AML-associated surface markers that could be used for further CAR technology. Finally, we describe strategies that might address the current issues of employing CAR T cell therapy in AML.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Science.gov (United States)

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Analysis of the surface membrane of iodinated leukemic cells by SDS-polyacrylamide gel electrophoresis

International Nuclear Information System (INIS)

Ishitani, Kunihiko; Ikeda, Akira; Tamura, Minoru; Takeuchi, Hidekazu; Ihara, Koji

1980-01-01

Surface proteins of human leukemic cells were labeled selectively by lactoperoxydase catalysed-iodination and examined by SDS-polyacrylamide gel electrophoresis. The electrophoretic pattern of the surface membranes of cells from a patients with chronic mylogeneous leukemia in blast crisis was of B cell type and showed Ia like antigen. Leukemic cells from a patient with hairly cell leukemia also expressed the pattern of B cell type when tested by this method the technique of iodinating cell surface with lactoperoxidase is useful in characterization of leukemia cells for diagnosis and monitoring of clinical course. (author)

Cancer antigens are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid cell line

International Nuclear Information System (INIS)

Shiraishi, Yukimasa; Soma, Hiroaki

1988-01-01

The authors have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the panning method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer

Docking of B-cell epitope antigen to specific hepatitis B antibody

Indian Academy of Sciences (India)

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

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.

Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe

Directory of Open Access Journals (Sweden)

Wang S

2016-01-01

Full Text Available Shuo Wang, Wanming Li, Dezheng Yuan, Jindan Song, Jin Fang Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, People’s Republic of China Abstract: The large external antigen (LEA is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605 conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05, indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of

Classical dendritic cells are required for dietary antigen-mediated peripheral regulatory T cell and tolerance induction

Science.gov (United States)

Esterházy, Daria; Loschko, Jakob; London, Mariya; Jove, Veronica; Oliveira, Thiago Y.; Mucida, Daniel

2016-01-01

Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development. PMID:27019226

Large, but not small, antigens require time- and temperature-dependent processing in accessory cells before they can be recognized by T cells

DEFF Research Database (Denmark)

Buus, S; Werdelin, O

1986-01-01

We have studied if antigens of different size and structure all require processing in antigen-presenting cells of guinea-pigs before they can be recognized by T cells. The method of mild paraformaldehyde fixation was used to stop antigen-processing in the antigen-presenting cells. As a measure...... of antigen presentation we used the proliferative response of appropriately primed T cells during a co-culture with the paraformaldehyde-fixed and antigen-exposed presenting cells. We demonstrate that the large synthetic polypeptide antigen, dinitrophenyl-poly-L-lysine, requires processing. After an initial......-dependent and consequently energy-requiring. Processing is strongly inhibited by the lysosomotrophic drug, chloroquine, suggesting a lysosomal involvement in antigen processing. The existence of a minor, non-lysosomal pathway is suggested, since small amounts of antigen were processed even at 10 degrees C, at which...

Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors.

Science.gov (United States)

Jindal, Vishal; Arora, Ena; Gupta, Sorab

2018-05-05

Chimeric antigen receptor (CAR) T cell therapy is a novel and innovative immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptor and co-stimulatory molecule which can activate an immune response to a cancer specific antigen. This therapy showed great results in hematological malignancies but were unable to prove their worth in solid tumors. Likely reasons for their failure are lack of antigens, poor trafficking, and hostile tumor microenvironment. Excessive amount of research is going on to improve the efficacy of CAR T cell therapy in solid tumors. In this article, we will discuss the challenges faced in improving the outcome of CAR T cell therapy in solid tumors and various strategies adopted to curb them.

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

Science.gov (United States)

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines

Directory of Open Access Journals (Sweden)

Cleo Goyvaerts

2015-01-01

Full Text Available In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

Developmental expression of a cell surface protein involved in sea urchin skeleton formation

International Nuclear Information System (INIS)

Farach, M.C.; Valdizan, M.; Park, H.R.; Decker, G.L.; Lennarz, W.J.

1986-01-01

The authors have previously used a monoclonal antibody (1223) to identify a 130 Kd cell surface protein involved in skeleton formation is sea urchin embryos. In the current study the authors have examined the expression of the 1223 antigen over the course of development of embryos of two species, Strongylocentrotus purpuratus and Lytechinus pictus. The 130 Kd protein is detected in S. purp eggs on immunoblots. Labeling with [ 3 H] leucine and immunoaffinity chromatography show that it also is synthesized shortly after fertilization. Immunofluroescence reveals that at this early stage the 1223 antigen is uniformly distributed on all of the cells. Synthesis decreases to a minimum by the time of hatching (18 h), as does the total amount of antigen present in the embryo. A second period of synthesis commences at the mesenchyme blastula stage, when the spicule-forming primary mesenchyme cells (PMCs) have appeared. During this later stage, synthesis and cell surface expression are restricted to the PMCs. In contrast to S. purp., in L. pictus the 130 Kd protein does not appear until the PMCs are formed. Hybrid embryos demonstrate a pattern of expression of the maternal species. These results suggest that early expression of 1223 antigen in S. purp. is due to utilization of maternal transcripts present in the egg. In both species later expression in PMCs appears to be the result of cell-type specific synthesis, perhaps encoded by embryonic transcripts

Chicken major histocompatibility complex-encoded B-G antigens are found on many cell types that are important for the immune system

DEFF Research Database (Denmark)

Salomonsen, J; Dunon, D; Skjødt, K

1991-01-01

B-G antigens are a polymorphic multigene family of cell surface molecules encoded by the chicken major histocompatibility complex (MHC). They have previously been described only on cells of the erythroid lineage. By using flow cytometry, section staining, and immunoprecipitation with monoclonal a...

Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens

Directory of Open Access Journals (Sweden)

Gao J

2017-02-01

Full Text Available Jie Gao,1–3 Lukasz J Ochyl,1,3 Ellen Yang,4 James J Moon1,3,5 1Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA; 2Department of Pharmaceutical Sciences, School of Pharmacy, Second Military Medical University, Shanghai, People’s Republic of China; 3Biointerfaces Institute, 4Department of Chemistry, 5Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA Abstract: Cationic liposomes (CLs have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs, and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation – the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I. However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs, antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3β-[N-(N',N'-dimethylaminoethane-carbamoyl] cholesterol (DC-Chol and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine

Production of prostate-specific antigen by a breast cancer cell line, Sk-Br-3

International Nuclear Information System (INIS)

Kamali Sarvestani, E.; Ghaderi, A.

2002-01-01

Prostate-specific antigen is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce prostate-specific antigen and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study prostate-specific antigen production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB-2. For this purpose we investigated the ability of prostate-specific antigen production in five different cell lines, including two breast cancer cell lines, Sk-Br-3 and MDA-MB-453. The prostate-specific antigen in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-prostate-specific antigen producer breast cancer cell line, MDA-MB-453. Progesterone receptor was expressed by both prostate-specific antigen-positive and -negative cell lines and only the intensity of staining and the number of positive cells in Sk-Br-3 population was higher than MDA-MB-453. According to our findings prostate-specific antigen can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations

CD8(+)NKT-like cells regulate the immune response by killing antigen-bearing DCs.

Science.gov (United States)

Wang, Chao; Liu, Xi; Li, Zhengyuan; Chai, Yijie; Jiang, Yunfeng; Wang, Qian; Ji, Yewei; Zhu, Zhongli; Wan, Ying; Yuan, Zhenglong; Chang, Zhijie; Zhang, Minghui

2015-09-15

CD1d-dependent NKT cells have been extensively studied; however, the function of CD8(+)NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8(+)NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8(+)NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8(+)NKT-like cell development is normal in CD1d(-/-) mice, which suggests that CD8(+)NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8(+)NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

CD8+NKT-like cells regulate the immune response by killing antigen-bearing DCs

Science.gov (United States)

Wang, Chao; Liu, Xi; Li, Zhengyuan; Chai, Yijie; Jiang, Yunfeng; Wang, Qian; Ji, Yewei; Zhu, Zhongli; Wan, Ying; Yuan, Zhenglong; Chang, Zhijie; Zhang, Minghui

2015-01-01

CD1d-dependent NKT cells have been extensively studied; however, the function of CD8+NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8+NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8+NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8+NKT-like cell development is normal in CD1d−/− mice, which suggests that CD8+NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8+NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8+NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8+NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens. PMID:26369936

Adult T-cell leukemia-associated antigen (ATLA): detection of a glycoprotein in cell- and virus-free supernatant.

Science.gov (United States)

Yamamoto, N; Schneider, J; Hinuma, Y; Hunsmann, G

1982-01-01

A glycoprotein of an apparent molecular mass of 46,000, gp 46, was enriched by affinity chromatography from the virus- and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells. gp 46 was specifically precipitated with sera from patients with adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.

Immunohistochemical detection and correlation between MHC antigen and cell-mediated immune system in recurrent glioma by APAAP method.

Science.gov (United States)

Miyagi, K; Ingram, M; Techy, G B; Jacques, D B; Freshwater, D B; Sheldon, H

1990-09-01

As part of an on-going clinical trial of immunotherapy for recurrent malignant gliomas, using alkaline phosphatase-anti-alkaline phosphatase method with monoclonal antibodies, we investigated the correlation between expression of the major histocompatibility complex (MHC) and the subpopulation of tumor-infiltrating lymphocytes (TILs) in 38 glioma specimens (20 grade IV, 11 grade III, and 7 grade II) from 33 patients. Thirty specimens (78.9%) were positive to class I MHC antigen and 20 (52.6%) were positive to class II MHC antigen. The correlations between class I MHC antigen expression and the number of infiltrating T8 (p less than 0.01), and also between class II MHC antigen expression and the number of infiltrating T4 (p less than 0.05) were significant. We conclude that TILs are the result of immunoreaction (host-defense mechanism). 31.6% of specimens had perivascular infiltration of T cells. The main infiltrating lymphocyte subset in moderate to marked perivascular cuffing was T4. Our results may indicate that lack of MHC antigen on the glioma cell surface has a share in the poor immunogenicity in glioma-bearing patients. In addition, considering the effector/target ratio, the number of infiltrating lymphocytes against glioma cells was too small, so the immunological intervention seems to be essential in glioma therapy. Previous radiation therapy and chemotherapy, including steroid therapy, did not influence lymphocyte and macrophage infiltration.

Evidence against suppressor cell involvement in naturally acquired tolerance of a minor histocompatibility antigen

International Nuclear Information System (INIS)

Johnson, L.L.

1991-01-01

The hypothesis was investigated that suppressor cells may be responsible for maintenance of immunologic tolerance of a minor H3 antigen in mice that express the antigen naturally. Lymphoid cell populations from B6.C-H-24c (HW54) mice, a congenic-resistant strain histoincompatible with H-24b-expressing C57BL/6 (B6) mice only with respect to the H-24 locus, were examined in cell-transfer experiments to see if they contained naturally arising H-24c-specific suppressor cells. The H-24 antigen was chosen for these studies because, unlike most other minor and major histocompatibility (H) antigens, it is not detectable on mature lymphoid cells by any of several functional criteria. Thus transfer of HW54 lymphoid cells to B6 hosts could be done without the complication of inducing hyporesponsiveness de novo in the host, as occurs with other minor H antigens that are expressed on lymphocytes. B6 hosts were given HW54 skin grafts along with HW54 lymphoid cells to assess their tolerance of the H-24c-encoded antigen. The hosts were either (1) normal, nonimmune B6 mice; (2) B6 mice rendered immunodeficient by thymectomy and irradiation (TxB) and repopulated with H-24c-immune B6 lymphocytes; or (3) TxB B6 hosts repopulated with nonimmune B6 lymphocytes. In each case it was found that the additionally infused HW54 lymphoid cells did not suppress the ability of these hosts to reject HW54 skin grafts. In other words, HW54 lymphoid cells appear not to possess suppressive activity specific for the H-24c antigen that might maintain antigen-specific natural tolerance. Additional experiments were performed to determine whether HW54 lymphoid cells can inhibit the ability of sublethally irradiated B6 mice to regain the capacity to reject HW54 skin

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

Science.gov (United States)

Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

2009-10-01

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Prevalence of Hepatitis B surface antigen in children with sickle cell anemia

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Baba Jibrin

2014-01-01

Full Text Available Background: Hepatitis B virus is known to be endemic in Africa. The seroepidemiological studies of HBV have shown that infection commonly occurs in childhood in Africa resulting in an increased tendency to chronicity. This cross-sectional study was undertaken to determine the seroprevalence of hepatitis B surface antigen among pediatric patients with homozygous hemoglobin S. Materials and Methods: Three hundred sickle cell anemia children aged 6 months-15 years (both in steady state and in crises attending the SCA clinic and on admission in emergency pediatrics unit and pediatrics medical ward, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria, were screened for hepatitis B infection using HBsAg as marker of infection. The sensitive enzyme linked immunosorbent assay method was used for detection of the marker. Three hundred children with minor illness attending pediatrics outpatient department and on admission in EPU/PMW for various treatment in the same hospital served as gender- and age-marched controls cohorts. Results: The sero-prevalence of HBsAg seropositivity for hepatitis B virus infection among SCA children was 17.3% (52/300 compared to 10.7% (32/300 of the control (P = 0.0875. The peak prevalence age group for HBV infection among SCA children was in the age group 1.1-5.0 years (6% compared to 10.1-15.0 years (4.7% in the control. Risk factors for HBV infection such as blood transfusion, traditional scarification/circumcision/uvulectomy, and tattooing did not significantly affect the prevalence of HBV infection in both SCA children and controls. Conclusion: Hepatitis B infection is common in Sokoto. The need for strict adherence to HBV immunization and further community-based studies on the risk factors are recommended.

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

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Jennifer Gordon

Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

The distribution of blood group antigens in experimentally produced carcinomas of rat palate

DEFF Research Database (Denmark)

Reibel, J; Philipsen, H P; Fisker, A V

1986-01-01

palate induced by a chemical carcinogen (4NQO). The H antigen, normally expressed on spinous cells in rats, was absent in malignant epithelium, whereas staining for the B antigen, normally expressed on basal cells, was variable. These changes are equivalent to those seen in human squamous cell carcinomas....... The blood group antigen staining pattern in experimentally produced verrucous carcinomas showed an almost normal blood group antigen expression. This may have diagnostic significance. Localized areas of hyperplastic palatal epithelium with slight dysplasia revealed loss of H antigen and the presence of B...... antigen in suprabasal strata equivalent to the pattern seen in human premalignant epithelium. We conclude from these findings, that the rat model is well suited to study changes in cell surface carbohydrates during chemical carcinogenesis....

Co-delivery of antigen and a lipophilic anti-inflammatory drug to cells via a tailorable nanocarrier emulsion.

Science.gov (United States)

Chuan, Yap Pang; Zeng, Bi Yun; O'Sullivan, Brendan; Thomas, Ranjeny; Middelberg, Anton P J

2012-02-15

Nanotechnology promises new drug carriers that can be tailored to specific applications. Here we report a new approach to drug delivery based on tailorable nanocarrier emulsions (TNEs), motivated by a need to co-deliver a protein antigen and a lipophilic drug for specific inhibition of nuclear factor kappa B (NF-κB) in antigen presenting cells (APCs). Co-delivery for NF-κB inhibition holds promise as a strategy for the treatment of rheumatoid arthritis. We used a highly surface-active peptide (SAP) to prepare a nanosized emulsion having defined surface properties predictable from the SAP sequence. Incorporating the lipophilic drug into the oil phase at the time of emulsion formation enabled its facile packaging. The SAP is depleted from bulk during emulsification, allowing simple subsequent addition of the drug-loaded oil-in-water emulsion to a solution of protein antigen. Decoration of emulsion surface with antigen was achieved via electrostatic deposition. In vitro data showed that the TNE prepared this way was internalized and well-tolerated by model APCs, and that good suppression of NF-κB expression was achieved. This work reports a new type of nanotechnology-based carrier, a TNE, which can potentially be tailored for co-delivery of multiple therapeutic components, and can be made using simple methods using only biocompatible materials. Copyright © 2011 Elsevier Inc. All rights reserved.

Studies on the surface antigenicity and susceptibility to antibody-dependent killing of developing schistosomula using sera from chronically infected mice and mice vaccinated with irradiated cercariae

International Nuclear Information System (INIS)

Bickle, Q.D.; Ford, M.J.

1982-01-01

Changes in the surface antigenicity and susceptibility to in vitro killing during development of schistosomula of Schistosoma mansoni were studied using serum from chronically infected mice (CIS) and from mice vaccinated with highly irradiated (20 krad) cercariae (VS). Binding of these sera was quantitated by counting the number of P388D 1 cells (a transformed, macrophage-like cell of mouse origin, bearing Fc receptors for IgG) binding to the parasite surface. Compared with schistosomula derived in vitro by mechanical transformation (MS), schistosomula recovered 3 hr after skin penetration in vitro (SS) showed a significant loss in surface binding of CIS. Schistosomula recovered 3 hr after skin penetration in vivo (SRS) showed even less binding, and this trend continued such that parasites recovered from the lungs 5 days after infection (LS) showed only minimal binding, and 10-day-old worms from the portal system showed no significant binding. In contrast, VS, which bound significantly less well to MS than CIS, showed enhanced binding to SS, and in the face of their declining antigenicity with respect to CIS, 3- to 24-hr SRS maintained this raised level of antigenicity. Although there appeared to be a decline in binding of VS thereafter, LS remained antigenic, still binding as many cells as MS did despite the fact that they also expressed host antigens detected usng antisera raised against mouse RBC. In spite of this persistence of VS binding up to the lung stage, resistance to eosinophil-mediated killing in vitro had developed by 48 hr post-infection, and LS were totally resistant to both eosinophil- and C-mediated killing

Studies on antigenic competition. Efforts to identify the cellular basis of competition using a cell transfer system

International Nuclear Information System (INIS)

McArthur, W.P.; Siskind, G.W.; Thorbecke, G.J.

1974-01-01

Antigenic competition was studied in a cell transfer system. The effect of reconstituting lethally irradiated mice with various numbers of thymus or bone marrow cells on the extent of antigenic competition was evaluated. Brucella or burro erythrocytes both caused inhibition of the immune response (Plaque-forming cell) to sheep erythrocytes when given two days prior to the test antigen. Varying the dose of thymus cells, or of bone marrow cells did not alter the degree of competition. Competition was observed even when the competing antigen was injected with bone marrow cells alone two days before the test antigen and thymus cells. The results suggest that mechanisms other than T-cell activation alone must be considered as operative in some models of antigenic competition

Allopurinol reduces antigen-specific and polyclonal activation of human T cells

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Damián ePérez-Mazliah

2012-09-01

Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

Science.gov (United States)

Gardell, Jennifer L; Parker, David C

2017-01-01

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.

Transfer plate radioassay using cell monolayers to detect anti-cell surface antibodies synthesized by lymphocyte hybridomas

International Nuclear Information System (INIS)

Schneider, M.D.; Eisenbarth, G.S.

1979-01-01

A solid phase [ 125 I] Protein A radioassay for anti-cell surface antibodies is described, which employs target cell monolayers cultured on fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plate are lowered into receptacles containing washing buffer on test sera. To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired tissue specificity. (Auth.)

Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses

NARCIS (Netherlands)

Dolen, Y.; Kreutz, M.; Gileadi, U.; Tel, J.; Vasaturo, A.; Dinther, E.A.W. van; Hout-Kuijer, M.A. van; Cerundolo, V.; Figdor, C.G.

2016-01-01

Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here,

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

Science.gov (United States)

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

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Thompson Joanne

2007-05-01

Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

Novel Antitumor Strategy Utilizing a Plasmid Expressing a Mycobacterium tuberculosis Antigen as a “Danger Signal” to Block Immune Escape of Tumor Cells

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Yoshiyuki Koyama

2015-07-01

Full Text Available Immune escape of tumor cells is one of the main obstacles hindering the effectiveness of cancer immunotherapy. We developed a novel strategy to block immune escape by transfecting tumor cells in vivo with genes of pathogenic antigens from Mycobacterium tuberculosis (TB. This induces presentation of the TB antigen on tumor cell surfaces, which can be recognized by antigen presenting cells (APCs as a “danger signal” to stimulate antitumor immune response. This strategy is also expected to amplify the immune response against tumor-associated antigens, and block immune escape of the tumor. DNA/PEI/chondroitin sulfate ternary complex is a highly effective non-viral gene vector system for in vivo transfection. A therapeutic complex was prepared using a plasmid encoding the TB antigen, early secretory antigenic target-6 (ESAT-6. This was injected intratumorally into syngeneic tumor-bearing mice, and induced significant tumor growth suppression comparable to or higher than similar complexes expressing cytokines such as interleukin-2 (IL-2 and interleukin-12 (IL-12. Co-transfection of the cytokine-genes and the ESAT-6-gene enhanced the antitumor efficacy of either treatment alone. In addition, complete tumor regression was achieved with the combination of ESAT-6 and IL-2 genes.

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

Science.gov (United States)

Schmitz, H

1981-01-01

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

The role of Plasmodium falciparum variant surface antigens in protective immunity and vaccine development

DEFF Research Database (Denmark)

Hviid, Lars

2010-01-01

There is substantial immuno-epidemiological evidence that the parasite-encoded, so-called variant surface antigens (VSAs) such as PfEMP1 on the surface of infected erythrocytes (IEs) are important-in some cases probably decisive-determinants of clinical outcome of P. falciparum malaria. The evide...... of VSAs, and how vaccines based on this type of antigens fit into the current global strategy to reduce, eliminate and eventually eradicate the burden of malaria....

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

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Craig A Gedye

Full Text Available Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Science.gov (United States)

Gedye, Craig A; Hussain, Ali; Paterson, Joshua; Smrke, Alannah; Saini, Harleen; Sirskyj, Danylo; Pereira, Keira; Lobo, Nazleen; Stewart, Jocelyn; Go, Christopher; Ho, Jenny; Medrano, Mauricio; Hyatt, Elzbieta; Yuan, Julie; Lauriault, Stevan; Meyer, Mona; Kondratyev, Maria; van den Beucken, Twan; Jewett, Michael; Dirks, Peter; Guidos, Cynthia J; Danska, Jayne; Wang, Jean; Wouters, Bradly; Neel, Benjamin; Rottapel, Robert; Ailles, Laurie E

2014-01-01

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

NARCIS (Netherlands)

Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

2012-01-01

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is

Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

OpenAIRE

Weerkamp, Anton H.; Jacobs, Ton

1982-01-01

Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel el...

Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

Science.gov (United States)

Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

2001-10-01

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

Do ABO blood group antigens hamper the therapeutic efficacy of mesenchymal stromal cells?

Science.gov (United States)

Moll, Guido; Hult, Annika; von Bahr, Lena; Alm, Jessica J; Heldring, Nina; Hamad, Osama A; Stenbeck-Funke, Lillemor; Larsson, Stella; Teramura, Yuji; Roelofs, Helene; Nilsson, Bo; Fibbe, Willem E; Olsson, Martin L; Le Blanc, Katarina

2014-01-01

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

Prevalence of hepatitis b virus surface antigens (HBsag) and ...

African Journals Online (AJOL)

The prevalences of hepatitis B virus surface antigen (HBsAg) and hepatitis C virus (HCV) antibodies were determined in 560 blood donors sera using ELISA kits (DIALAB., Austria). Forty eight (8.57%) of these were positive for hepatitis B virus infection, while 33(5.89%) were positive to hepatitis C virus antibodies. The sex ...

Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

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Toshiki Ochi

2010-01-01

Full Text Available The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1, and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

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Seydoux E

2014-08-01

Full Text Available Emilie Seydoux,1,2 Barbara Rothen-Rutishauser,1,3 Izabela M Nita,1 Sandor Balog,3 Amiq Gazdhar,1 Philip A Stumbles,4,5 Alke Petri-Fink,3,6 Fabian Blank,1,* Christophe von Garnier1,*1Department of Respiratory Medicine, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; 3Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 4School of Veterinary and Life Sciences, Molecular and Biomedical Sciences, Murdoch University, Perth, WA, Australia; 5Telethon Kids Institute, Perth, WA, Australia; 6Department of Chemistry, University of Fribourg, Fribourg, Switzerland*These authors contributed equally to the manuscriptIntroduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrow–derived DCs (BMDCs were exposed in vitro to 20 or 1,000 nm polystyrene (PS particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particle–positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity

Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity.

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Janin Chandra

Full Text Available Antigen presenting cells (APCs in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.

Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

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Benencia Fabian

2008-04-01

Full Text Available Abstract Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV B radiation using a convenient tumor model expressing human papilloma virus (HPV E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.

Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

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Sergio Cepeda

2018-01-01

Full Text Available Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs, such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

Skin-Resident T Cells Drive Dermal Dendritic Cell Migration in Response to Tissue Self-Antigen.

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Ali, Niwa; Zirak, Bahar; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

2018-05-01

Migratory dendritic cell (DC) subsets deliver tissue Ags to draining lymph nodes (DLNs) to either initiate or inhibit T cell-mediated immune responses. The signals mediating DC migration in response to tissue self-antigen are largely unknown. Using a mouse model of inducible skin-specific self-antigen expression, we demonstrate that CD103 + dermal DCs (DDCs) rapidly migrate from skin to skin DLN (SDLNs) within the first 48 h after Ag expression. This window of time was characterized by the preferential activation of tissue-resident Ag-specific effector T cells (Teffs), with no concurrent activation of Ag-specific Teffs in SDLNs. Using genetic deletion and adoptive transfer approaches, we show that activation of skin-resident Teffs is required to drive CD103 + DDC migration in response to tissue self-antigen and this Batf3-dependent DC population is necessary to mount a fulminant autoimmune response in skin. Conversely, activation of Ag-specific Teffs in SDLNs played no role in DDC migration. Our studies reveal a crucial role for skin-resident T cell-derived signals, originating at the site of self-antigen expression, to drive DDC migration during the elicitation phase of an autoimmune response. Copyright © 2018 by The American Association of Immunologists, Inc.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

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Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

Antigen-Specific lgA B Memory Cell Responses to Shigella Antigens Elicited in Volunteers Immunized with Live Attenuated Shigella flexneri 2a Oral Vaccine Candidates

Science.gov (United States)

2011-01-01

167. [10] E.V. Oaks, T.L. Hale, S.B. Formal, Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella ...cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates J.K. Simona,b... Shigella ;. B cell memory; Immunoglobulin lgA; Mucosal immunity Abstract We studied the induction of antigen-specific lgA memory B cells (BM) in

The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell

DEFF Research Database (Denmark)

Møller, Morten; Rath, Martin F; Klein, David C

2006-01-01

The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal g...... for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

Regional variations of cell surface carbohydrates in human oral stratified epithelium

DEFF Research Database (Denmark)

Vedtofte, P; Dabelsteen, Erik; Hakomori, S

1984-01-01

The distribution of blood group carbohydrate chains with antigen A, B, H type 2 chain (A and B precursor), and N-acetyllactosamine (H type 2 precursor) specificity was studied in human oral epithelium from different anatomical regions. These represented various epithelial differentiation patterns...... epithelium from nine blood group A, two blood group B, and nine blood group O individuals. The blood group carbohydrate chains were examined in tissue sections by immunofluorescence microscopy. The A and B blood group antigens were detected by human blood group sera, and antigen H type 2 chains and N...... antigen H type 2 chains in metaplastically keratinized buccal epithelium was found to differ significantly from that seen in normal non-keratinized buccal epithelium. The regional variations demonstrated in cell surface carbohydrates are suggested to reflect differences in tissue differentiation....

Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

NARCIS (Netherlands)

Ananias, Hildo J. K.; van den Heuvel, Marius C.; Helfrich, Wijnand; de Jong, Igle J.

2009-01-01

OBJECTIVE. Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

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Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Stem Cell Physics. Laser Manipulation of Blood Types: Laser-Stripping-Away of Red Blood Cell Surface Antigens

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Stefan, V. Alexander

2014-03-01

A novel mechanism of importance for the transfusion medicine[2] is proposed. The interaction of ultrashort wavelength multilaser beams with the flowing blood thin films can lead to a conversion of blood types A, B, and AB into O type.[3] The stripping away of antigens is done by the scanning-multiple-lasers of a high repetition rate in the blue-purple frequency domain. The guiding-lasers are in the red-green frequency domain. The laser force, (parametric interaction with the antigen eigen-oscillation),[4] upon the antigen protein molecule must exceed its weight. Supported by Nikola Tesla Labs, La Jolla, CA.

Treatment of Schistosoma mansoni with miltefosine in vitro enhances serological recognition of defined worm surface antigens.

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Marwa H El-Faham

2017-08-01

Full Text Available Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro.In an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH. Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA, Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection.This study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ, whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug's anti-parasite efficacy and be involved in the development

Human Parvovirus B19 Induced Apoptotic Bodies Contain Altered Self-Antigens that are Phagocytosed by Antigen Presenting Cells

Science.gov (United States)

Thammasri, Kanoktip; Rauhamäki, Sanna; Wang, Liping; Filippou, Artemis; Kivovich, Violetta; Marjomäki, Varpu; Naides, Stanley J.; Gilbert, Leona

2013-01-01

Human parvovirus B19 (B19V) from the erythrovirus genus is known to be a pathogenic virus in humans. Prevalence of B19V infection has been reported worldwide in all seasons, with a high incidence in the spring. B19V is responsible for erythema infectiosum (fifth disease) commonly seen in children. Its other clinical presentations include arthralgia, arthritis, transient aplastic crisis, chronic anemia, congenital anemia, and hydrops fetalis. In addition, B19V infection has been reported to trigger autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. However, the mechanisms of B19V participation in autoimmunity are not fully understood. B19V induced chronic disease and persistent infection suggests B19V can serve as a model for viral host interactions and the role of viruses in the pathogenesis of autoimmune diseases. Here we investigate the involvement of B19V in the breakdown of immune tolerance. Previously, we demonstrated that the non-structural protein 1 (NS 1) of B19V induces apoptosis in non-permissive cells lines and that this protein can cleave host DNA as well as form NS1-DNA adducts. Here we provide evidence that through programmed cell death, apoptotic bodies (ApoBods) are generated by B19V NS1 expression in a non-permissive cell line. Characterization of purified ApoBods identified potential self-antigens within them. In particular, signature self-antigens such as Smith, ApoH, DNA, histone H4 and phosphatidylserine associated with autoimmunity were present in these ApoBods. In addition, when purified ApoBods were introduced to differentiated macrophages, recognition, engulfment and uptake occurred. This suggests that B19V can produce a source of self-antigens for immune cell processing. The results support our hypothesis that B19V NS1-DNA adducts, and nucleosomal and lysosomal antigens present in ApoBods created in non-permissive cell lines, are a source of self-antigens. PMID:23776709

Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

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Kerstin Trautwein-Weidner

2015-10-01

Full Text Available Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.

Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen

NARCIS (Netherlands)

ter Borg, F.; ten Kate, F. J.; Cuypers, H. T.; Leentvaar-Kuijpers, A.; Oosting, J.; Wertheim-van Dillen, P. M.; Honkoop, P.; Rasch, M. C.; de Man, R. A.; van Hattum, J.; Chamuleau, R. A.; Reesink, H. W.; Jones, E. A.

1998-01-01

BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA

Antigen presenting cells costimulatory signaling during pre-implantation pregnancy 

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Anna Sławek

2012-09-01

Full Text Available  Success of pregnancy depends on many factors. Three phenomena inducing immune tolerance against semi-allogeneic conceptus may play a crucial role in the pre-implantation period of pregnancy: influence of sex hormones in sex cycle, presence of oocyte or embryo and the presence of semen in the female reproductive tract. On the other hand dendritic cells are the most effective antigen-presenting cells in regulation of immune phenomena and also are considered as potent participants in inducing immune tolerance in the pregnancy. They communicate with T cells in cell contact-dependent manner or via cytokines. During cell-cell contacts, costimulatory molecules play a key role and their expression is often dependent on cytokines milieu. Both costimulatory molecules and cytokines influence generation of T regulatory cells. Interactions of these molecules are closely related. In this paper we would like to pay attention to the importance of antigen presenting cells costimulatory potency in immune regulation during a pre-implantation period of pregnancy.

A rhamnose-rich O-antigen mediates adhesion, virulence, and host colonization for the xylem-limited phytopathogen Xylella fastidiosa.

Science.gov (United States)

Clifford, Jennifer C; Rapicavoli, Jeannette N; Roper, M Caroline

2013-06-01

Xylella fastidiosa is a gram-negative, xylem-limited bacterium that causes a lethal disease of grapevine called Pierce's disease. Lipopolysaccharide (LPS) composes approximately 75% of the outer membrane of gram-negative bacteria and, because it is largely displayed on the cell surface, it mediates interactions between the bacterial cell and its surrounding environment. LPS is composed of a conserved lipid A-core oligosaccharide component and a variable O-antigen portion. By targeting a key O-antigen biosynthetic gene, we demonstrate the contribution of the rhamnose-rich O-antigen to surface attachment, cell-cell aggregation, and biofilm maturation: critical steps for successful infection of the host xylem tissue. Moreover, we have demonstrated that a fully formed O-antigen moiety is an important virulence factor for Pierce's disease development in grape and that depletion of the O-antigen compromises its ability to colonize the host. It has long been speculated that cell-surface polysaccharides play a role in X. fastidiosa virulence and this study confirms that LPS is a major virulence factor for this important agricultural pathogen.

A novel and effective cancer immunotherapy mouse model using antigen-specific B cells selected in vitro.

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Tatsuya Moutai

Full Text Available Immunotherapies such as adoptive transfer of T cells or natural killer cells, or monoclonal antibody (MoAb treatment have recently been recognized as effective means to treat cancer patients. However, adoptive transfer of B cells or plasma cells producing tumor-specific antibodies has not been applied as a therapy because long-term culture and selective expansion of antigen-specific B cells has been technically very difficult. Here, we describe a novel cancer immunotherapy that uses B-cell adoptive transfer. We demonstrate that germinal-center-like B cells (iGB cells induced in vitro from mouse naïve B cells become plasma cells and produce IgG antibodies for more than a month in the bone marrow of non-irradiated recipient mice. When transferred into mice, iGB cells producing antibody against a surrogate tumor antigen suppressed lung metastasis and growth of mouse melanoma cells expressing the same antigen and prolonged survival of the recipients. In addition, we have developed a novel culture system called FAIS to selectively expand antigen-specific iGB cells utilizing the fact that iGB cells are sensitive to Fas-induced cell death unless their antigen receptors are ligated by membrane-bound antigens. The selected iGB cells efficiently suppressed lung metastasis of melanoma cells in the adoptive immunotherapy model. As human blood B cells can be propagated as iGB cells using culture conditions similar to the mouse iGB cell cultures, our data suggest that it will be possible to treat cancer-bearing patients by the adoptive transfer of cancer-antigen-specific iGB cells selected in vitro. This new adoptive immunotherapy should be an alternative to the laborious development of MoAb drugs against cancers for which no effective treatments currently exist.

IL-4Rα-associated antigen processing by B cells promotes immunity in Nippostrongylus brasiliensis infection.

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William G C Horsnell

2013-10-01

Full Text Available In this study, B cell function in protective T(H2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα⁻/⁻ mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4⁺ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88⁻/⁻ B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4⁺ T cell-mediated protective immunity against N. brasiliensis infection.

Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model

DEFF Research Database (Denmark)

Foged, Camilla; Brodin, Birger; Frøkjær, Sven

2005-01-01

Current vaccine development includes optimization of antigen delivery to antigen presenting cells, such as dendritic cells (DC). Particulate systems have attracted increasing attention in the development of vaccine delivery systems. In the present study, we investigated DC uptake of model...... fluorescent polystyrene particles with a broad size range and variable surface properties. Localization of particles was investigated using confocal laser scanning microscopy and uptake was quantified by flow cytometry. Immature DC were generated from mononuclear cells isolated from human blood...

Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells

Science.gov (United States)

Boivin, Nicolas; Baillargeon, Joanie; Doss, Prenitha Mercy Ignatius Arokia; Roy, Andrée-Pascale; Rangachari, Manu

2015-01-01

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals. PMID:25885435

Dissection and manipulation of antigen-specific T cell responses

NARCIS (Netherlands)

Schepers, Koen

2006-01-01

T cells recognize pathogen-derived antigens and are crucial for fighting pathogens such as viruses and bacteria. In addition, T cells are able to recognize and attack certain types of tumors, in particular virally induced tumors. In this thesis we aimed 1) to obtain more insight into

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

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Kurosawa Nobuyuki

2012-09-01

Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression.

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Chikara Furusawa

Full Text Available The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.

Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression.

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Furusawa, Chikara; Yamaguchi, Tomoyuki

The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.

Functional antigen binding by the defective B cells of CBA/N mice.

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Snippe, H; Merchant, B; Lizzio, E F; Inman, J K

1982-01-01

CBA/N mice have an X-linked B cell defect which prevents them from responding to nonmitogenic thymic independent (TI-2) antigens such as dinitrophenylated DNP-Ficoll (1,2). The F1 male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F1 males) could not respond to DNP-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F1 male recipients as expected. In contrast, normal CBA/N X C3H/HeN F1 female spleen cells could respond and effect a "rescue"; they mounted strong plaque-forming cell responses 7 days after in vitro exposure to DNP-Ficoll and subsequent transfer into irradiated F1 male recipients. Defective F1 male spleen cells, however, could bind significant quantities of 125I-DNP-Ficoll after in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-Ficoll-exposed and washed F1 male spleen cells could, after transfer, aid normal untreated F1 female cells in their rescue function. The defective F1 male spleen cells could convey immunogenic quantities of DNP-Ficoll to the "rescuing" F1 female cells. Mitomycin treatment of F1 male cells did not interfere with their conveyor function. Goat anti-mouse mu serum impeded the passive antigen conveyor function of defective F1 male cells as did prior exposure to high concentrations of free DNP hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F1 male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.

Quantitative analysis of rat Ig (sub)classes binding to cell surface antigens

International Nuclear Information System (INIS)

Nilsson, R.; Brodin, T.; Sjoegren, H.-O.

1982-01-01

An indirect 125 I-labeled protein A assay for detection of cell surface-bound rat immunoglobulins is presented. The assay is quantitative and rapid and detects as little as 1 ng of cell surface-bound Ig. It discriminates between antibodies belonging to different IgG subclasses, IgM and IgA. The authors describe the production and specificity control of the reagents used and show that the test can be used for quantitative analysis. A large number of sera from untreated rats are tested to evaluate the frequency of falsely positive responses and variation due to age, sex and strain of rat. With this test it is relatively easy to quantitate the binding of classes and subclasses of rat immunoglobulins in a small volume (6 μl) of untreated serum. (Auth.)

Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals.

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Benson, Micah J; Elgueta, Raul; Schpero, William; Molloy, Michael; Zhang, Weijun; Usherwood, Edward; Noelle, Randolph J

2009-08-31

The hypothesis that bystander inflammatory signals promote memory B cell (B(MEM)) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220(+)IgG(+) B(MEM) toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent B(MEM) clonally expand. Surprisingly, proliferating B(MEM) do not acquire germinal center (GC) B cell markers before generating daughter B(MEM) and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of B(MEM) proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of B(MEM) occurred. The absence of a B(MEM) response to nonspecific inflammatory signals clearly shows that B(MEM) proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

Three-dimensional structure of a glycosylated cell surface antigen from D. discoideum: a primordial adhesion motif

International Nuclear Information System (INIS)

Mabbutt, B.C.; Swarbrick, J.; Cubeddu, L.; Hill, A.

1999-01-01

Full text: We have determined the solution structure of pre-spore specific antigen (PsA), a predominant cell surface glycoprotein from the slime mould Dictyostelium discoideum. The structure and function of this protein suggests that it serves as a molecular signal for multicellular organisation, and that it may also be an adhesion motif mediating direct cell-cell contact. PsA consists of a 90-residue N-terminal globular domain tethered to the cell membrane via a heavily O-glycosylated stalk and a GPI anchor. No homologous sequences have been identified for the N-terminal domain. At Macquarie University, the D. discoideum organism has been well developed as a eukaryotic expression host for glycosylated proteins. For NMR, we have engineered a soluble form of PsA (residues 1-122) containing the globular 'head' and the glycopeptide linker. 15 N- and 15 N/ 13 C-labelled PsA was generated in this organism via a protocol that is readily adaptable for the cost-effective production of milligram quantities of other isotopically labelled recombinant proteins. Using 3D heteronuclear NMR, we have solved the three-dimensional structure of the PsA glycoprotein. It defines an eight stranded β-sandwich of five-on-three topology in a unique arrangement. A long loop is constrained by a cis proline residue and a disulphide bond to form an opening across one end of the sandwich, exposing portions of the hydrophobic interior. We postulate that this distortion of the sandwich fold structures a binding site. Structural and dynamics information was also obtained concerning the intact glycopeptide linker of the protein, which comprises a repeating P-T-V-T motif. In our recombinant form, each Thr residue is modified by a single GlcNAc sugar. This simple structure yields interpretable NMR spectra, which show the glycosylated linker to be in extended conformation, and undergoing distinctly different mobility from the globular domain. These same sugar residues provide an ideal attachment

Use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels

Energy Technology Data Exchange (ETDEWEB)

McMichael, J C; Greisiger, L M; Millman, I [Institute for Cancer Research, Philadelphia, PA (USA). Fox Chase Cancer Center

1981-08-28

Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). /sup 125/I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 ..mu..l or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, the authors observed two bands of HBsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck hepatitis virus surface antigen (WHsAg).

Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

Science.gov (United States)

Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

2016-11-01

To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p exosomes significantly suppressed (p exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

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.

The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells.

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Nadja Kettern

Full Text Available The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS. Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs. CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA. On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS in other mammalian cell types.

Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells

International Nuclear Information System (INIS)

Suhrbier, A.; Winger, L.A.; Castellano, E.; Sinden, R.E.

1990-01-01

Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

International Nuclear Information System (INIS)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

2016-01-01

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

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Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

2016-04-22

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

Modulation of antigen presenting cell functions during chronic HPV infection

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Abate Assefa Bashaw

2017-12-01

Full Text Available High-risk human papillomaviruses (HR-HPV infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity.

77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

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2012-01-24

... Exclusive License: Development of T Cell Receptors and Chimeric Antigen Receptors Into Therapeutics for.... 61/473,409 entitled ``Anti-epidermal growth factor receptor variant III chimeric antigen receptors... EGFRvIII chimeric antigen (CARs) and methods of using these engineered T cells to treat and/or prevent...

Antigen-specific T8+ human clone of cells with a nonspecific augmenting function on the T4 cell-B cell helper interaction

International Nuclear Information System (INIS)

Brines, R.D.; Sia, D.Y.; Lehner, T.

1987-01-01

The authors isolated a T8 + T3 + Ia + clone of cells from the peripheral blood mononuclear cells of a healthy subject. The clone was expanded and maintained with autologous feed cells, interleukin 2, and a streptococcal antigen. The T8 + clone of cells responded specifically to the streptococcal antigen, in the absence of accessory cells,and released a soluble factor. Both the cloned cells and the corresponding soluble factor expressed augmenting helper but not suppressor activity. The augmenting helper activity for B cell antibody synthesis was demonstrable only in the presence of autologous T 4 cells. Radioimmunoassay was used to measure antibodies. Although stimulation of the T8 + cloned cells was antigen-specific, the resulting soluble factor elicited nonspecific antibody synthesis in the presence of T4 and B cells. The T8 + cloned cell-derived factor was adsorbed by B cells but not by T4 cells. Preliminary studies suggest that the factor has the properties of a B cell growth factor. They suggest that the T8 + population consists of functionally heterogeneous cell subsets, some that have suppressor function and others that augment the T4 + helper-inducer activity in B cell antibody synthesis

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

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Ian A Cockburn

2010-05-01

Full Text Available Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

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Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Prevalence of Hepatitis-B Surface Antigen among Blood Donors in ...

African Journals Online (AJOL)

Information is scarce on the prevalence of Hepatitis-B Virus (HBV) infection among blood donors in Taraba State. Hepatitis-B surface antigen (HBsAg) ELISA [Gudans Industrial Hong 2 Kou, China] was used to determine the prevalence of HBsAg among 804 blood donors aged between 11 and 65 years in Federal Medical ...

Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

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Schendel Dolores J

2010-09-01

Full Text Available Abstract Background Antigen-loaded dendritic cells (DC are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility. Methods In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC compared with conventional DC prepared in seven days (7d mDC, which represent the most common form of DC used for vaccines to date. Results Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivtRNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA. Conclusions This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

NARCIS (Netherlands)

Brakenhoff, R H; Gerretsen, M; Knippels, E M; van Dijk, M.; van Essen, H; Weghuis, D O; Sinke, R J; Snow, G B; van Dongen, G A

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose

Regional Delivery of Chimeric Antigen Receptor (CAR) T-Cells for Cancer Therapy.

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Sridhar, Praveen; Petrocca, Fabio

2017-07-18

Chimeric Antigen Receptor (CAR) T-cells are T-cells with recombinant receptors targeted to tumor antigens. CAR-T cell therapy has emerged as a mode of immunotherapy and is now being extensively explored in hematologic cancer. In contrast, CAR-T cell use in solid tumors has been hampered by multiple obstacles. Several approaches have been taken to circumvent these obstacles, including the regional delivery of CAR-T cells. Regional CAR-T cell delivery can theoretically compensate for poor T-cell trafficking and tumor antigen specificity while avoiding systemic toxicity associated with intravenous delivery. We reviewed completed clinical trials for the treatment of glioblastoma and metastatic colorectal cancer and examined the data in these studies for safety, efficacy, and potential advantages that regional delivery may confer over systemic delivery. Our appraisal of the available literature revealed that regional delivery of CAR-T cells in both glioblastoma and hepatic colorectal metastases was generally well tolerated and efficacious in select instances. We propose that the regional delivery of CAR-T cells is an area of potential growth in the solid tumor immunotherapy, and look towards future clinical trials in head and neck cancer, mesothelioma, and peritoneal carcinomatosis as the use of this technique expands.

Regional Delivery of Chimeric Antigen Receptor (CAR T-Cells for Cancer Therapy

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Praveen Sridhar

2017-07-01

Full Text Available Chimeric Antigen Receptor (CAR T-cells are T-cells with recombinant receptors targeted to tumor antigens. CAR-T cell therapy has emerged as a mode of immunotherapy and is now being extensively explored in hematologic cancer. In contrast, CAR-T cell use in solid tumors has been hampered by multiple obstacles. Several approaches have been taken to circumvent these obstacles, including the regional delivery of CAR-T cells. Regional CAR-T cell delivery can theoretically compensate for poor T-cell trafficking and tumor antigen specificity while avoiding systemic toxicity associated with intravenous delivery. We reviewed completed clinical trials for the treatment of glioblastoma and metastatic colorectal cancer and examined the data in these studies for safety, efficacy, and potential advantages that regional delivery may confer over systemic delivery. Our appraisal of the available literature revealed that regional delivery of CAR-T cells in both glioblastoma and hepatic colorectal metastases was generally well tolerated and efficacious in select instances. We propose that the regional delivery of CAR-T cells is an area of potential growth in the solid tumor immunotherapy, and look towards future clinical trials in head and neck cancer, mesothelioma, and peritoneal carcinomatosis as the use of this technique expands.

Shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells.

Science.gov (United States)

Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

1990-07-01

The accurate identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of acute lymphoblastic leukemia (ALL). We demonstrated that soluble CD9 antigen was shed into CSF obtained from children with ALL, using enzyme-linked immunosorbent assay (ELISA), which used the activity of CD9 antigen to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4, simultaneously. Using RCA1/SJ-9A4 ELISA, CD9 antigen was detectable in CSF but not in plasma from 12 cases of CD9+ ALL in central nervous system (CNS) relapse. However, CD9 antigen was not released into CSF from 11 cases of CD9- ALL with CNS involvement, 136 cases of CD9+ ALL in complete remission (CR), 29 cases of CD9- ALL in CR, or 21 cases of aseptic meningitis. Interestingly, the levels of CD9 antigen were elevated in CSF from 7 of 10 CD9+ ALL patients without cytologically proven CNS involvement at diagnosis, with subsequent return to undetectable levels after initial induction chemotherapy was begun. In addition, sequential analysis of CSF from a 5-year-old boy with CD9+ ALL in CNS relapse showed that levels of CD9 antigen correlated well with the number of leukemic cells in CSF. Serial quantitative analysis of CD9 antigen in CSF could be useful to detect the proliferation of residual leukemic cells before the clinical manifestation.

Identification of a surface antigen on Theileria parva sporozoites by monoclonal antibody.

OpenAIRE

Dobbelaere, D A; Shapiro, S Z; Webster, P

1985-01-01

A mouse monoclonal antibody (mAbD1) that neutralizes sporozoites of different stocks of the protozoan parasite Theileria parva has been used to localize and identify a sporozoite antigen. Protein A-colloidal gold was used to localize bound mAbD1 in immunoelectron microscopic studies. mAbD1 bound to sporozoite antigen, which was evenly spread over the surface of all sporozoites. Immune complexes were obtained by incubation of sporozoite suspensions with mAbD1 followed by Zwittergent 3-14 extra...

Protein antigenic structures recognized by T cells: potential applications to vaccine design.

Science.gov (United States)

Berzofsky, J A; Cease, K B; Cornette, J L; Spouge, J L; Margalit, H; Berkower, I J; Good, M F; Miller, L H; DeLisi, C

1987-08-01

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic

Characterization of plant plasma membrane antigens: [Annual] progress report

International Nuclear Information System (INIS)

Galbraith, D.W.; Afonso, C.L.; Meyer, D.; Harkins, K.R.

1987-01-01

Protoplast plasma membranes were used to raise antibodies in mice to cell surface antigens. Monoclonal antibodies were selected from those produced and used for indirect immunofluorescence microscopic analysis of N. tabacum cells. In parallel studies cDNA expression libraries were prepared. (DT)

Effects of radiation on the expression of antigens on the membranes of human adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato; Imai, Kozo; Oouchi, Atsushi; Shidou, Mitsuo; Takahashi, Hiroki; Koshiba, Hirohumi; Yachi, Akira; Morita, Kazuo

1992-01-01

We have investigated the effects of irradiation on the membranes of tumor cells. X-ray irradiation caused remarkable increases in the expression of tumor-associated antigens (YH206, CEA) and c-erbB-2 protein by flow cytometry, whereas IFN had no obvious effect on the expression of tumor-associated antigens and c-erbB-2 protein. On the other hand, the expression of MHC Class I and ICAM-1 antigen on the membrane by flow cytometry was enhanced by both irradiation and IFN. In addition, the irradiated cells, when analyzed using a CEA specific probe, showed remarkable increases in the CEA mRNA compared to IFN-treated cells. It is possible that enhancement of the expression of tumor-associated antigens and c-erbB-2 protein, together with the enhancement of that of MHC-Class I and ICAM-1, would help cytotoxic killer cells recognize the tumor cells. (author)

Characterization and Classification of Mesenchymal Stem Cells in Several Species Using Surface Markers for Cell Therapy Purposes.

Science.gov (United States)

Ghaneialvar, Hori; Soltani, Leila; Rahmani, Hamid Reza; Lotfi, Abbas Sahebghadam; Soleimani, Masoud

2018-01-01

Mesenchymal stem cells are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesenchymal tissue lineages such as osteocytes, adipocytes and chondrocytes. Such lineages can then be used in cell therapy. The aim of present study was to characterize bone marrow derived mesenchymal stem cells in four different species, including: sheep, goat, human and mouse. Human bone-marrow mesenchymal stem cells were purchased, those of sheep and goat were isolated from fetal bone marrow, and those of mouse were collected by washing bone cavity of femur and tibia with DMEM/F12. Using flow-cytometry, they were characterized by CD surface antigens. Furthermore, cells of third passage were examined for their osteogenic and adipogenic differentiation potential by oil red and alizarin red staining respectively. According to the results, CD markers studied in the four groups of mesenchymal stem cells showed a different expression. Goat and sheep expressed CD44 and CD166, and weakly expressed CD34, CD45, CD105 and CD90. Similarly, human and mouse mesenchymal cells expressed CD44, CD166, CD105 and CD90 whereas the expression of CD34 and CD45 was negative. In conclusion, although all mesenchymal stem cells display plastic adherence and tri-lineage differentiation, not all express the same panel of surface antigens described for human mesenchymal stem cells. Additional panel of CD markers are necessary to characterize regenerative potential and possible application of these stem cells in regenerative medicine and implantology.

Healthy human T-Cell Responses to Aspergillus fumigatus antigens.

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Neelkamal Chaudhary

2010-02-01

Full Text Available Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H1-T(H17 and destructive allergic (T(H2 immunity. How Aspergillus allergens (Asp f proteins participate in the development of allergic sensitization is unknown.To determine whether Asp f proteins are strictly associated with T(H2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17 to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H1-responses to Asp f3 (a putative peroxismal membrane protein, Asp f9/16 (cell wall glucanase, Asp f11 (cyclophilin type peptidyl-prolyl isomerase and Asp f22 (enolase. Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals.Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3 identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

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Claude Oeuvray

1994-01-01

Full Text Available We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.

Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes.

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Carlos Alfaro

Full Text Available Dendritic cells (DC are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs as a result of being co-attracted by interleukin-8 (IL-8, for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA protein, were able to cross-present the antigen to CD8 (OT-1 and CD4 (OT-2 TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d are coinjected in the footpad of mice with autologous DC (H-2(b. In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.

IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate

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Noviski, Mark; Mueller, James L; Satterthwaite, Anne; Garrett-Sinha, Lee Ann; Brombacher, Frank

2018-01-01

Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells. PMID:29521626

Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

OpenAIRE

Stroncek, David F.; Lee, Daniel W.; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N.; Kaplan, Rosandra N.; Fry, Terry J.; Mackall, Crystal L.

2017-01-01

Background Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Methods Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We ...

Memory control by the B cell antigen receptor.

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Engels, Niklas; Wienands, Jürgen

2018-05-01

The generation of memory B cells (MBCs) that have undergone immunoglobulin class switching from IgM, which dominates primary antibody responses, to other immunoglobulin isoforms is a hallmark of immune memory. Hence, humoral immunological memory is characterized by the presence of serum immunoglobulins of IgG subtypes known as the γ-globulin fraction of blood plasma proteins. These antibodies reflect the antigen experience of B lymphocytes and their repeated triggering. In fact, efficient protection against a previously encountered pathogen is critically linked to the production of pathogen-specific IgG molecules even in those cases where the primary immune response required cellular immunity, for example, T cell-mediated clearance of intracellular pathogens such as viruses. Besides IgG, also IgA and IgE can provide humoral immunity depending on the microbe's nature and infection route. The molecular mechanisms underlying the preponderance of switched immunoglobulin isotypes during memory antibody responses are a matter of active and controversial debate. Here, we summarize the phenotypic characteristics of distinct MBC subpopulations and discuss the decisive roles of different B cell antigen receptor isotypes for the functional traits of class-switched B cell populations. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from Toxoplasma gondii

International Nuclear Information System (INIS)

Grujic, Ognjen; Grigg, Michael E.; Boulanger, Martin J.

2008-01-01

Preliminary X-ray diffraction studies of the bradyzoite-specific surface antigen BSR4 from T. gondii are described. Toxoplasma gondii is an important global pathogen that infects nearly one third of the world’s adult population. A family of developmentally expressed structurally related surface-glycoprotein adhesins (SRSs) mediate attachment to and are utilized for entry into host cells. The latent bradyzoite form of T. gondii persists for the life of the host and expresses a distinct family of SRS proteins, of which the bradyzoite-specific antigen BSR4 is a prototypical member. Structural studies of BSR4 were initiated by first recombinantly expressing BSR4 in insect cells, which was followed by crystallization and preliminary X-ray data collection to 1.95 Å resolution. Data processing showed that BSR4 crystallized with one molecule in the asymmetric unit of the P4 1 2 1 2 or P4 3 2 1 2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 Å 3 Da −1

Generation and characterization of antigenic variants induced by exposure of tumor cells to UV radiation in vitro