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Sample records for cell receptors mhc

  1. Can Selective MHC Downregulation Explain the Specificity and Genetic Diversity of NK Cell Receptors?

    NARCIS (Netherlands)

    Carrillo-Bustamante, Paola; Kesmir, Can; de Boer, Rob J

    2015-01-01

    Natural killer (NK) cells express inhibiting receptors (iNKRs), which specifically bind MHC-I molecules on the surface of healthy cells. When the expression of MHC-I on the cell surface decreases, which might occur during certain viral infections and cancer, iNKRs lose inhibiting signals and the

  2. Thermodynamics of T cell receptor – peptide/MHC interactions: progress and opportunities

    Science.gov (United States)

    Armstrong, Kathryn M.; Insaidoo, Francis K.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCR) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van’t Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize commonly disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but for understanding specifics of individual interactions and the engineering of T cell receptors with desired molecular recognition properties. PMID:18496839

  3. Probing natural killer cell education by Ly49 receptor expression analysis and computational modelling in single MHC class I mice.

    Directory of Open Access Journals (Sweden)

    Sofia Johansson

    Full Text Available Murine natural killer (NK cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for "missing self" recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an "educating impact" on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.

  4. Identification of the cognate peptide-MHC target of T cell receptors using molecular modeling and force field scoring

    DEFF Research Database (Denmark)

    Lanzarotti, Esteban; Marcatili, Paolo; Nielsen, Morten

    2018-01-01

    Interactions of T cell receptors (TCR) to peptides in complex with MHC (p:MHC) are key features that mediate cellular immune responses. While MHC binding is required for a peptide to be presented to T cells, not all MHC binders are immunogenic. The interaction of a TCR to the p:MHC complex holds...... terms. Building a benchmark of TCR:p:MHC complexes where epitopes and non-epitopes are modelled using state-of-the-art molecular modelling tools, scoring p:MHC to a given TCR using force-fields, optimized in a cross-validation setup to evaluate TCR inter atomic interactions involved with each p:MHC, we...... and model the TCR:p:MHC complex structure. In summary, we conclude that it is possible to identify the TCR cognate target among different candidate peptides by using a force-field based model, and believe this works could lay the foundation for future work within prediction of TCR:p:MHC interactions....

  5. Peptide-MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices

    Science.gov (United States)

    Singha, Santiswarup; Shao, Kun; Yang, Yang; Clemente-Casares, Xavier; Solé, Patricia; Clemente, Antonio; Blanco, Jesús; Dai, Qin; Song, Fayi; Liu, Shang Wan; Yamanouchi, Jun; Umeshappa, Channakeshava Sokke; Nanjundappa, Roopa Hebbandi; Detampel, Pascal; Amrein, Matthias; Fandos, César; Tanguay, Robert; Newbigging, Susan; Serra, Pau; Khadra, Anmar; Chan, Warren C. W.; Santamaria, Pere

    2017-07-01

    We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

  6. 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.

  7. Distinct spatial relationship of interleukin-9 receptor with IL-2R and MHC glycoproteins in human T lymphoma cells

    OpenAIRE

    Nizsalóczki, Enikő; Csomós, István; Nagy, Péter; Fazekas, Zsolt; Goldman, Carolyn K.; Waldmann, Thomas A.; Damjanovich, Sándor; Vámosi, György; Mátyus, László; Bodnár, Andrea

    2014-01-01

    The IL-9R consists of the α-subunit and the γc-chain shared with other cytokine receptors, including IL-2R, an important regulator of T cells. We have previously shown that IL-2R is expressed in common clusters with MHC glycoproteins in lipid rafts of human T lymphoma cells raising the question what the relationship between clusters of IL-2R/MHC and IL-9R is. Confocal microscopic co-localization and FRET experiments capable of detecting membrane protein organization at different size scales r...

  8. A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

    Energy Technology Data Exchange (ETDEWEB)

    Sethi, D.K.; Heroux, A.; Schubert, D. A.; Anders, A.-K.; Bonsor, D. A.; Thomas, C. P.; Sundberg, E. J.; Pyrdol, J.; Wucherpfennig, K. W.

    2011-01-17

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  9. A Highly Tilted Binding Mode by a Self-Reactive T Cell Receptor Results in Altered Engagement of Peptide and MHC

    Energy Technology Data Exchange (ETDEWEB)

    D Sethi; D Schubert; A Anders; A Heroux; D Bonsor; C Thomas; E Sundberg; J Pyrdol; K Wucherpfennig

    2011-12-31

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  10. Crystal structure of a Gammadelta T-cell Receptor Specific for the Human MHC class I Homolog MICA

    Energy Technology Data Exchange (ETDEWEB)

    B Xu; J Pizarro; M Holmes; C McBeth; V Groh; T Spies; R Strong

    2011-12-31

    {gamma}{delta} T cells play important roles in bridging innate and adaptive immunity, but their recognition mechanisms remain poorly understood. Human {gamma}{delta} T cells of the V{sub {delta}}1 subset predominate in intestinal epithelia and respond to MICA and MICB (MHC class I chain-related, A and B; MIC) self-antigens, mediating responses to tumorigenesis or viral infection. The crystal structure of an MIC-reactive V{sub {delta}}1 {gamma}{delta} T-cell receptor (TCR) showed expected overall structural homology to antibodies, {alpha}{beta}, and other {gamma}{delta} TCRs, but complementary determining region conformations and conservation of V{sub {delta}}1 use revealed an uncharacteristically flat potential binding surface. MIC, likewise, serves as a ligand for the activating immunoreceptor natural killer group 2, D (NKG2D), also expressed on {gamma}{delta} T cells. Although MIC recognition drives both the TCR-dependent stimulatory and NKG2D-dependent costimulatory signals necessary for activation, interaction analyses showed that MIC binding by the two receptors was mutually exclusive. Analysis of relative binding kinetics suggested sequential recognition, defining constraints for the temporal organization of {gamma}{delta} T-cell/target cell interfaces.

  11. Crystal structure of a gammadelta T-cell receptor specific for the human MHC class I homolog MICA.

    Science.gov (United States)

    Xu, Bin; Pizarro, Juan C; Holmes, Margaret A; McBeth, Christine; Groh, Veronika; Spies, Thomas; Strong, Roland K

    2011-02-08

    γδ T cells play important roles in bridging innate and adaptive immunity, but their recognition mechanisms remain poorly understood. Human γδ T cells of the V(δ)1 subset predominate in intestinal epithelia and respond to MICA and MICB (MHC class I chain-related, A and B; MIC) self-antigens, mediating responses to tumorigenesis or viral infection. The crystal structure of an MIC-reactive V(δ)1 γδ T-cell receptor (TCR) showed expected overall structural homology to antibodies, αβ, and other γδ TCRs, but complementary determining region conformations and conservation of V(δ)1 use revealed an uncharacteristically flat potential binding surface. MIC, likewise, serves as a ligand for the activating immunoreceptor natural killer group 2, D (NKG2D), also expressed on γδ T cells. Although MIC recognition drives both the TCR-dependent stimulatory and NKG2D-dependent costimulatory signals necessary for activation, interaction analyses showed that MIC binding by the two receptors was mutually exclusive. Analysis of relative binding kinetics suggested sequential recognition, defining constraints for the temporal organization of γδ T-cell/target cell interfaces.

  12. Human NK cells selective targeting of colon cancer-initiating cells: A role for natural cytotoxicity receptors and MHC class i molecules

    KAUST Repository

    Tallerico, Rossana

    2013-01-23

    Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma- derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors. Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved.

  13. Human NK cells selective targeting of colon cancer-initiating cells: A role for natural cytotoxicity receptors and MHC class i molecules

    KAUST Repository

    Tallerico, Rossana; Todaro, Matilde; Di Franco, Simone; MacCalli, Cristina; Garofalo, Cinzia; Sottile, Rosa; Palmieri, Camillo; Tirinato, Luca; Pangigadde, Pradeepa N.; La Rocca, Rosanna; Mandelboim, Ofer; Stassi, Giorgio; Di Fabrizio, Enzo M.; Parmiani, Giorgio; Moretta, Alessandro; Dieli, Francesco; Kã rre, Klas; Carbone, Ennio

    2013-01-01

    Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma- derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors. Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved.

  14. Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

    Science.gov (United States)

    Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

    2006-01-01

    The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

  15. Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies

    Science.gov (United States)

    Bentley, Carol; Yates, Jenna; Salimi, Maryam; Greig, Jenny; Wiblin, Sarah; Hassanali, Tasneem; Banham, Alison H.

    2017-01-01

    Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy. PMID:28448627

  16. Innate lymphoid cells and the MHC.

    Science.gov (United States)

    Robinette, M L; Colonna, M

    2016-01-01

    Innate lymphoid cells (ILCs) are a new class of immune cells that include natural killer (NK) cells and appear to be the innate counterparts to CD4(+) helper T cells and CD8(+) cytotoxic T cells based on developmental and functional similarities. Like T cells, both NK cells and other ILCs also show connections to the major histocompatibility complex (MHC). In human and mouse, NK cells recognize and respond to classical and nonclassical MHC I molecules as well as structural homologues, whereas mouse ILCs have recently been shown to express MHC II. We describe the history of MHC I recognition by NK cells and discuss emerging roles for MHC II expression by ILC subsets, making comparisons between both mouse and human when possible. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Williams

    2017-07-01

    Full Text Available The discovery of naturally occurring T cell receptors (TCRs that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds rather than synergy (total CD8 cooperation alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our

  18. Leukocyte Ig-Like Receptors – a model for MHC class I disease associations

    Directory of Open Access Journals (Sweden)

    Rachel Louise Allen

    2016-07-01

    Full Text Available MHC class I (MHC-I polymorphisms are associated with the outcome of some viral infections and autoimmune diseases. MHC-I proteins present antigenic peptides and are recognised by receptors on Natural Killer cells and Cytotoxic T lymphocytes, thus enabling the immune system to detect self-antigens and eliminate targets lacking self or expressing foreign antigens. Recognition of MHC-I, however, extends beyond receptors on cytotoxic leukocytes. Members of the Leukocyte Ig-like receptor (LILR family are expressed on monocytic cells and can recognise both classical and non-classical MHC-I alleles. Despite their relatively broad specificity when compared to the T Cell Receptor or Killer Ig-like Receptors, variations in the strength of LILR binding between different MHC-I alleles have recently been shown to correlate with control of HIV infection. We suggest that LILR recognition may mediate MHC-I disease association in a manner that does not depend on a binary discrimination of self/non-self by cytotoxic cells. Instead, the effects of LILR activity following engagement by MHC-I may represent a degrees of self model, whereby strength of binding to different alleles determines the degree of influence exerted by these receptors on immune cell functions. LILR are expressed by myelomonocytic cells and lymphocytes, extending their influence across antigen presenting cell subsets including dendritic cells, macrophages and B cells. They have been identified as important players in the response to infection, inflammatory diseases and cancer, with recent literature to indicate that MHC-I recognition by these receptors and consequent allelic effects could extend an influence beyond the immune system.

  19. MHC class II molecules deliver costimulatory signals in human T cells through a functional linkage with IL-2-receptors

    DEFF Research Database (Denmark)

    Odum, Niels; Kanner, S B; Ledbetter, J A

    1993-01-01

    MHC class II-positive T cells are found in tissues involved in autoimmune and infectious disorders. Because stimulation of class II molecules by mAb or bacterial superantigens induces protein tyrosine phosphorylation through activation of PTK3 in T cells, we hypothesized that class II signals play...... tyrosine phosphorylation of specific substrates including PLC-gamma 1. Combined stimulation of IL-2R and class II molecules had an additive effect on tyrosine phosphorylation. Pretreatment of T cells with a protein tyrosine kinase inhibitor, herbimycin A, inhibited IL-2 and class II-induced proliferation...... a regulatory function in T cell activation. Here, we show that cross-linking HLA-DR and -DP but not -DQ molecules by immobilized mAb enhanced proliferative T cell responses to IL-2. In contrast, class II stimulation had no effect on IL-4-induced proliferation. The costimulatory effect was most pronounced...

  20. Structure of a Pheromone Receptor-Associated Mhc Molecule With An Open And Empty Groove

    Energy Technology Data Exchange (ETDEWEB)

    Olson, R.; Huey-Tubman, K.E.; Dulac, C.; Bjorkman, P.J.; /Caltech /Harvard U.

    2006-10-06

    Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

  1. Structure of a pheromone receptor-associated MHC molecule with an open and empty groove.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available Neurons in the murine vomeronasal organ (VNO express a family of class Ib major histocompatibility complex (MHC proteins (M10s that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

  2. H pylori receptor MHC class II contributes to the dynamic gastric epithelial apoptotic response

    Science.gov (United States)

    Bland, David A; Suarez, Giovanni; Beswick, Ellen J; Sierra, Johanna C; Reyes, Victor E

    2006-01-01

    AIM: To investigate the role of MHC class II in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. METHODS: After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class II and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. RESULTS: Pretreatment of N87 cells with the anti-MHC class II IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class II resulted in a marked reduced caspase activation, while simple ligation of MHC class II did not. Crosslinking of MHC class II also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class II IgM blocked the recruitment of FADD to the cell surface. CONCLUSION: The results presented here demonstrate that the ability of MHC class II to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class II signaling can be pro-apoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic effects during the earlier time points of H pylori infection. This effect is possibly mediated by the ability of MHC class II to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by H pylori-infected gastric epithelial cells. PMID:16981259

  3. MHC class I is functionally associated with antigen receptors in human T and B lymphomas

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Jacoby, B F; Skov, S

    1996-01-01

    lines the increase in [Ca2+]i after MHC-I cross-linking caused upregulation of CD69, an early marker of activation. When studying the effect of MHC-I cross-linking on the TCR- and B cell antigen receptor (BCR)- mediated increase in [Ca2+]i, respectively, we observed that MHC-I had a costimulatory effect...... on the TCR-mediated increase in [Ca2+]i in Jurkat cells but not on the anti-IgM-mediated activity of Solubo cells. Studies of subpopulations of Jurkat and Solubo cells expressing different levels of MHC-I on their cell surfaces revealed that the TCR- and BCR-mediated increases in [Ca2+]i, respectively, were...

  4. Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice

    International Nuclear Information System (INIS)

    Bix, M.; Nanshih Liao; Raulet, D.; Zijlstra, M.; Loring, J.; Jaenisch, R.

    1991-01-01

    Irradiated MHC-heterozygous mice often reject bone marrow cells transplanted from one of the homozygous parental strains, a phenomenon ('hybrid resistance') that appears to violate the laws of transplantation. Rejection of parental and allogeneic marrow cells also differs from conventional T cell-mediated rejection mechanisms as it is effected by NK1.1 + cells. To account for the unusual specificity of bone marrow rejection, it has been proposed that NK1.1 + cells destroy marrow cells that fail to express the full complement of self MHC class I (MHC-I) molecules. We show here that NK1.1 + cells in normal mice reject haemopoietic transplants from mice that are deficient for normal cell-surface MHC-I expression because of a targeted mutation in the β 2 -microglobulin gene. These findings demonstrate that deficient expression of MHC-I molecules renders marrow cells susceptible to rejection. (author)

  5. MHC class II polymorphisms, autoreactive T-cells and autoimmunity

    Directory of Open Access Journals (Sweden)

    Sue eTsai

    2013-10-01

    Full Text Available Major histocompatibility complex (MHC genes, also known as human leukocyte antigen genes (HLA in humans, are the prevailing contributors of genetic susceptibility to autoimmune diseases such as Type 1 Diabetes (T1D, Multiple Sclerosis (MS, and Rheumatoid arthritis (RA, among others (Todd and Wicker, 2001;MacKay et al., 2002;Hafler et al., 2007. Although the pathways through which MHC molecules afford autoimmune risk or resistance remain to be fully mapped out, it is generally accepted that they do so by shaping the central and peripheral T cell repertoires of the host towards autoimmune proclivity or resistance, respectively. Disease-predisposing MHC alleles would both spare autoreactive thymocytes from central tolerance and bias their development towards a pathogenic phenotype. Protective MHC alleles, on the other hand, would promote central deletion of autoreactive thymocytes and skew their development towards non-pathogenic phenotypes. This interpretation of the data is at odds with two other observations: that in MHC-heterozygous individuals, resistance is dominant over susceptibility; and that it is difficult to understand how deletion of one or a few clonal autoreactive T cell types would suffice to curb autoimmune responses driven by hundreds if not thousands of autoreactive T cell specificities. This review provides an update on current advances in our understanding of the mechanisms underlying MHC class II-associated autoimmune disease susceptibility and/or resistance and attempts to reconcile these seemingly opposing concepts.

  6. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... mobilization of intracellular free calcium compared with MHC-I crosslinking of wild-type DT40 cells. Thus, expression of BCR at the cell surface is likely to be important for the signal cascade initiated by MHC-I crosslinking. Our data suggest that signal transduction initiated through ligation of the MHC...

  7. Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells.

    Science.gov (United States)

    Luoma, Adrienne M; Castro, Caitlin D; Mayassi, Toufic; Bembinster, Leslie A; Bai, Li; Picard, Damien; Anderson, Brian; Scharf, Louise; Kung, Jennifer E; Sibener, Leah V; Savage, Paul B; Jabri, Bana; Bendelac, Albert; Adams, Erin J

    2013-12-12

    The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Distinctive CD8+ T cell and MHC class I signatures in polycythemia vera patients.

    Science.gov (United States)

    Cardoso, Elsa M; Esgalhado, André J; Patrão, Luís; Santos, Mónica; Neves, Vasco Pinto; Martinez, Jorge; Patto, Maria Assunção Vaz; Silva, Helena; Arosa, Fernando A

    2018-05-22

    Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by overproduction of red blood cells. We have performed a comprehensive characterization of blood immune cells for expression of naïve and memory receptors as well as β 2 m-associated and β 2 m-free MHC class I heavy chains, also known as closed and open conformers, respectively, in PV patients and age-matched controls (CTR). We show that the peripheral CD3 + CD8 + T cell pool in PV patients is clearly divided into two discrete populations, a more granular CD3 + CD8 high T cell population enriched in effector-memory CD45RA + T cells (CD8 + TEMRA) when compared to CTR (P e., CD3 + CD8 int CD28 int . While the percentage of CD3 + CD8 int TN cells correlated positively with the number of erythrocytes, the percentage of CD3 + CD8 int TEMRA correlated negatively with the number of platelets. Finally, we report that PV patients' lymphocytes and monocytes display lower levels of closed (W6/32 + ) MHC-I conformers at the cell surface while exhibiting increased amounts of open (HC-10 + ) MHC-I conformers. The implications of this distinctive immune signature are discussed.

  9. Molecular Basis of Natural Killer Cell Tumor Target Recognition: The NKr/MHC Class I Complex

    National Research Council Canada - National Science Library

    Hasemann, Charles

    1999-01-01

    .... We have pursued this via the heterologous expression of wild type and mutant NK receptors for the purpose of the determination of the atomic structure of an NK receptor/ class I MHC complex via X-ray crystallography...

  10. MHC class II molecules regulate growth in human T cells

    DEFF Research Database (Denmark)

    Nielsen, M; Odum, Niels; Bendtzen, K

    1994-01-01

    MHC-class-II-positive T cells are found in tissues involved in autoimmune disorders. Stimulation of class II molecules by monoclonal antibodies (mAbs) or bacterial superantigens induces protein tyrosine phosphorylation through activation of protein tyrosine kinases in T cells, and class II signals...... lines tested. Only one of three CD4+, CD45RAhigh, ROhigh T cells responded to class II costimulation. There was no correlation between T cell responsiveness to class II and the cytokine production profile of the T cell in question. Thus, T cell lines producing interferon (IFN)-gamma but not IL-4 (TH1...... modulate several T cell responses. Here, we studied further the role of class II molecules in the regulation of T cell growth. Costimulation of class II molecules by immobilized HLA-DR mAb significantly enhanced interleukin (IL)-2-supported T cell growth of the majority of CD4+, CD45RAlow, ROhigh T cell...

  11. Chemotherapeutics and radiation stimulate MHC class I expression through elevated interferon-beta signaling in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Shan Wan

    Full Text Available Low doses of anticancer drugs have been shown to enhance antitumor immune response and increase the efficacy of immunotherapy. The molecular basis for such effects remains elusive, although selective depletion of T regulatory cells has been demonstrated. In the current studies, we demonstrate that topotecan (TPT, a topoisomerase I-targeting drug with a well-defined mechanism of action, stimulates major histocompatibility complex class I (MHC I expression in breast cancer cells through elevated expression/secretion of interferon-β (IFN-β and activation of type I IFN signaling. First, we show that TPT treatment elevates the expression of both total and cell-surface MHC I in breast cancer cells. Second, conditioned media from TPT-treated breast cancer ZR-75-1 cells induce elevated expression of cell-surface MHC I in drug-naïve recipient cells, suggesting the involvement of cytokines and/or other secreted molecules. Consistently, TPT-treated cells exhibit elevated expression of multiple cytokines such as IFN-β, TNF-α, IL-6 and IL-8. Third, either knocking down the type I interferon receptor subunit 1 (IFNAR1 or addition of neutralizing antibody against IFN-β results in reduced MHC I expression in TPT-treated cells. Together, these results suggest that TPT induces increased IFN-β autocrine/paracrine signaling through type I IFN receptor, resulting in the elevated MHC I expression in tumor cells. Studies have also demonstrated that other chemotherapeutic agents (e.g. etoposide, cisplatin, paclitaxel and vinblastine similarly induce increased IFN-β secretion and elevated MHC I expression. In addition, conditioned media from γ-irradiated donor cells are shown to induce IFN-β-dependent MHC I expression in unirradiated recipient cells. In the aggregate, our results suggest that many cancer therapeutics induce elevated tumor antigen presentation through MHC I, which could represent a common mechanism for enhanced antitumor immune response through

  12. Prediction of antigenic epitopes and MHC binders of neurotoxin ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... scorpion chlorotoxin-like short-chain neurotoxins (SCNs). *Corresponding ... Protein sequence analysis. Here we ... MHC/peptide binding is a log-transformed value related to the IC50 values in nM ..... porter. Adducts of MHC and peptide complexes are the ligands for T cell receptors (TCR) (Table-1). MHC.

  13. Schwann cells promote post-traumatic nerve inflammation and neuropathic pain through MHC class II.

    Science.gov (United States)

    Hartlehnert, Maike; Derksen, Angelika; Hagenacker, Tim; Kindermann, David; Schäfers, Maria; Pawlak, Mathias; Kieseier, Bernd C; Meyer Zu Horste, Gerd

    2017-10-02

    The activation of T helper cells requires antigens to be exposed on the surface of antigen presenting cells (APCs) via MHC class II (MHC-II) molecules. Expression of MHC-II is generally limited to professional APCs, but other cell types can express MHC-II under inflammatory conditions. However, the importance of these conditional APCs is unknown. We and others have previously shown that Schwann cells are potentially conditional APCs, but the functional relevance of MHC-II expression by Schwann cells has not been studied in vivo. Here, we conditionally deleted the MHC-II β-chain from myelinating Schwann cells in mice and investigated how this influenced post-traumatic intraneural inflammation and neuropathic pain using the chronic constriction injury (CCI) model. We demonstrate that deletion of MHC-II in myelinating Schwann cells reduces thermal hyperalgesia and, to a lesser extent, also diminishes mechanical allodynia in CCI in female mice. This was accompanied by a reduction of intraneural CD4+ T cells and greater preservation of preferentially large-caliber axons. Activation of T helper cells by MHC-II on Schwann cells thus promotes post-traumatic axonal loss and neuropathic pain. Hence, we provide experimental evidence that Schwann cells gain antigen-presenting function in vivo and modulate local immune responses and diseases in the peripheral nerves.

  14. Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity.

    Science.gov (United States)

    Hacker, Ulrich T; Schildhauer, Ines; Barroso, Margarita Céspedes; Kofler, David M; Gerner, Franz M; Mysliwietz, Josef; Buening, Hildegard; Hallek, Michael; King, Susan B S

    2006-05-01

    The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.

  15. MHC class II+ (HLA-DP-like) cells in the cow reproductive tract: II. Immunolocalization of MHC class II+ cells in oviduct and vagina.

    Science.gov (United States)

    Eren, U; Kum, S; Sandikçi, M; Eren, V; Ilhan, F

    2009-08-01

    The aim of this study was to determine and examine the distribution of major frequency MHC II+ cells in the oviduct and vagina of cows during the oestrous and dioestrus phases. Right oviduct (ampulla, isthmus) and vaginal samples taken from a total of twenty seven multiparous cows were used. Tissue samples were processed to obtain both cryostat and paraffin sections. Sections were stained immunocytochemically using StreptABC method using a specific monoclonal antibody to MHC II+ cell population. Intra-epithelial and subepithelial areas along with lamina propria, muscularis mucosae and serosa of both ampulla and isthmus and intra-epithelial/subepithelial areas and mucosae of vagina were examined for the presence of MHC II+ cells. The density of immune positive cells was determined using a subjective scoring system. MHC II+ cells were demonstrated in all areas examined in both oestrus and dioestrus. In oestrus, the density of MHC II+ cells decreased in subepithelial areas (in between the epithelial cells and the basal membrane) of isthmus, whereas the density of immune positive cells was increased in muscularis mucosae of isthmus (P < 0.05), lamina propria and muscularis mucosae of ampulla (P < 0.05) as well as in the mucosae of vagina (P MHC II+ cells observed in the oviduct and vagina increases in the majority of areas examined due to the effect of oestrogen.

  16. Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells

    OpenAIRE

    Walseng, Even; Furuta, Kazuyuki; Bosch, Berta; Weih, Karis A.; Matsuki, Yohei; Bakke, Oddmund; Ishido, Satoshi; Roche, Paul A.

    2010-01-01

    The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface e...

  17. Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC

    Science.gov (United States)

    Tai, Lee-Hwa; Goulet, Marie-Line; Belanger, Simon; Toyama-Sorimachi, Noriko; Fodil-Cornu, Nassima; Vidal, Silvia M.; Troke, Angela D.; McVicar, Daniel W.; Makrigiannis, Andrew P.

    2008-01-01

    Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2Kb. Conversely, CpG-ODN–dependent IFN-α production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2Kb ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo. PMID:19075287

  18. Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

    DEFF Research Database (Denmark)

    Tungatt, Katie; Bianchi, Valentina; Crowther, Michael D

    2015-01-01

    Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multim...

  19. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes

    DEFF Research Database (Denmark)

    Schmidt, Esben G W; Buus, Soren; Thorn, Mette

    2009-01-01

    to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear...

  20. Structural and Functional Characterization of a Single-chain Peptide-MHC Molecule that Modulates both Naive and Activated CD8plus T Cells

    Energy Technology Data Exchange (ETDEWEB)

    D Samanta; G Mukherjee; U Ramagopal; R Chaparro; S Nathenson; T DiLorenzo; S Almo

    2011-12-31

    Peptide-MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8{sup +} T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8{sup +} T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scK{sup d}.IGRP) by using the class I MHC molecule H-2K{sup d} and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP{sub 206-214}), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scK{sup d}.IGRP tetramers bound specifically to cognate CD8{sup +} T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-{gamma} response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8{sup +} T cells makes them a potential intervention strategy in early and late stages of disease.

  1. Epitopes associated with MHC restriction site of T cells. III. I-J epitope on MHC-restricted T helper cells

    International Nuclear Information System (INIS)

    Asano, Y.; Nakayama, T.; Kubo, M.; Yagi, J.; Tada, T.

    1987-01-01

    I-J epitopes were found to be associated with the functional site of the class II MHC-restricted helper T (Th) cells: Virtually all of the H-2k-restricted Th cell function of H-2kxbF1 T cells was inhibited by the anti-I-Jk mAb, leaving the H-2b-restricted function unaffected. The I-Jk epitope was inducible in Th cells of different genotype origin according to the environmental class II antigens present in the early ontogeny of T cells. Although above results suggested that I-J is the structure reflecting the inducible MHC restriction specificity, further studies revealed some interesting controversies: First, the I-J phenotype did not always correlate with the class II restriction specificity, e.g., I-Ab-restricted Th from 5R was I-Jk-positive, whereas I-Ak-restricted Th of 4R was not. Second, there was no trans expression of parental I-J phenotypes and restriction specificities in F1 Th, e.g., the I-J phenotype was detected only on I-Ab-restricted Th of (4R X 5R)F1, whereas it was absent on I-Ak-restricted Th. This strict linkage between the restriction specificity and I-J phenotype was also found on Th cells developed in bone marrow chimera constructed with intra-H-2-recombinant mice. The expression of I-Jk was always associated with the restriction specificity of the relevant host. Thus, the restriction specificity of Th cells followed the host type, and the I-J expression on Th was exactly the same as that expressed by the host haplotype. These results indicate that I-J is an isomorphic structure adaptively expressed on Th cells that is involved in the unidirectional regulatory cell interactions, and that the polymorphism cannot be explained merely by the restriction specificity of the conventional T cell receptor heterodimer

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ole Audun Werner Haabeth

    2014-04-01

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

  5. Haplotype specific alteration of diabetes MHC risk by olfactory receptor gene polymorphism.

    Science.gov (United States)

    Jahromi, Mohamed M

    2012-12-01

    Evidence for genes associated with risk for Type 1 diabetes (T1D) in the extended region of the major histocompatibility complex (MHC) genes is accumulating. The aim of this study was to investigate the association pattern of the extended MHC region with T1D susceptibility to identify effects independent of well established DR/DQ genes. A total of 394 Europid families with T1D were genotyped for the single nucleotide polymorphism (SNP) in the olfactory receptor family 14, subfamily J, member 1 (OR14J1) gene, rs9257691, in the MHC telomeric region. The OR provides "an internal depiction of our external world" through the capture of odorant molecules in the main OR system by several large families of G-protein coupled receptors (GPCR). These receptors transduce and chemosignals into the central nervous system (CNS). This SNP was chosen to identify its association with T1D. Interestingly, OR14J1C allele was significantly associated with T1D that seems to go with DRB1*0401, Χ(2)=10.9, p=0.0003. However, by fixing both genes of DR*0401-DQB1*0302, high risk, the association of T1D with OR14J1C still existed, Χ(2)=7.4, p=0.005. The occurrence of association of the OR14J1C allele with T1D patients with DRB1*401/DQB1*0302 is an independent risk for T1D. As an accumulative report suggests the role of OR in the pathogenesis of diabetic microvascular and other diabetic complications, undoubtedly, this haplotype specific alteration of T1D risk is an independent risk for the disease and can address the promising MHC-linked gene other than DR/DQ. Moreover, there is nothing to hinder for that this might be a signal that identifies the role of OR gene in the pathogenesis of T1D in patients who are prone to diabetic complications. Copyright © 2012. Published by Elsevier B.V.

  6. 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....

  7. Confinement of activating receptors at the plasma membrane controls natural killer cell tolerance.

    Science.gov (United States)

    Guia, Sophie; Jaeger, Baptiste N; Piatek, Stefan; Mailfert, Sébastien; Trombik, Tomasz; Fenis, Aurore; Chevrier, Nicolas; Walzer, Thierry; Kerdiles, Yann M; Marguet, Didier; Vivier, Eric; Ugolini, Sophie

    2011-04-05

    Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

  8. Cell surface appearance of unexpected host MHC determinants on thymocytes from radiation bone marrow chimeras

    International Nuclear Information System (INIS)

    Sharrow, S.O.; Mathieson, B.J.; Singer, A.

    1981-01-01

    The phenotypic appearance of cell surface antigens on murine thymocytes from long-term radiation bone marrow chimeras was analyzed using indirect immunofluorescence and flow microfluorometry. Cells maturing in the thymi of these mice were typed for MHC (Kk, I-Ak, H-2b, Kb, and Ib) and non-MHC (Lty 1, Ly 9, and TL) determinants. All cells were of donor origin as determined by non-MHC (Ly) phenotype in P1 leads to P2, P1 x P2 leads to P1, and P1 leads to P2 radiation chimeras. In contrast, the MHC phenotypes of these thymocytes were markedly affected by the host environment. Specifically, H-2 and I-A determinants of both parental phenotypes were detected on thymocytes from P1 leads to P1 x P2 chimeras; I-A determinants of host phenotype were present, whereas I-A determinants of donor phenotype were reduced on thymocytes from P1 x P2 leads to P1 chimeras; and thymocytes from P1 leads to P2 chimeras possessed H-2 and I-A determinants of host phenotype but showed reduction of donor I-A phenotype determinants. The appearance of host cell surface H-2 and I-A determinants on thymocytes from chimeras closely parallels the functional recognition of MHC determinants by T cells from chimeric mice and thus may be significantly related to the development of the self-recognition repertoire by maturing T cells

  9. Epigenetic mechanisms regulate MHC and antigen processing molecules in human embryonic and induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Beatriz Suárez-Alvarez

    2010-04-01

    Full Text Available Human embryonic stem cells (hESCs are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM components and NKG2D ligands (NKG2D-L in hESCs, induced pluripotent stem cells (iPSCs and NTera2 (NT2 teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1 and tapasin (TPN components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of beta2-microglobulin (beta2m light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and beta2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs. Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.

  10. Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

    Science.gov (United States)

    Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

    2010-01-01

    Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance

  11. Features of target cell lysis by class I and class II MHC restricted cytolytic T lymphocytes

    International Nuclear Information System (INIS)

    Maimone, M.M.; Morrison, L.A.; Braciale, V.L.; Braciale, T.J.

    1986-01-01

    The lytic activity of influenza virus-specific muvine cytolytic T lymphocyte (CTL) clones that are restricted by either H-2K/D (class I) or H-2I (class II) major histocompatibility (MHC) locus products was compared on an influenza virus-infected target cell expressing both K/D and I locus products. With the use of two in vitro measurements of cytotoxicity, conventional 51 Cr release, and detergent-releasable radiolabeled DNA (as a measure of nuclear disintegration in the early post-lethal hit period), the authors found no difference between class I and class II MHC-restricted CTL in the kinetics of target cell destruction. In addition, class II MHC-restricted antiviral CTL failed to show any lysis of radiolabeled bystander cells. Killing of labeled specific targets by these class II MHC-restricted CTL was also efficiently inhibited by unlabeled specific competitor cells in a cold target inhibition assay. In sum, these data suggest that class I and class II MHC-restricted CTL mediate target cell destruction by an essentially similar direct mechanism

  12. Improved pan-specific prediction of MHC class I peptide binding using a novel receptor clustering data partitioning strategy

    DEFF Research Database (Denmark)

    Mattsson, Andreas Holm; Kringelum, Jens Vindahl; Garde, C.

    2016-01-01

    Pan-specific prediction of receptor-ligand interaction is conventionally done using machine-learning methods that integrates information about both receptor and ligand primary sequences. To achieve optimal performance using machine learning, dealing with overfitting and data redundancy is critical....... Most often so-called ligand clustering methods have been used to deal with these issues in the context of pan-specific receptor-ligand predictions, and the MHC system the approach has proven highly effective for extrapolating information from a limited set of receptors with well characterized binding...

  13. Thymic repopulation following intrathymic injection of mouse bone marrow cells in MHC matched and mismatched recipients

    International Nuclear Information System (INIS)

    Chervenak, R.

    1986-01-01

    T cell precursors (pre-T cells) have traditionally been detected by their ability to repopulate the thymus of heavily irradiated mice following intravenous injection. Recently, Goldschneider et. al. developed an assay system which involves the direct injection of pre-T cells into the thymus. The authors used this technique to evaluate the ability of bone marrow cells to repopulate thymuses in various donor-host strain combinations. Sub-lethally irradiated (600R) mice were injected intrathymically with 2 x 10 6 bone marrow cells which differed from the recipient with respect to their Thy 1 allotype. The percentage of thymus cells expressing either the donor or recipient type Thy 1 marker was determined 14 to 21 days after injection. These experiments showed that in MHC matched donor-host combinations (AKR/cum → AKR/J and CBA/J → AKR/J), cells derived from the donor inoculum accounted for 40% to 75% of the total thymus population. MHC mismatched donor-host combinations (C57BL/6J → AKR/J and Balb/c → AKR/J) resulted in significantly less donor-type repopulation of the thymus. In these cases, donor repopulation typically ranged from 0% to 4%. The ability of the pre-T cells detected by intrathymic injection to proliferate in the thymic environment, therefore, appears to be influenced by the MHC. This may reflect commitment of pre-T cells to MHC haplotype recognition prior to their migration to the thymus

  14. Human CD4+ T cells lyse target cells via granzyme/perforin upon circumvention of MHC class II restriction by an antibody-like immunoreceptor.

    Science.gov (United States)

    Hombach, Andreas; Köhler, Heike; Rappl, Gunter; Abken, Hinrich

    2006-10-15

    Immune elimination of tumor cells requires the close cooperation between CD8+ CTL and CD4+ Th cells. We circumvent MHC class II-restriction of CD4+ T cells by expression of a recombinant immunoreceptor with an Ab-derived binding domain redirecting specificity. Human CD4+ T cells grafted with an immunoreceptor specific for carcinoembryonic Ag (CEA) are activated to proliferate and secrete cytokines upon binding to CEA+ target cells. Notably, redirected CD4+ T cells mediate cytolysis of CEA+ tumor cells with high efficiencies. Lysis by redirected CD4+ T cells is independent of death receptor signaling via TNF-alpha or Fas, but mediated by perforin and granzyme because cytolysis is inhibited by blocking the release of cytotoxic granules, but not by blocking of Fas ligand or TNF-alpha. CD4+ T cells redirected by Ab-derived immunoreceptors in a MHC class II-independent fashion substantially extend the power of an adoptive, Ag-triggered immunotherapy not only by CD4+ T cell help, but also by cytolytic effector functions. Because cytolysis is predominantly mediated via granzyme/perforin, target cells that are resistant to death receptor signaling become sensitive to a cytolytic attack by engineered CD4+ T cells.

  15. MHC-I Ligand Discovery Using Targeted Database Searches of Mass Spectrometry Data: Implications for T-Cell Immunotherapies

    DEFF Research Database (Denmark)

    Murphy, J. Patrick; Konda, Prathyusha; Kowalewski, Daniel J.

    2017-01-01

    Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I pept...

  16. Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

    Science.gov (United States)

    Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

    2017-01-15

    The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

  17. CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma.

    Science.gov (United States)

    Zhou, Yong; Zha, Jie; Lin, Zhijuan; Fang, Zhihong; Zeng, Hanyan; Zhao, Jintao; Luo, Yiming; Li, Zhifeng; Xu, Bing

    2018-01-15

    Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4 + T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19 + cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19 + cells than patients who did not show recurrence. Examining cytotoxic CD4 + T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4 + T cells. Also, frequency of cytotoxic CD4 + T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4 + T cells against autologous CD19 + cells was investigated. We found that the cytotoxic potential of CD4 + T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19 + cells presented a significant reduction after longer incubation with cytotoxic CD4 + T cells, suggesting that cytotoxic CD4 + T cells preferentially eliminated MHC II-expressing CD19 + cells. Blocking MHC II on CD19 + cells significantly reduced the cytolytic capacity of CD4 + T cells. Despite these discoveries, the frequency of cytotoxic CD4 + T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4 + T cells presented an MHC II-dependent cytotoxic potential against autologous CD19 + cells and could potentially represent a future treatment option for DLBCL. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

    DEFF Research Database (Denmark)

    La Rocca, Rosanna; Tallerico, Rossana; Hassan, Almosawy Talib

    2014-01-01

    In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were...... treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells...... between 700–1800 cm-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased...

  19. Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

    DEFF Research Database (Denmark)

    Joosten, I; Wauben, M H; Holewijn, M C

    1994-01-01

    New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC-peptide-TCR interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptides one...... characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides...

  20. MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

    Science.gov (United States)

    Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

    2015-01-01

    Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

  1. MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

    Science.gov (United States)

    Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

    2015-01-01

    Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

  2. MHC class II expression through a hitherto unknown pathway supports T helper cell-dependent immune responses: implications for MHC class II deficiency

    NARCIS (Netherlands)

    Buch, Thorsten; Polic, Bojan; Clausen, Björn E.; Weiss, Susanne; Akilli-Ozturk, Ozlem; Chang, Cheong-Hee; Flavell, Richard; Schulz, Ansgar; Jonjic, Stipan; Waisman, Ari; Förster, Irmgard

    2006-01-01

    MHC class II (MHCII) deficiency or bare lymphocyte syndrome (BLS) is a severe immunodeficiency characterized by deficient T helper (Th)-cell-dependent immunity. The disease is caused by defects of the MHCII promoter complex resulting in low or absent MHCII expression. We demonstrate in a murine

  3. T lymphocytes from irradiation chimeras repopulated with 13-day fetal liver cells recognize antigens only in association with self-MHC products

    International Nuclear Information System (INIS)

    Nisbet-Brown, E.; Diener, E.

    1986-01-01

    The restriction specificities of maturing thymocytes are determined by the Class II MHC antigens expressed by non-lymphoid thymic tissues. The proliferative response of mature T lymphocytes to antigen-presenting cells (APC) and antigen requires that the APC express the same MHC antigens as the thymus in which the T cells differentiated. Thus, in the two-way bone marrow chimera [A + B----(A x B)F1], T lymphocyte populations of A and B haplotypes have each acquired the potential to recognize antigens associated with either parental haplotype. In spite of the large body of work on MHC restriction, we still do not have a clear understanding of the mechanisms which impose self restriction. The chimeric model systems used previously to study MHC restriction have used adult bone marrow cells as the source of lymphoid precursors. During normal ontogeny, T cells are derived from precursors in the fetal liver and we felt that a direct comparison of T cells from fetal liver and bone marrow-repopulated animals would shed light on the development of MHC restriction specificities during T cell ontogeny in the thymus or prethymically. We found that parental T lymphocyte populations isolated from two-way fetal liver chimeras cooperated only with syngeneic APC, while those from bone marrow chimeras cooperated with APC of either parental haplotype. This suggests that fetal liver and bone marrow may not be equivalent sources of stem cells. Our results may be due to fundamental differences between thymocyte precursors in fetal liver and bone marrow, including the time course of their expression of T cell receptor gene products

  4. Evolution of MHC-based technologies used for detection of antigen-responsive T cells

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Hadrup, Sine Reker

    2017-01-01

    T cell-mediated recognition of peptide-major histocompatibility complex (pMHC) class I and II molecules is crucial for the control of intracellular pathogens and cancer, as well as for stimulation and maintenance of efficient cytotoxic responses. Such interactions may also play a role in the deve...

  5. High-throughput discovery of T cell epitopes in type 1 diabetes using DNA barcode labelledpeptide-MHC multimers

    DEFF Research Database (Denmark)

    Lyngaa, Rikke Birgitte; Bentzen, Amalie Kai; Overgaard, A. Julie

    2016-01-01

    applying a novel technology where the selection of MHC-multimer binding T cells is followed by amplification and sequencing of MHC multimer-associated DNA barcodes revealing their recognition. This technique enables simultaneous detection of >1000 specificities. Identifying post translational modifications...

  6. Design of glycopeptides used to investigate class II MHC binding and T-cell responses associated with autoimmune arthritis.

    Directory of Open Access Journals (Sweden)

    Ida E Andersson

    Full Text Available The glycopeptide fragment CII259-273 from type II collagen (CII binds to the murine A(q and human DR4 class II Major Histocompatibility Complex (MHC II proteins, which are associated with development of murine collagen-induced arthritis (CIA and rheumatoid arthritis (RA, respectively. It has been shown that CII259-273 can be used in therapeutic vaccination of CIA. This glycopeptide also elicits responses from T-cells obtained from RA patients, which indicates that it has an important role in RA as well. We now present a methodology for studies of (glycopeptide-receptor interactions based on a combination of structure-based virtual screening, ligand-based statistical molecular design and biological evaluations. This methodology included the design of a CII259-273 glycopeptide library in which two anchor positions crucial for binding in pockets of A(q and DR4 were varied. Synthesis and biological evaluation of the designed glycopeptides provided novel structure-activity relationship (SAR understanding of binding to A(q and DR4. Glycopeptides that retained high affinities for these MHC II proteins and induced strong responses in panels of T-cell hybridomas were also identified. An analysis of all the responses revealed groups of glycopeptides with different response patterns that are of high interest for vaccination studies in CIA. Moreover, the SAR understanding obtained in this study provides a platform for the design of second-generation glycopeptides with tuned MHC affinities and T-cell responses.

  7. Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

    Science.gov (United States)

    Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

    2016-02-01

    Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

  8. NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets

    DEFF Research Database (Denmark)

    Nielsen, Morten; Andreatta, Massimo

    2016-01-01

    Background: Binding of peptides to MHC class I molecules (MHC-I) is essential for antigen presentation to cytotoxic T-cells.Results: Here, we demonstrate how a simple alignment step allowing insertions and deletions in a pan-specific MHC-I binding machine-learning model enables combining informat...... specificities and ligand length scales, and demonstrated how this approach significantly improves the accuracy for prediction of peptide binding and identification of MHC ligands. The method is available at www.cbs.dtu.dk/services/NetMHCpan-3.0....

  9. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Directory of Open Access Journals (Sweden)

    Krishna Das

    Full Text Available In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY, were transfected with an expression plasmid encoding a β2m-specific single guide (sgRNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO clones did not give rise to tumors in syngeneic mice (C57BL/6N, unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  10. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    2008-09-01

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  11. Understanding TR binding to pMHC complexes: how does a TR scan many pMHC complexes yet preferentially bind to one.

    Directory of Open Access Journals (Sweden)

    Javed Mohammed Khan

    Full Text Available Understanding the basis of the binding of a T cell receptor (TR to the peptide-MHC (pMHC complex is essential due to the vital role it plays in adaptive immune response. We describe the use of computed binding (free energy (BE, TR paratope, pMHC epitope, molecular surface electrostatic potential (MSEP and calculated TR docking angle (θ to analyse 61 TR/pMHC crystallographic structures to comprehend TR/pMHC interaction. In doing so, we have successfully demonstrated a novel/rational approach for θ calculation, obtained a linear correlation between BE and θ without any "codon" or amino acid preference, provided an explanation for TR ability to scan many pMHC ligands yet specifically bind one, proposed a mechanism for pMHC recognition by TR leading to T cell activation and illustrated the importance of the peptide in determining TR specificity, challenging the "germline bias" theory.

  12. A T-Cell Receptor Breaks the Rules | Center for Cancer Research

    Science.gov (United States)

    Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their parents; and the antigen fragment, called a peptide epitope, is excised from one of thousands of possible proteins—originally part of an invading pathogen or a cancer cell—that T cells are capable of identifying and attacking. The framework of an MHC molecule holding a centrally displayed or “presented” peptide is what engages the TCR and triggers T-cell action. This role of MHC molecules presenting antigens to the TCR is a central tenet of immunology, with the fit between a TCR and the MHC framework actually “hardwired” into their three-dimensional structures.

  13. Expression of rat class I major histocompatibility complex (MHC) alloantigens and hepatocytes and hepatoma cells

    International Nuclear Information System (INIS)

    Hunt, J.M.; Desai, P.A.; Chakraborty, S.

    1986-01-01

    Altered expression of Class I MHC alloantigens has been reported for murine tumors, and may be associated with the tumorigenic phenotype of tumor cells. To characterize MHC Class I alloantigen expression on a chemically-induced transplantable rat hepatoma cell line, 17X, derived from a (WF x F344) F 1 rat, polyvalent anti-F344 and anti-WF rat alloantisera were first used to immunoprecipitate the rat RT1.A Class I MHC alloantigens expressed on primary (WF x F344) F 1 hepatocyptes in short-term monolayer cultures. Two-dimensional isoelectric focusing and SDS-PAGE of immunoprecipitates from 35 S-methionine-labeled (WF x F344) F 1 hepatocytes clearly resolved the RT1.A/sup u/ (WF) and RT1.A/sup LvI/ (F344) parental alloantigens. Identical radiolabeling and immunoprecipitation failed to detect either parental alloantigen on the 17X hepatoma cells. However, indirect immunofluorescence and immunoblot analyses demonstrated the presence of parental alloantigens on the 17X cells. Immunization of F344 rats but not of WF rats with 17X cells resulted in antibodies cytotoxic for normal (WF X F344) F 1 spleen cells in the presence of complement. These findings indicate that a combination of detection techniques will be necessary to characterize altered alloantigen expression on rat hepatoma cells

  14. Specific blockade by CD54 and MHC II of CD40-mediated signaling for B cell proliferation and survival

    DEFF Research Database (Denmark)

    Doyle, I S; Hollmann, C A; Crispe, I N

    2001-01-01

    Regulation of B lymphocyte proliferation is critical to maintenance of self-tolerance, and intercellular interactions are likely to signal such regulation. Here, we show that coligation of either the adhesion molecule ICAM-1/CD54 or MHC II with CD40 inhibited cell cycle progression and promoted...... these effects. Addition of BCR or IL-4 signals did not overcome the effect of ICAM-1 or MHC II on CD40-induced proliferation. FasL expression was not detected in B cell populations. These results show that MHC II and ICAM-1 specifically modulate CD40-mediated signaling, so inhibiting proliferation...

  15. Additional file 4: of MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis

    OpenAIRE

    Lipski, Deborah; Dewispelaere, RÊmi; Foucart, Vincent; Caspers, Laure; Defrance, Matthieu; Bruyns, Catherine; Willermain, François

    2017-01-01

    Figure S4. MHC class II expression in the retina during classical EAU. Three weeks after immunization, eye cryosections were prepared and stained for MHC class II (green) and IBA1 (red) or endoglin (magenta) detection. Cell nuclei were stained with Hoechst (blue). Each picture was chosen as representative of an experiment conducted on six or more animals. A. MHC class II and IBA1 expression. B. MHC class II and endoglin expression. (PPTX 7276 kb)

  16. 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.

  17. Immunotherapy of MHC class I-deficient tumours and epigenetic upregulation of MHC class I molecules on tumour cells

    Czech Academy of Sciences Publication Activity Database

    Reiniš, Milan; Manning, Jasper; Indrová, Marie; Přibylová, Hana; Bieblová, Jana; Šímová, Jana; Bubeník, Jan

    2007-01-01

    Roč. 20, Suppl. 1 (2007), S29-S29 ISSN 1107-3756. [World Congress on Advances in Oncology /12./ and International Symposium on Molecular Medicine /10./. 11.10.2007-12.10.2007, Hernissos] R&D Projects: GA ČR GA301/04/0492; GA ČR GA301/07/1410 Institutional research plan: CEZ:AV0Z50520514 Keywords : Immunotherapy * MHC I-deficient tumours * epigenetics Subject RIV: EB - Genetics ; Molecular Biology

  18. Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

    KAUST Repository

    La Rocca, Rosanna

    2014-12-26

    In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm−1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

  19. Mechanical stress downregulates MHC class I expression on human cancer cell membrane.

    Directory of Open Access Journals (Sweden)

    Rosanna La Rocca

    Full Text Available In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal, depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK cells cytotoxic recognition.

  20. IMGT unique numbering for MHC groove G-DOMAIN and MHC superfamily (MhcSF) G-LIKE-DOMAIN

    DEFF Research Database (Denmark)

    Lefranc, Marie-Paule; Duprat, E.; Kaas, Quentin

    2005-01-01

    IMGT, the international ImMunoGeneTics information system® (http://imgt.cines.fr) provides a common access to expertly annotated data on the genome, proteome, genetics and structure of immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC), and related proteins...

  1. Methods for quantifying T cell receptor binding affinities and thermodynamics

    Science.gov (United States)

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  2. High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells

    DEFF Research Database (Denmark)

    Bottley, G; Watherston, O G; Hiew, Y-L

    2007-01-01

    a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16- and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural...... killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy...

  3. 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.

  4. MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection

    Science.gov (United States)

    Rozanov, Dmitri V.; Rozanov, Nikita D.; Chiotti, Kami; Reddy, Ashok; Wilmarth, Phillip A.; David, Larry L.; Cha, Seung W.; Woo, Sunghee; Pevzner, Pavel; Bafna, Vineet; Burrows, Gregory G.; Rantala, Juha K.; Levin, Trevor; Anur, Pavana; Johnson-Camacho, Katie; Tabatabaei, Shaadi; Munson, Daniel J.; Bruno, Tullia C.; Slansky, Jill E.; Kappler, John W.; Hirano, Naoto; Boegel, Sebastian; Fox, Bernard A.; Egelston, Colt; Simons, Diana L.; Jimenez, Grecia; Lee, Peter P.; Gray, Joe W.; Spellman, Paul T.

    2018-01-01

    Breast cancer therapy based on amplifying a patient’s antitumor immune response depends on the availability of appropriate MHC class I-restricted, breast cancer-specific epitopes. To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cell lines. We determined the sequence of 3,196 MHC class I-bound peptides representing 1,921 proteins from a panel of 20 breast cancer cell lines including basal, luminal, and claudin-low subtypes. The data has been deposited to the ProteomeXchange with identifier PXD006406. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2,740. Of the unique peptides eluted, more than 1,750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, only 3 of these immunogenic peptides have been identified in breast cancer cells in earlier studies. MHC class I binding probability of eluted peptides was used to plot the distribution of MHC class I allele-specific peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. PMID:29331515

  5. MHC molecules protect T cell epitopes against proteolytic destruction

    DEFF Research Database (Denmark)

    Mouritsen, S; Meldal, M; Werdelin, O

    1992-01-01

    There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures...

  6. MHC class II restricted innate-like double negative T cells contribute to optimal primary and secondary immunity to Leishmania major.

    Science.gov (United States)

    Mou, Zhirong; Liu, Dong; Okwor, Ifeoma; Jia, Ping; Orihara, Kanami; Uzonna, Jude Ezeh

    2014-09-01

    Although it is generally believed that CD4(+) T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3(+)CD4(-)CD8(-) (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.

  7. The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

    Science.gov (United States)

    Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

    2014-03-01

    Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Impact of clonal competition for peptide-MHC complexes on the CD8[superscript +] T-cell repertoire selection in a persistent viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Wynn, Katherine K.; Fulton, Zara; Cooper, Leanne; Silins, Sharon L.; Gras, Stephanie; Archbold, Julia K.; Tynan, Fleur E.; Miles, John J.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie; Khanna, Rajiv (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

    2008-04-29

    CD8{sup +} T-cell responses to persistent viral infections are characterized by the accumulation of an oligoclonal T-cell repertoire and a reduction in the naive T-cell pool. However, the precise mechanism for this phenomenon remains elusive. Here we show that human cytomegalovirus (HCMV)-specific CD8{sup +} T cells recognizing distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T-cell repertoire or a private, diverse T-cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public T-cell receptor (TCR) was coincident with an atypical major histocompatibility complex (MHC)-peptide structure, in that the epitope adopted a helical conformation that bulged from the peptide-binding groove, while a diverse TCR profile was observed in response to the epitope that formed a flatter, more 'featureless' landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared with the T cells expressing diverse TCR. These findings provide new insights into our understanding on how the biology of antigen presentation in addition to the structural features of the pMHC-I might shape the T-cell repertoire and its phenotype.

  9. Virus encoded MHC-like decoys diversify the inhibitory KIR repertoire.

    Directory of Open Access Journals (Sweden)

    Paola Carrillo-Bustamante

    Full Text Available Natural killer (NK cells are circulating lymphocytes that play an important role in the control of viral infections and tumors. Their functions are regulated by several activating and inhibitory receptors. A subset of these receptors in human NK cells are the killer immunoglobulin-like receptors (KIRs, which interact with the highly polymorphic MHC class I molecules. One important function of NK cells is to detect cells that have down-regulated MHC expression (missing-self. Because MHC molecules have non polymorphic regions, their expression could have been monitored with a limited set of monomorphic receptors. Surprisingly, the KIR family has a remarkable genetic diversity, the function of which remains poorly understood. The mouse cytomegalovirus (MCMV is able to evade NK cell responses by coding "decoy" molecules that mimic MHC class I. This interaction was suggested to have driven the evolution of novel NK cell receptors. Inspired by the MCMV system, we develop an agent-based model of a host population infected with viruses that are able to evolve MHC down-regulation and decoy molecules. Our simulations show that specific recognition of MHC class I molecules by inhibitory KIRs provides excellent protection against viruses evolving decoys, and that the diversity of inhibitory KIRs will subsequently evolve as a result of the required discrimination between host MHC molecules and decoy molecules.

  10. Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability

    Energy Technology Data Exchange (ETDEWEB)

    Burrows, Scott R.; Chen, Zhenjun; Archbold, Julia K.; Tynan, Fleur E.; Beddoe, Travis; Kjer-Nielsen, Lars; Miles, John J.; Khanna, Rajiv; Moss, Denis J.; Liu, Yu Chih; Gras, Stephanie; Kostenko, Lyudmila; Brennan, Rebekah M.; Clements, Craig S.; Brooks, Andrew G.; Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

    2010-07-07

    {alpha}{beta} T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

  11. 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.

  12. MHC protocols

    National Research Council Canada - National Science Library

    Powis, Stephen H; Vaughan, Robert W

    2003-01-01

    ... because it contains genes encoding components of the complement pathway. The entire human MHC has recently been sequenced (1) and each subregion is now known to contain many other genes, a number of which have immunological functions. The study of polymorphism within the MHC is well established, because the region contains the highly polymorphic HLA genes. HLA polym...

  13. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

    Energy Technology Data Exchange (ETDEWEB)

    Manz, Boryana N. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Univ. of California, Berkeley, CA (United States); Jackson, Bryan L. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Petit, Rebecca S. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Dustin, Michael L. [New York School of Medicine, New York, NY (United States); Groves, Jay [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2011-05-31

    T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.

  14. Conformational variation of surface class II MHC proteins during myeloid dendritic cell differentiation accompanies structural changes in lysosomal MIIC

    Czech Academy of Sciences Publication Activity Database

    Potolicchio, I.; Chitta, S.; Xu, X.; Fonseca, D.; Crisi, G.; Hořejší, Václav; Strominger, J. L.; Stern, L. J.; Raposo, G.; Santambrogio, L.

    2005-01-01

    Roč. 175, č. 8 (2005), s. 4935-4947 ISSN 0022-1767 Institutional research plan: CEZ:AV0Z50520514 Keywords : MHC II * HLA-DR * dendritic cell Subject RIV: EC - Immunology Impact factor: 6.387, year: 2005

  15. MHC class I signaling in T cells leads to tyrosine kinase activity and PLC-gamma 1 phosphorylation

    DEFF Research Database (Denmark)

    Skov, S; Odum, Niels; Claesson, M H

    1995-01-01

    phosphorylation and the subsequent calcium response. The early tyrosine kinase activity was found to be dependent on expression of the TCR/CD3 complex and the CD45 molecule on the surface of the T cells. Furthermore, MHC-I cross-linking was shown to tyrosine phosphorylate PLC-gamma 1 (phospholipase C-gamma 1...

  16. Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4+T cells in natural SIV infection.

    Directory of Open Access Journals (Sweden)

    Michael Schindler

    2008-07-01

    Full Text Available Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500 and 15 animals with low (<500 CD4+ T-cells/microl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL-2 and programmed death (PD-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.

  17. MHC class II expression through a hitherto unknown pathway supports T helper cell-dependent immune responses: implications for MHC class II deficiency.

    Science.gov (United States)

    Buch, Thorsten; Polic, Bojan; Clausen, Björn E; Weiss, Susanne; Akilli-Ozturk, Ozlem; Chang, Cheong-Hee; Flavell, Richard; Schulz, Ansgar; Jonjic, Stipan; Waisman, Ari; Förster, Irmgard

    2006-02-15

    MHC class II (MHCII) deficiency or bare lymphocyte syndrome (BLS) is a severe immunodeficiency characterized by deficient T helper (Th)-cell-dependent immunity. The disease is caused by defects of the MHCII promoter complex resulting in low or absent MHCII expression. We demonstrate in a murine model of MHCII deficiency (RFX5- or CIITA-deficient mice) that residual MHCII expression by professional antigen-presenting cells (APCs) is sufficient to support activation of adoptively transferred Th cells. Furthermore, upon transplantation of WT thymic epithelium, we observed development of endogenous Th cells with restoration of Th-cell-dependent antibody responses and immunity to cytomegalovirus infection, thus opening the possibility of an alternative treatment regimen for BLS. Residual MHCII expression was further induced by the presence of Th cells and also other stimuli. Analysis of CIITA/RFX5 double-deficient animals revealed that this inducible MHCII expression is genetically independent of the known promoter complex and thus constitutes an alternative MHCII expression pathway. In these experiments, we also detected a novel repressive function of the RFX complex in the absence of CIITA.

  18. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.

    2014-01-01

    Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usa...

  19. Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

    2016-01-01

    -major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive...... cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer...

  20. Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

    DEFF Research Database (Denmark)

    Lamberth, K; Claesson, M H

    2001-01-01

    Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP......-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP...

  1. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    Science.gov (United States)

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  2. Generation and characterization of peptide-specific, MHC-restricted cytotoxic T lymphocyte (CTL) and helper T cell lines from unprimed T cells under microculture conditions.

    Science.gov (United States)

    Sambhara, S R; Upadhya, A G; Miller, R G

    1990-06-12

    We describe a microculture system for the generation of CTL and T helper cells against peptides. Tryptic digest and cyanogen bromide fragments of chicken ovalbumin and synthetic peptides of ovalbumin (323-339) and influenza virus (NP 365-380) were used to generate CTL and T helper lines from unprimed T cells. These lines were both peptide-specific and MHC-restricted. The relative ease of generating peptide-specific, MHC-restricted CTL and helper T cell lines with as few as 10(6) unprimed lymphocytes can be an efficient method of detecting potential immunogenic determinants of an antigen.

  3. Multiparametric analysis of host response to murine cytomegalovirus in MHC class I-disparate mice reveals primacy of Dk-licensed Ly49G2+ NK cells in viral control.

    Science.gov (United States)

    Prince, Jessica; Lundgren, Alyssa; Stadnisky, Michael D; Nash, William T; Beeber, Amira; Turner, Stephen D; Brown, Michael G

    2013-11-01

    MHC class I D(k) and Ly49G2 (G2) inhibitory receptor-expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without D(k), G2(+) NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, D(k) licenses G2(+) NK cells for effector activity. These data suggested that D(k)-licensed G2(+) NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/My × C57L offspring segregating D(k) were examined before infection and postinfection, including Ly49(+) NK subsets, receptor expression features, and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using the Pearson correlation, followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that G2 expression on naive blood NK cells was predictive of MCMV resistance. However, rapid G2(+) NK cell expansion following viral exposure occurred selectively in D(k) offspring; this response was more highly correlated with MCMV control than all other immune cell features. We infer that D(k)-licensed G2(+) NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. Therefore, MHC polymorphism regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.

  4. NLRC5/MHC class I transactivator is a target for immune evasion in cancer.

    Science.gov (United States)

    Yoshihama, Sayuri; Roszik, Jason; Downs, Isaac; Meissner, Torsten B; Vijayan, Saptha; Chapuy, Bjoern; Sidiq, Tabasum; Shipp, Margaret A; Lizee, Gregory A; Kobayashi, Koichi S

    2016-05-24

    Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.

  5. The role of NK1.1+ cells in the protection against MHC class I+ HPV16-associated tumours

    Czech Academy of Sciences Publication Activity Database

    Šímová, Jana; Bubeník, Jan; Bieblová, Jana; Jandlová, Táňa

    2004-01-01

    Roč. 50, č. 6 (2004), s. 200-202 ISSN 0015-5500 R&D Projects: GA AV ČR IAA5052203; GA AV ČR KSK5011112; GA MZd NC7148; GA MZd NR7807 Institutional research plan: CEZ:AV0Z5052915 Keywords : HPV16 * MHC class I expression * NK1.1+ cells Subject RIV: EC - Immunology Impact factor: 0.507, year: 2004

  6. Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

    DEFF Research Database (Denmark)

    Lamberth, K; Claesson, M H

    2001-01-01

    Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti-apoptotic......Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP......-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP...

  7. Neurons are MHC class I-dependent targets for CD8 T cells upon neurotropic viral infection.

    Directory of Open Access Journals (Sweden)

    Grégoire Chevalier

    2011-11-01

    Full Text Available Following infection of the central nervous system (CNS, the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL because they do not express major histocompatibility class I (MHC I molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV, in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and

  8. Pan-specific prediction of peptide-MHC Class I complex stability, a correlate of T cell immunogenicity

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Fenoy, Emilio; Harndahl, Mikkel

    2016-01-01

    Binding of peptides to MHC class I (MHC-I) molecules is the most selective event in the processing and presentation of Ags to CTL, and insights into the mechanisms that govern peptide-MHC-I binding should facilitate our understanding of CTL biology. Peptide-MHC-I interactions have traditionally b...

  9. MHC class II tetramers made from isolated recombinant α and β chains refolded with affinity-tagged peptides

    DEFF Research Database (Denmark)

    Braendstrup, Peter; Justesen, Sune Frederik Lamdahl; Osterbye, Thomas

    2013-01-01

    Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking...... effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding...... a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides...

  10. Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells1

    Science.gov (United States)

    Vyas, Jatin M.; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J. Christopher; Van der Veen, Annemarthe G.; Ploegh, Hidde L.

    2009-01-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. PMID:17513769

  11. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

    Science.gov (United States)

    Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

    2007-06-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

  12. Dimeric MHC-peptides inserted into an immunoglobulin scaffold as new immunotherapeutic agents

    Science.gov (United States)

    Goldberg, Burt; Bona, Constantin

    2011-01-01

    Abstract The interactions of the T cell receptor (TCR) with cognate MHC-peptide and co-stimulatory molecules expressed at surface of antigen presenting cells (APC) leads to activation or tolerance of T cells. The development of molecular biological tools allowed for the preparation of soluble MHC-peptide molecules as surrogate for the APC. A decade ago a monomeric class II MHC molecule in which the peptide was covalently linked to β-chain of class II molecule was generated. This type of molecule had a low-binding affinity and did not cause the multimerization of TCR. The requirement of multimerization of TCR led to development of a new class of reagents, chimeric peptides covalently linked to MHC that was dimerized via Fc fragment of an immunoglobulin and linked to 3′ end of the β-chain of MHC class II molecule. These soluble dimerized MHC-peptide chimeric molecules display high affinity for the TCR and caused multimerization of TCR without processing by an APC. Because dimeric molecules are devoid of co-stimulatory molecules interacting with CD28, a second signal, they induce anergy rather the activation of T cells. In this review, we compare the human and murine dimerized MHC class II-peptides and their effect on CD4+ T cells, particularly the generation of T regulatory cells, which make these chimeric molecules an appealing approach for the treatment of autoimmune diseases. PMID:21435177

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

    OpenAIRE

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

    2003-01-01

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

  14. The role of CD4+ T cells in cell-mediated immunity to LCMV: studies in MHC class I and class II deficient mice

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Marker, O; Thomsen, Allan Randrup

    1994-01-01

    Parameters of the virus-specific T-cell response were analysed in order to dissect the contribution of CD4+ and CD8+ T cells to cell-mediated immunity to lymphocytic choriomeningitis virus. In MHC class II deficient mice, initial T-cell responsiveness was not impaired, but virus clearance...... was delayed, and virus-specific Td activity declined more rapidly. Furthermore, class I restricted Tc memory appeared to be impaired in these mice. To directly evaluate the role of CD4+ cells in virus clearance and T-cell mediated inflammation, MHC class I deficient mice were also studied. No virus...... exudate. This low-grade response was associated with some degree of virus control as organ titres were lower in these animals than in matched T-cell deficient nu/nu mice or class I deficient mice treated with anti-CD4 monoclonal antibody. This confirms that CD4+ cells are not needed to induce a virus...

  15. Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

    KAUST Repository

    La Rocca, Rosanna; Tallerico, Rossana; Talib Hassan, Almosawy; Das, Gobind; Tadepally, Lakshmikanth; Matteucci, Marco; Liberale, Carlo; Mesuraca, Maria; Scumaci, Domenica; Gentile, Francesco; Cojoc, Gheorghe; Perozziello, Gerardo; Ammendolia, Antonio; Gallo, Adriana; Kä rre, Klas; Cuda, Giovanni; Candeloro, Patrizio; Di Fabrizio, Enzo M.; Carbone, Ennio

    2014-01-01

    bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm−1, indicated a relative concentration variation of MHC class I. PCA analysis was also

  16. Human umbilical cord mesenchymal stromal cells suppress MHC class II expression on rat vascular endothelium and prolong survival time of cardiac allograft

    Science.gov (United States)

    Qiu, Ying; Yun, Mark M; Han, Xia; Zhao, Ruidong; Zhou, Erxia; Yun, Sheng

    2014-01-01

    Background: Human umbilical cord mesenchymal stromal cells (UC-MSCs) have low immunogenicity and immune regulation. To investigate immunomodulatory effects of human UC-MSCs on MHC class II expression and allograft, we transplanted heart of transgenic rats with MHC class II expression on vascular endothelium. Methods: UC-MSCs were obtained from human umbilical cords and confirmed with flow cytometry analysis. Transgenic rat line was established using the construct of human MHC class II transactivator gene (CIITA) under mouse ICAM-2 promoter control. The induced MHC class II expression on transgenic rat vascular endothelial cells (VECs) was assessed with immunohistological staining. And the survival time of cardiac allograft was compared between the recipients with and without UC-MSC transfusion. Results: Flow cytometry confirmed that the human UC-MSCs were positive for CD29, CD44, CD73, CD90, CD105, CD271, and negative for CD34 and HLA-DR. Repeated infusion of human UC-MSCs reduced MHC class II expression on vascular endothelia of transplanted hearts, and increased survival time of allograft. The UC-MSCs increased regulatory cytokines IL10, transforming growth factor (TGF)-β1 and suppressed proinflammatory cytokines IL2 and IFN-γ in vivo. The UC-MSC culture supernatant had similar effects on cytokine expression, and decreased lymphocyte proliferation in vitro. Conclusions: Repeated transfusion of the human UC-MSCs reduced MHC class II expression on vascular endothelia and prolonged the survival time of rat cardiac allograft. PMID:25126177

  17. FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas

    DEFF Research Database (Denmark)

    Brown, P J; Wong, K K; Felce, S L

    2016-01-01

    The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II......) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes......, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (PABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone...

  18. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O

    1996-01-01

    Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

  19. MHC class I Dk locus and Ly49G2+ NK cells confer H-2k resistance to murine cytomegalovirus.

    Science.gov (United States)

    Xie, Xuefang; Stadnisky, Michael D; Brown, Michael G

    2009-06-01

    Essential NK cell-mediated murine CMV (MCMV) resistance is under histocompatibility-2(k) (H-2(k)) control in MA/My mice. We generated a panel of intra-H2(k) recombinant strains from congenic C57L.M-H2(k/b) (MCMV resistant) mice for precise genetic mapping of the critical interval. Recombination breakpoint sites were precisely mapped and MCMV resistance/susceptibility traits were determined for each of the new lines to identify the MHC locus. Strains C57L.M-H2(k)(R7) (MCMV resistant) and C57L.M-H2(k)(R2) (MCMV susceptible) are especially informative; we found that allelic variation in a 0.3-megabase interval in the class I D locus confers substantial difference in MCMV control phenotypes. When NK cell subsets responding to MCMV were examined, we found that Ly49G2(+) NK cells rapidly expand and selectively acquire an enhanced capacity for cytolytic functions only in C57L.M-H2(k)(R7). We further show that depletion of Ly49G2(+) NK cells before infection abrogated MCMV resistance in C57L.M-H2(k)(R7). We conclude that the MHC class I D locus prompts expansion and activation of Ly49G2(+) NK cells that are needed in H-2(k) MCMV resistance.

  20. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    Science.gov (United States)

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  1. Circumsporozoite Protein-Specific Kd-Restricted CD8+ T Cells Mediate Protective Antimalaria Immunity in Sporozoite-Immunized MHC-I-Kd Transgenic Mice

    Directory of Open Access Journals (Sweden)

    Jing Huang

    2014-01-01

    Full Text Available Although the roles of CD8+ T cells and a major preerythrocytic antigen, the circumsporozoite (CS protein, in contributing protective antimalaria immunity induced by radiation-attenuated sporozoites, have been shown by a number of studies, the extent to which these players contribute to antimalaria immunity is still unknown. To address this question, we have generated C57BL/6 (B6 transgenic (Tg mice, expressing Kd molecules under the MHC-I promoter, called MHC-I-Kd-Tg mice. In this study, we first determined that a single immunizing dose of IrPySpz induced a significant level of antimalaria protective immunity in MHC-I-Kd-Tg mice but not in B6 mice. Then, by depleting various T-cell subsets in vivo, we determined that CD8+ T cells are the main mediator of the protective immunity induced by IrPySpz. Furthermore, when we immunized (MHC-I-Kd-Tg × CS-Tg F1 mice with IrPySpz after crossing MHC-I-Kd-Tg mice with PyCS-transgenic mice (CS-Tg, which are unable to mount PyCS-specific immunity, we found that IrPySpz immunization failed to induce protective antimalaria immunity in (MHC-I-Kd-Tg × CS-Tg F1 mice, thus indicating the absence of PyCS antigen-dependent immunity in these mice. These results indicate that protective antimalaria immunity induced by IrPySpz in MHC-I-Kd-Tg mice is mediated by CS protein-specific, Kd-restricted CD8+ T cells.

  2. How nonuniform contact profiles of T cell receptors modulate thymic selection outcomes

    Science.gov (United States)

    Chen, Hanrong; Chakraborty, Arup K.; Kardar, Mehran

    2018-03-01

    T cell receptors (TCRs) bind foreign or self-peptides attached to major histocompatibility complex (MHC) molecules, and the strength of this interaction determines T cell activation. Optimizing the ability of T cells to recognize a diversity of foreign peptides yet be tolerant of self-peptides is crucial for the adaptive immune system to properly function. This is achieved by selection of T cells in the thymus, where immature T cells expressing unique, stochastically generated TCRs interact with a large number of self-peptide-MHC; if a TCR does not bind strongly enough to any self-peptide-MHC, or too strongly with at least one self-peptide-MHC, the T cell dies. Past theoretical work cast thymic selection as an extreme value problem and characterized the statistical enrichment or depletion of amino acids in the postselection TCR repertoire, showing how T cells are selected to be able to specifically recognize peptides derived from diverse pathogens yet have limited self-reactivity. Here, we investigate how the diversity of the postselection TCR repertoire is modified when TCRs make nonuniform contacts with peptide-MHC. Specifically, we were motivated by recent experiments showing that amino acids at certain positions of a TCR sequence have large effects on thymic selection outcomes, and crystal structure data that reveal a nonuniform contact profile between a TCR and its peptide-MHC ligand. Using a representative TCR contact profile as an illustration, we show via simulations that the statistical enrichment or depletion of amino acids now varies by position according to the contact profile, and, importantly, it depends on the implementation of nonuniform contacts during thymic selection. We explain these nontrivial results analytically. Our study has implications for understanding the selection forces that shape the functionality of the postselection TCR repertoire.

  3. Immunization with MHC class I-negative but not -positive HPV16-associated tumour cells inhibits growth of MHC class I-negative tumours

    Czech Academy of Sciences Publication Activity Database

    Reiniš, Milan; Šímová, Jana; Indrová, Marie; Bieblová, Jana; Přibylová, Hana; Moravcová, Simona; Jandlová, Táňa; Bubeník, Jan

    2007-01-01

    Roč. 30, č. 4 (2007), s. 1011-1017 ISSN 1019-6439 R&D Projects: GA MZd NR7807 EU Projects: European Commission(XE) 18933 - CLINIGENE Grant - others:Liga proti rakovině, Praha(CZ) XX Institutional research plan: CEZ:AV0Z50520514 Keywords : HPV16 * MHC class I-deficient tumours * immunologic crossreaction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.295, year: 2007

  4. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

  5. Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. I. Recognition by two alpha beta T cell receptors

    DEFF Research Database (Denmark)

    Rognan, D; Stryhn, A; Fugger, L

    2000-01-01

    dynamics. Next, three-dimensional models of two different T cell receptors (TCRs) both specific for the Ha255-262/Kk complex were generated based on previously published TCR X-ray structures. Finally, guided by the recently published X-ray structures of ternary TCR/peptide/MHC-I complexes, the TCR models...... the models. They were found to account well for the experimentally obtained data, lending considerable support to the proposed models and suggesting a universal docking mode for alpha beta TCRs to MHC-peptide complexes. Such models may also be useful in guiding future rational experimentation....

  6. A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates

    Science.gov (United States)

    Rosner, Cornelia; Neff, Jennifer; Roos, Christian; Eberle, Manfred; Aujard, Fabienne; Münch, Claudia; Schempp, Werner; Carrington, Mary; Shiina, Takashi; Inoko, Hidetoshi; Knaust, Florian; Coggill, Penny; Sehra, Harminder; Beck, Stephan; Abi-Rached, Laurent; Reinhardt, Richard; Walter, Lutz

    2009-01-01

    There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire. PMID:19834558

  7. MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Johansen, B

    1999-01-01

    B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine...

  8. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains

    DEFF Research Database (Denmark)

    Lefranc, Marie-Paule; Pommié, Christelle; Kaas, Quentin

    2005-01-01

    IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr) provides a common access to expertly annotated data on the genome, proteome, genetics and structure of immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC), and related proteins...

  9. A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells

    DEFF Research Database (Denmark)

    Andersen, P S; Stryhn, A; Hansen, B E

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might ...

  10. Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings

    Directory of Open Access Journals (Sweden)

    Cory M. Ayres

    2017-08-01

    Full Text Available Structural biology of peptides presented by class I and class II MHC proteins has transformed immunology, impacting our understanding of fundamental immune mechanisms and allowing researchers to rationalize immunogenicity and design novel vaccines. However, proteins are not static structures as often inferred from crystallographic structures. Their components move and breathe individually and collectively over a range of timescales. Peptides bound within MHC peptide-binding grooves are no exception and their motions have been shown to impact recognition by T cell and other receptors in ways that influence function. Furthermore, peptides tune the motions of MHC proteins themselves, which impacts recognition of peptide/MHC complexes by other proteins. Here, we review the motional properties of peptides in MHC binding grooves and discuss how peptide properties can influence MHC motions. We briefly review theoretical concepts about protein motion and highlight key data that illustrate immunological consequences. We focus primarily on class I systems due to greater availability of data, but segue into class II systems as the concepts and consequences overlap. We suggest that characterization of the dynamic “energy landscapes” of peptide/MHC complexes and the resulting functional consequences is one of the next frontiers in structural immunology.

  11. T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

    Directory of Open Access Journals (Sweden)

    Keith R Miller

    Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

  12. MHC class II expression in lung cancer.

    Science.gov (United States)

    He, Yayi; Rozeboom, Leslie; Rivard, Christopher J; Ellison, Kim; Dziadziuszko, Rafal; Yu, Hui; Zhou, Caicun; Hirsch, Fred R

    2017-10-01

    Immunotherapy is an exciting development in lung cancer research. In this study we described major histocompatibility complex (MHC) Class II protein expression in lung cancer cell lines and patient tissues. We studied MHC Class II (DP, DQ, DR) (CR3/43, Abcam) protein expression in 55 non-small cell lung cancer (NSCLC) cell lines, 42 small cell lung cancer (SCLC) cell lines and 278 lung cancer patient tissues by immunohistochemistry (IHC). Seven (12.7%) NSCLC cell lines were positive for MHC Class II. No SCLC cell lines were found to be MHC Class II positive. We assessed 139 lung cancer samples available in the Hirsch Lab for MHC Class II. There was no positive MHC Class II staining on SCLC tumor cells. MHC Class II expression on TILs in SCLC was significantly lower than that on TILs in NSCLC (P<0.001). MHC Class II was also assessed in an additional 139 NSCLC tumor tissues from Medical University of Gdansk, Poland. Patients with positive staining of MHC Class II on TILs had longer regression-free survival (RFS) and overall survival (OS) than those whose TILs were MHC Class II negative (2.980 years, 95% CI 1.628-4.332 vs. 1.050 years, 95% CI 0.556-1.554, P=0.028) (3.230 years, 95% CI 2.617-3.843 vs. 1.390 years, 95% CI 0.629-2.151, P=0.014). MHC Class II was expressed both in NSCLC cell lines and tissues. However, MHC Class II was not detected in SCLC cell lines or tissue tumor cells. MHC Class II expression was lower on SCLC TILs than on NSCLC TILs. Loss of expression of MHC Class II on SCLC tumor cells and reduced expression on SCLC TILs may be a means of escaping anti-cancer immunity. Higher MHC Class II expression on TILs was correlated with better prognosis in patients with NSCLC. Copyright © 2017. Published by Elsevier B.V.

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

    Science.gov (United States)

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

    2017-11-01

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

  14. Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies

    Directory of Open Access Journals (Sweden)

    Jennifer D. Stone

    2013-08-01

    Full Text Available Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional alpha-beta T cell receptor (TCR against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR consisting of a single-chain antibody as an Fv fragment (scFv linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the alpha-beta TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

  15. NK cells and missing self recognition : genetic control, mhc class i dependent education and potential use in cancer therapy

    OpenAIRE

    Wickström, Stina L

    2015-01-01

    NK cells belong to the innate immune system and are important in the defense against virus infections and malignant cells. They mediate their effector functions via release of cytotoxic granules and by cytokine production which can influence the status of other (immune) cells. NK cells are regulated by germline encoded receptors, both activating and inhibitory, recognizing molecules that are induced upon infection or cellular stress and self ligands respectively. Ly49 receptors (Ly49r) make u...

  16. A novel protective MHC-I haplotype not associated with dominant Gag-specific CD8+ T-cell responses in SIVmac239 infection of Burmese rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Naofumi Takahashi

    Full Text Available Several major histocompatibility complex class I (MHC-I alleles are associated with lower viral loads and slower disease progression in human immunodeficiency virus (HIV and simian immunodeficiency virus (SIV infections. Immune-correlates analyses in these MHC-I-related HIV/SIV controllers would lead to elucidation of the mechanism for viral control. Viral control associated with some protective MHC-I alleles is attributed to CD8+ T-cell responses targeting Gag epitopes. We have been trying to know the mechanism of SIV control in multiple groups of Burmese rhesus macaques sharing MHC-I genotypes at the haplotype level. Here, we found a protective MHC-I haplotype, 90-010-Id (D, which is not associated with dominant Gag-specific CD8+ T-cell responses. Viral loads in five D+ animals became significantly lower than those in our previous cohorts after 6 months. Most D+ animals showed predominant Nef-specific but not Gag-specific CD8+ T-cell responses after SIV challenge. Further analyses suggested two Nef-epitope-specific CD8+ T-cell responses exerting strong suppressive pressure on SIV replication. Another set of five D+ animals that received a prophylactic vaccine using a Gag-expressing Sendai virus vector showed significantly reduced viral loads compared to unvaccinated D+ animals at 3 months, suggesting rapid SIV control by Gag-specific CD8+ T-cell responses in addition to Nef-specific ones. These results present a pattern of SIV control with involvement of non-Gag antigen-specific CD8+ T-cell responses.

  17. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  18. 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.

  19. MHC motif viewer

    DEFF Research Database (Denmark)

    Rapin, Nicolas Philippe Jean-Pierre; Hoof, Ilka; Lund, Ole

    2008-01-01

    . Algorithms that predict which peptides MHC molecules bind have recently been developed and cover many different alleles, but the utility of these algorithms is hampered by the lack of tools for browsing and comparing the specificity of these molecules. We have, therefore, developed a web server, MHC motif....... A special viewing feature, MHC fight, allows for display of the specificity of two different MHC molecules side by side. We show how the web server can be used to discover and display surprising similarities as well as differences between MHC molecules within and between different species. The MHC motif...

  20. Characterization of the Canine MHC Class I DLA-88*50101 Peptide Binding Motif as a Prerequisite for Canine T Cell Immunotherapy.

    Directory of Open Access Journals (Sweden)

    Sharon M Barth

    Full Text Available There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients.

  1. Donor T cells primed on leukemia lysate-pulsed recipient APCs mediate strong graft-versus-leukemia effects across MHC barriers in full chimeras

    OpenAIRE

    Ghosh, Arnab; Koestner, Wolfgang; Hapke, Martin; Schlaphoff, Verena; Länger, Florian; Baumann, Rolf; Koenecke, Christian; Cornberg, Markus; Welte, Karl; Blazar, Bruce R.; Sauer, Martin G.

    2009-01-01

    Antigen-presenting cells (APCs) of host origin drive graft-versus-leukemia (GVL) effects but can also trigger life-threatening graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) across major histocompatibility complex (MHC) barriers. We show that in vitro priming of donor lymphocytes can circumvent the need of recipient-derived APCs in vivo for mediating robust GVL effects and significantly diminishes the risk of severe GVHD. In vitro, generated and expanded T cel...

  2. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    Science.gov (United States)

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

  3. C57BL/6 mice need MHC class II Aq to develop collagen-induced arthritis dependent on autoreactive T cells.

    Science.gov (United States)

    Bäcklund, Johan; Li, Cuiqin; Jansson, Erik; Carlsen, Stefan; Merky, Patrick; Nandakumar, Kutty-Selva; Haag, Sabrina; Ytterberg, Jimmy; Zubarev, Roman A; Holmdahl, Rikard

    2013-07-01

    Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified. To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII. Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic. The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.

  4. A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.

    Science.gov (United States)

    Ross, Peter; Holmes, Jennifer C; Gojanovich, Gregory S; Hess, Paul R

    2012-12-15

    Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. NK1.1+ cells are important for the development of protective immunity against MHC I-deficient, HPV16-associated tumours

    Czech Academy of Sciences Publication Activity Database

    Indrová, Marie; Šímová, Jana; Bieblová, Jana; Bubeník, Jan; Reiniš, Milan

    2011-01-01

    Roč. 25, č. 1 (2011), s. 281-288 ISSN 1021-335X R&D Projects: GA AV ČR IAA500520807 Grant - others:EU-FP6-NOE(XE) Project 018933 Institutional research plan: CEZ:AV0Z50520514 Keywords : MHC class I-deficient tumours * CD8+, CD4+, NK1.1+cell subpopulations * interferon gamma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.835, year: 2011

  6. CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

    Science.gov (United States)

    Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo

    2014-11-19

    Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired

  7. Effect of Increased Cyclic AMP Concentration on Muscle Protein Synthesis and Beta-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture

    Science.gov (United States)

    Young, R. B.; Vaughn, J. R.; Bridge, K. Y.; Smith, C. K.

    1998-01-01

    Analogies of epinephrine are known to cause hypertrophy of skeletal muscle when fed to animals. These compounds presumably exert their physiological action through interaction with the P-adrenergic receptor. Since the intracellular signal generated by the Beta-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation of cAMP by treatment with forskolin would alter muscle protein metabolism and P-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 micrometers forskolin for a total of three days. At the end of the treatment period, both the concentration of cAMP and the quantity of myosin heavy chain (MHC) were measured. Concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. In contrast, the quantity of MHC was increased approximately 50% above control cells at 0.2 micrometers forskolin, but exhibited a gradual decline at higher levels of forskolin so that the quantity of MHC in cells treated with 30 micrometers forskolin was not significantly different from controls. Curiously, the intracellular concentration of cAMP which elicited the maximum increase in the quantity of MHC was only 40% higher than cAMP concentration in control cells.

  8. A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation

    NARCIS (Netherlands)

    Paul, Petra; van den Hoorn, Tineke; Jongsma, Marlieke L. M.; Bakker, Mark J.; Hengeveld, Rutger; Janssen, Lennert; Cresswell, Peter; Egan, David A.; van Ham, Marieke; ten Brinke, Anja; Ovaa, Huib; Beijersbergen, Roderick L.; Kuijl, Coenraad; Neefjes, Jacques

    2011-01-01

    MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and

  9. Immune receptors involved in Streptococcus suis recognition by dendritic cells.

    Directory of Open Access Journals (Sweden)

    Marie-Pier Lecours

    Full Text Available Streptococcus suis is an important swine pathogen and an emerging zoonotic agent of septicemia and meningitis. Knowledge on host immune responses towards S. suis, and strategies used by this pathogen for subversion of these responses is scarce. The objective of this study was to identify the immune receptors involved in S. suis recognition by dendritic cells (DCs. Production of cytokines and expression of co-stimulatory molecules by DCs were shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize S. suis and become activated mostly through Toll-like receptor (TLR signaling. Supporting this fact, TLR2(-/- DCs were severely impaired in the release of several cytokines and the surface expression of CD86 and MHC-II. The release of IL-12p70 and CXC10, and the expression of CD40 were found to depend on signaling by both TLR2 and TLR9. The release of IL-23 and CXCL1 were partially dependent on NOD2. Finally, despite the fact that MyD88 signaling was crucial for DC activation and maturation, MyD88-dependent pathways were not implicated in S. suis internalization by DCs. This first study on receptors involved in DC activation by S. suis suggests a major involvement of MyD88 signaling pathways, mainly (but not exclusively through TLR2. A multimodal recognition involving a combination of different receptors seems essential for DC effective response to S. suis.

  10. MHC class II ligation induces CD58 (LFA-3)-mediated adhesion in human T cells

    DEFF Research Database (Denmark)

    Nielsen, M; Gerwien, J; Geisler, C

    1998-01-01

    ligation induces homotypic adhesion in both beta2-integrin-positive and negative, CD4-positive T cell lines. Anti-CD18 monoclonal antibody (mAb) weakly inhibited the adhesion response in beta2-integrin-positive T cells and had no effect on beta2-integrin-negative T cells. In contrast, an anti-CD58 (LFA-3...

  11. Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex

    OpenAIRE

    1984-01-01

    Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) show...

  12. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B

    DEFF Research Database (Denmark)

    Skov, Søren; Pedersen, Marianne Terndrup; Andresen, Lars

    2005-01-01

    We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either...

  13. Antigen recognition by MHC-incompatible cells of a human mismatched chimera

    NARCIS (Netherlands)

    Roncarolo, M. G.; Yssel, H.; Touraine, J. L.; Bacchetta, R.; Gebuhrer, L.; de Vries, J. E.; Spits, H.

    1988-01-01

    Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones

  14. Identification of SIV Nef CD8(+) T cell epitopes restricted by a MHC class I haplotype associated with lower viral loads in a macaque AIDS model.

    Science.gov (United States)

    Nomura, Takushi; Yamamoto, Hiroyuki; Takahashi, Naofumi; Naruse, Taeko K; Kimura, Akinori; Matano, Tetsuro

    2014-07-25

    Virus-specific CD8(+) T-cell responses are crucial for the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. Multiple studies on HIV-infected individuals and SIV-infected macaques have indicated association of several major histocompatibility complex class I (MHC-I) genotypes with lower viral loads and delayed AIDS progression. Understanding of the viral control mechanism associated with these MHC-I genotypes would contribute to the development of intervention strategy for HIV control. We have previously reported a rhesus MHC-I haplotype, 90-120-Ia, associated with lower viral loads after SIVmac239 infection. Gag206-216 and Gag241-249 epitope-specific CD8(+) T-cell responses have been shown to play a central role in the reduction of viral loads, whereas the effect of Nef-specific CD8(+) T-cell responses induced in all the 90-120-Ia(+) macaques on SIV replication remains unknown. Here, we identified three CD8(+) T-cell epitopes, Nef9-19, Nef89-97, and Nef193-203, associated with 90-120-Ia. Nef9-19 and Nef193-203 epitope-specific CD8(+) T-cell responses frequently selected for mutations resulting in viral escape from recognition by these CD8(+) T cells, indicating that these CD8(+) T cells exert strong suppressive pressure on SIV replication. Results would be useful for elucidation of the viral control mechanism associated with 90-120-Ia. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Docosahexaenoic acid modifies the clustering and size of lipid rafts and the lateral organization and surface expression of MHC class I of EL4 cells.

    Science.gov (United States)

    Shaikh, Saame Raza; Rockett, Benjamin Drew; Salameh, Muhammad; Carraway, Kristen

    2009-09-01

    An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

  16. 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.

  17. 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.

  18. MHC-based detection of antigen-specific CD8(+) T cell responses

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker; Schumacher, Nana Maria Pii

    2010-01-01

    The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different...

  19. Dendritic cells sensitize TCRs through self-MHC-mediated Src family kinase activation

    Czech Academy of Sciences Publication Activity Database

    Meraner, P.; Hořejší, Václav; Wolpl, A.; Fischer, G.F.; Stingl, G.; Maurer, D.

    2007-01-01

    Roč. 178, č. 4 (2007), s. 2262-2271 ISSN 0022-1767 Institutional research plan: CEZ:AV0Z50520514 Keywords : TCR * dendritic cells * Src kinases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.068, year: 2007

  20. Exhaustion of CTL memory and recrudescence of viremia in lymphocytic choriomeningitis virus-infected MHC class II-deficient mice and B cell-deficient mice

    DEFF Research Database (Denmark)

    Thomsen, Allan Randrup; Johansen, J; Marker, O

    1996-01-01

    To study the contribution of CD4+ T cells and B cells to antiviral immunity and long term virus control, MHC class II-deficient and B cell-deficient mice were infected with lymphocytic choriomeningitis virus. In class II-deficient mice, which lack CD4+ T cells, the primary CTL response is virtually...... this phenomenon could reflect participation of B cells and/or Abs in long term virus control, similar experiments were performed with mice that do not have mature B cells because of a disrupted membrane exon of the mu chain gene. In these mice, the cell-mediated immune response was slightly delayed, but transient...... and that in their absence, the virus-specific CTL potential becomes exhausted. Together our results indicate that while CD8+ cells play a dominant role in acute virus control, all three major components of the immune system are required for long term virus control....

  1. Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

    DEFF Research Database (Denmark)

    Werdelin, O; Mouritsen, S; Petersen, B L

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...... molecules are the questions discussed in this review. To us, the entire concept of processing has appeal not only because it explains some hitherto well-established, but poorly understood, phenomena such as the fact that T lymphocytes focus their attention entirely upon antigens on other cells. It has...

  2. Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16

    Czech Academy of Sciences Publication Activity Database

    Šmahel, M.; Sobotková, E.; Bubeník, Jan; Šímová, Jana; Žák, R.; Ludvíková, V.; Mikyšková, Romana; Kovařík, J.; Jelínek, F.; Povýšil, C.; Marinov, J.; Vonka, V.

    2001-01-01

    Roč. 84, č. 3 (2001), s. 374-380 ISSN 0007-0920 R&D Projects: GA MZd NC5900; GA MZd NC5526; GA ČR GA312/99/0542; GA ČR GA312/98/0826 Institutional research plan: CEZ:AV0Z5052915 Keywords : human papillomavirus * E6/E7 oncogenes * cell transformation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.942, year: 2001

  3. An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4+ T Cells Controls Obesity-Induced Inflammation

    Directory of Open Access Journals (Sweden)

    Kae Won Cho

    2014-10-01

    Full Text Available An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4+ T cells by unclear mechanisms. We have examined whether interactions between adipose tissue macrophages (ATMs and CD4+ T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen-dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHC II showed protection from diet-induced obesity. Deletion of MHC II expression in macrophages led to an adipose tissue-specific decrease in the effector/memory CD4+ T cells, attenuation of CD11c+ ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c+ ATMs in obese mice. These studies demonstrate the importance of MHCII-restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.

  4. Helper T cell epitope-mapping reveals MHC-peptide binding affinities that correlate with T helper cell responses to pneumococcal surface protein A.

    Directory of Open Access Journals (Sweden)

    Rajesh Singh

    2010-02-01

    Full Text Available Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC class II. We also characterized spleen- and cervical lymph node (CLN-derived helper T lymphocyte (HTL cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA(199-246 consistently caused the greatest IFN-gamma, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4(+ T cells isolated from S. pneumonia strain EF3030-challeged F(1 (B6xBALB/c mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA(199-246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.

  5. Molecular regulation of MHC class I chain-related protein A expression after HDAC-inhibitor treatment of Jurkat T cells

    DEFF Research Database (Denmark)

    Andresen, Lars; Jensen, Helle; Pedersen, Marianne T

    2007-01-01

    In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA......1 site from position -113 to -93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity...

  6. microRNA 125a Regulates MHC-I Expression on Esophageal Adenocarcinoma Cells, Associated With Suppression of Anti-tumor Immune Response and Poor Outcomes of Patients.

    Science.gov (United States)

    Mari, Luigi; Hoefnagel, Sanne J M; Zito, Domenico; van de Meent, Marian; van Endert, Peter; Calpe, Silvia; Sancho Serra, Maria Del Carmen; Heemskerk, Mirjam H M; van Laarhoven, Hanneke W M; Hulshof, Maarten C C M; Gisbertz, Susanne S; Medema, Jan Paul; van Berge Henegouwen, Mark I; Meijer, Sybren L; Bergman, Jacques J G H M; Milano, Francesca; Krishnadath, Kausilia K

    2018-06-07

    Immune checkpoint inhibition may affect growth or progression of highly aggressive cancers, such as esophageal adenocarcinoma (EAC). We investigated the regulation of expression of major histocompatibility complex, class 1 (MHC-I) proteins (encoded by HLA-A, HLA-B, and HLA-C) and the immune response to EACs in patient samples. We performed quantitative PCR array analyses of OE33 cells and OE19 cells, which express different levels of the ATP binding cassette subfamily B member 1 (TAP1) and TAP2, required for antigen presentation by MHC-I, to identify microRNAs that regulate their expression. We performed luciferase assays to validate interactions between microRNAs and potential targets. We overexpressed candidate microRNAs in OE33, FLO-1, and OACP4 C cell lines and performed quantitative PCR, immunoblot, and flow cytometry analyses to identify changes in mRNA and protein expression; we studied the effects of cytotoxic T cells. We performed microRNA in situ hybridization, RNA-sequencing, and immunohistochemical analyses of tumor tissues from 51 untreated patients with EAC in the Netherlands. Clinical and survival data were collected for patients, and EACs subtypes were determined. We found OE19 cells to have increased levels of 7 microRNAs. Of these, we found binding sites for microRNA 125a (MIR125a)-5p in the 3'UTR of the TAP2 mRNA and binding sites for MIR148a-3p in 3'UTRs of HLA-A, HLA-B, and HLA-C mRNAs. Overexpression of these microRNAs reduced expression of TAP2 in OE33, FLO-1, and OACP4 C cells, and reduced cell-surface levels of MHC-I. OE33 cells that expressed the viral peptide BZLF1 were killed by cytotoxic T cells, whereas OE33 that overexpressed MIR125a-5p or MIR 148a along with BZLF1 were not. In EAC and non-tumor tissues, levels of MIR125a-5p correlated inversely with levels of TAP2 protein. High expression of TAP1 by EAC correlated with significantly shorter overall survival times of patients. EACs that expressed high levels of TAP1 and genes involved

  7. Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.

    Directory of Open Access Journals (Sweden)

    Christoph Thelemann

    Full Text Available Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD and involve CD4(+ T cells, which are activated by major histocompatibility complex class II (MHCII molecules on antigen-presenting cells (APCs. However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC affects CD4(+ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL-10 receptor-blocking antibodies (anti-IL10R mAb. To assess the role of interferon (IFN-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+ T-helper type (Th1 cells - but not group 3 innate lymphoid cells (ILCs or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+ T cells and forkhead box P3 (FoxP3(+ regulatory T (Treg cells. IFN-γ produced mainly by CD4(+ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

  8. Binding of human beta 2-microglobulin to murine EL4 thymoma cells upregulates MHC class I heavy-chain epitopes, inhibits IL-2 secretion and induces resistance to killing by natural killer cells

    DEFF Research Database (Denmark)

    Claësson, M H; Nissen, Mogens Holst

    1994-01-01

    line (ABLS-8), X63 B-lymphoma cells and YAC cells did not bind h beta 2m. In two of the T lymphomas, EL4 and BW5147, binding of h beta 2m led to an increase in major histocompatibility complex class I (MHC-I) heavy-chain epitope expression as measured by anti-H-2K/D antibody binding and FACS analysis....... EL4 cells which had bound h beta 2m decreased their rate of constitutive IL-2 secretion and became resistant to activated natural killer (NK) cell killing. The present data suggest the binding of h beta 2m to mouse T cells leads to conformational changes of MHC-I heavy chains which influence both...

  9. Targeting Multiple Tumors Using T-Cells Engineered to Express a Natural Cytotoxicity Receptor 2-Based Chimeric Receptor

    Directory of Open Access Journals (Sweden)

    Vasyl Eisenberg

    2017-09-01

    Full Text Available Recent developments in cancer treatment are demonstrating the increasing and powerful potential of immunotherapeutic strategies. In this regard, the adoptive transfer of tumor-specific T-lymphocytes approaches can lead to tumor regression in cancer patients. More recently, the use of T-cells genetically engineered to express cancer-specific receptors such as the anti-CD19 chimeric antigen receptor (CAR continues to show promise for the treatment of hematological malignancies. Still, there is a crucial need to develop efficient CAR-T cell approaches for the treatment of solid tumors. It has been shown that other lymphocytes such as natural killer (NK cells can demonstrate potent antitumor function—nonetheless, their use in immunotherapy is rather limited due to difficulties in expanding these cells to therapeutically relevant numbers and to suppression by endogenous inhibitory mechanisms. Cancer recognition by NK cells is partly mediated by molecules termed natural cytotoxicity receptors (NCRs. In the present study, we hypothesize that it is possible to endow T-cells with an NK recognition pattern, providing them with a mean to recognize tumor cells, in a non-MHC restricted way. To test this, we genetically modified human T-cells with different chimeric receptors based on the human NCR2 molecule and then assessed their antitumor activity in vitro and in vivo. Our results show that expression in primary lymphocytes of an NCR2-derived CAR, termed s4428z, confers T-cells with the ability to specifically recognize heterogeneous tumors and to mediate tumor cytotoxicity in a mouse model. This study demonstrates the benefit of combining tumor recognition capability of NK cells with T cell effectiveness to improve cancer immunotherapy.

  10. A chemically inert drug can stimulate T cells in vitro by their T cell receptor in non-sensitised individuals

    International Nuclear Information System (INIS)

    Engler, Olivier B.; Strasser, Ingrid; Naisbitt, Dean J.; Cerny, Andreas; Pichler, Werner J.

    2004-01-01

    Drugs can interact with T cell receptors (TCR) after binding to peptide-MHC structures. This binding may involve the formation of a stable, covalent bond between a chemically reactive drug and MHC or the peptide embedded within. Alternatively, if the drug is chemically inert, the binding may be non-covalent and readily reversible. Both types of drug presentation account for a substantial number of adverse side effects to drugs. Presently no tests are available to predict the ability of chemically inert drugs to stimulate an immune response. Here we present data on the successful induction of a primary T cell immune response in vitro against a chemically inert drug using blood from healthy individuals, previously not exposed to the drug. Blood lymphocytes were stimulated by the chemically inert drug sulfamethoxazole and the protein-reactive drug-metabolite sulfamethoxazole-nitroso in the presence of IL-2. 9/10 individuals reacted in response to sulfamethoxazole-nitroso, but only three reacted to the chemically inert compound sulfamethoxazole. Drug reactive T cells could be detected after 14-35 days of cell culture by drug-specific proliferation or cytotoxicity, which was MHC-restricted. These cells were CD4, CD8 positive or CD4/CD8 double positive and T cell clones generated secreted Th0 type cytokines. Drug interaction lead to down-regulation of specific TCR. These data confirm the ability of chemically inert drugs to stimulate certain T cells by their TCR and may provide the opportunity to screen new drugs for their ability to interact with TCRs

  11. MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis

    DEFF Research Database (Denmark)

    Skov, S; Bregenholt, S; Claesson, Mogens Helweg

    1997-01-01

    Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate...... that the ZAP70 tyrosine kinase is tyrosine phosphorylated in Jurkat T cells and in purified peripheral T cells after MHC-I ligation. The tyrosine-phosphorylated ZAP70 kinase exhibits a particular phenotype with low affinities for proteins at 21, 40, 60, and 120 kDa, proteins normally co-precipitated with ZAP70...... after TCR/CD3 stimulation. The phosphorylation of ZAP70 after MHC-I ligation was dependent on TCR/CD3 surface expression. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex. MHC-I cross-linking induces a phosphorylated zeta-protein that migrates as a dimer at 42 k...

  12. It's the peptide-MHC affinity, stupid.

    Science.gov (United States)

    Kammertoens, Thomas; Blankenstein, Thomas

    2013-04-15

    Adoptively transferred T cells can reject large established tumors, but recurrence due to escape variants frequently occurs. In this issue of Cancer Cell, Engels et al. demonstrate that the affinity of the target peptide to the MHC molecule determines whether large tumors will relapse following adoptive T cell therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Effects of Thy-1+ cell depletion on the capacity of donor lymphoid cells to induce tolerance across an entire MHC disparity in sublethally irradiated adult hosts

    International Nuclear Information System (INIS)

    Pierce, G.E.; Watts, L.M.

    1989-01-01

    Thy-1+ cell depletion with anti-Thy-1.2 mAb and complement markedly reduced the capacity of C57BL/6J, H-2b bone marrow to establish mixed lymphoid chimerism and induce tolerance to C57BL/6J skin grafts across an entire MHC disparity in BALB/c, H-2d hosts conditioned with sublethal, fractionated 7.5 Gy total-body irradiation. In this model tolerance can be transferred to secondary irradiated BALB/c hosts only by cells of C57BL/6J donor, not host, genotype isolated from the spleens of tolerant hosts. Thy-1+ cell depletion abolished the capacity of C57BL/6J donor cells from tolerant BALB/c host spleens to transfer tolerance. The capacity of semiallogeneic BALB/c x C57BL/6J F1, H-2d/b donor BM and spleen cells to induce chimerism and tolerance to C57BL/6J skin grafts in BALB/c parental hosts was also reduced by Thy-1+ cell depletion. Thus the requirement for donor Thy-1+ cells cannot be explained simply on the basis of alloaggression. It is unlikely that the requisite Thy-1+ cells are nonspecific suppressor cells: Thy-1+ cell depletion had no effect on the slight but significant prolongation of third-party C3H/HeJ, H-2k skin grafts in irradiated BALB/c hosts injected with allogeneic C57BL/6J or semiallogeneic BALB/c x C57BL/6J F1 BM compared to irradiated controls injected with medium only. Furthermore, injections of semiallogeneic F1 spleen cells had no significant effect on the survival of the third-party grafts, although these cells were fully capable of inducing tolerance, and their capacity to induce tolerance was significantly reduced by Thy-1+ cell depletion. The requirement for a specific population of lymphoid cells, i.e. Thy-1+, remains unexplained but suggests that donor cells might play a role in the induction or maintenance of tolerance in this model other than merely providing a circulating source of donor antigens

  15. Peptide-loaded dendritic cells prime and activate MHC-class I-restricted T cells more efficiently than protein-loaded cross-presenting DC

    DEFF Research Database (Denmark)

    Met, Ozcan; Buus, Søren; Claesson, Mogens H

    2003-01-01

    -pulsed DC. Moreover, SIINFEKL-loaded DC were up to 50 times more efficient than DC-pulsed with OVA-protein for generation of an H-2K(b)-restricted response. Immunization of mice with SIINFEKL-loaded DC resulted in a much stronger H-2K(b)-restricted response than immunization with OVA-pulsed DC. These data......Undifferentiated and differentiated dendritic cells (uDC and dDC, respectively), derived from the bone marrow, were studied in vitro and in vivo. Ovalbumin (OVA) and two OVA-derived peptides binding to H-2K(b) and I-A(b), respectively, were used. Two IL-2 secreting T cell hybridomas specific...... for the OVA-derived epitopes were used in the in vitro read-out. The ability to cross-present the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL) was restricted to dDC, which express CD11c(+), CD86(+), and MHC-II(+). In vitro, the antigenicity of SIINFEKL-loaded DC declined at a slower rate than that of OVA...

  16. MHCcluster, a method for functional clustering of MHC molecules

    DEFF Research Database (Denmark)

    Thomsen, Martin Christen Frølund; Lundegaard, Claus; Buus, Søren

    2013-01-01

    The identification of peptides binding to major histocompatibility complexes (MHC) is a critical step in the understanding of T cell immune responses. The human MHC genomic region (HLA) is extremely polymorphic comprising several thousand alleles, many encoding a distinct molecule. The potentially...... binding specificity. The method has a flexible web interface that allows the user to include any MHC of interest in the analysis. The output consists of a static heat map and graphical tree-based visualizations of the functional relationship between MHC variants and a dynamic TreeViewer interface where...

  17. Uncoupling of T Cell Receptor Zeta Chain Function during the Induction of Anergy by the Superantigen, Staphylococcal Enterotoxin A

    Directory of Open Access Journals (Sweden)

    William D. Cornwell

    2010-06-01

    Full Text Available Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy. We characterized the components of the T cell receptor (TCR during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

  18. Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. II. Cross-reaction between a monoclonal antibody and two alpha beta T cell receptors

    DEFF Research Database (Denmark)

    Rognan, D; Engberg, J; Stryhn, A

    2000-01-01

    -peptide pair into the Fab combining site. Interestingly, the most energetically favored binding mode shows numerous analogies to the recently determined recognition of class I MHC-peptide complexes by alpha beta T cell receptors (TCRs). The pSAN13.4.1 also binds diagonally across the MHC binding groove......The recombinant antibody, pSAN13.4.1, has a unique T cell like specificity; it binds an Influenza Hemagglutinin octapeptide (Ha255-262) in an MHC (H-2Kk)-restricted manner, and a detailed comparison of the fine specificity of pSAN13.4.1 with the fine specificity of two Ha255-262-specific, H-2Kk......-restricted T cell hybridomas has supported this contention. A three-dimensional model of pSAN13.4.1 has been derived by homology modeling techniques. Subsequently, the structure of the pSAN13.4.1 antibody in complex with the antigenic Ha-Kk ligand was derived after a flexible and automated docking of the MHC...

  19. The MHC molecules of nonmammalian vertebrates

    DEFF Research Database (Denmark)

    Kaufman, J; Skjoedt, K; Salomonsen, J

    1990-01-01

    class II distribution. The axolotl has a very poor immune response (as though there are no helper T cells), a wide class II distribution and, for most animals, no cell surface class I molecule. It would be enlightening to understand both the mechanisms for the regulation of the MHC molecules during...

  20. Expression of GABAergic receptors in mouse taste receptor cells.

    Directory of Open Access Journals (Sweden)

    Margaret R Starostik

    Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

  1. The systems biology of MHC class II antigen presentation

    NARCIS (Netherlands)

    Paul, Petra

    2012-01-01

    Major histocompatibility class II molecules (MHC class II) are one of the key regulators of adaptive immunity because of their specific expression by professional antigen presenting cells (APC). They present peptides derived from endocytosed material to T helper lymphocytes. Consequently, MHC class

  2. The SysteMHC Atlas project.

    Science.gov (United States)

    Shao, Wenguang; Pedrioli, Patrick G A; Wolski, Witold; Scurtescu, Cristian; Schmid, Emanuel; Vizcaíno, Juan A; Courcelles, Mathieu; Schuster, Heiko; Kowalewski, Daniel; Marino, Fabio; Arlehamn, Cecilia S L; Vaughan, Kerrie; Peters, Bjoern; Sette, Alessandro; Ottenhoff, Tom H M; Meijgaarden, Krista E; Nieuwenhuizen, Natalie; Kaufmann, Stefan H E; Schlapbach, Ralph; Castle, John C; Nesvizhskii, Alexey I; Nielsen, Morten; Deutsch, Eric W; Campbell, David S; Moritz, Robert L; Zubarev, Roman A; Ytterberg, Anders Jimmy; Purcell, Anthony W; Marcilla, Miguel; Paradela, Alberto; Wang, Qi; Costello, Catherine E; Ternette, Nicola; van Veelen, Peter A; van Els, Cécile A C M; Heck, Albert J R; de Souza, Gustavo A; Sollid, Ludvig M; Admon, Arie; Stevanovic, Stefan; Rammensee, Hans-Georg; Thibault, Pierre; Perreault, Claude; Bassani-Sternberg, Michal; Aebersold, Ruedi; Caron, Etienne

    2018-01-04

    Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β2-Microglobulin through Distinct Binding Sites

    DEFF Research Database (Denmark)

    Merkle, Patrick S.; Irving, Melita; Hongjian, Song

    2017-01-01

    from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and......T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics...... are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR...

  4. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    Science.gov (United States)

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  5. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  6. A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.

    Science.gov (United States)

    Graham, Simon P; Honda, Yoshikazu; Pellé, Roger; Mwangi, Duncan M; Glew, E Jane; de Villiers, Etienne P; Shah, Trushar; Bishop, Richard; van der Bruggen, Pierre; Nene, Vishvanath; Taracha, Evans L N

    2007-02-09

    Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-gamma ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge. The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.

  7. Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

    Science.gov (United States)

    Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

    2016-01-01

    The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

  8. Characterization of a Nonclassical Class I MHC Gene in a Reptile, the Galápagos Marine Iguana (Amblyrhynchus cristatus)

    Science.gov (United States)

    Glaberman, Scott; Du Pasquier, Louis; Caccone, Adalgisa

    2008-01-01

    Squamates are a diverse order of vertebrates, representing more than 7,000 species. Yet, descriptions of full-length major histocompatibility complex (MHC) genes in this group are nearly absent from the literature, while the number of MHC studies continues to rise in other vertebrate taxa. The lack of basic information about MHC organization in squamates inhibits investigation into the relationship between MHC polymorphism and disease, and leaves a large taxonomic gap in our understanding of amniote MHC evolution. Here, we use both cDNA and genomic sequence data to characterize a class I MHC gene (Amcr-UA) from the Galápagos marine iguana, a member of the squamate subfamily Iguaninae. Amcr-UA appears to be functional since it is expressed in the blood and contains many of the conserved peptide-binding residues that are found in classical class I genes of other vertebrates. In addition, comparison of Amcr-UA to homologous sequences from other iguanine species shows that the antigen-binding portion of this gene is under purifying selection, rather than balancing selection, and therefore may have a conserved function. A striking feature of Amcr-UA is that both the cDNA and genomic sequences lack the transmembrane and cytoplasmic domains that are necessary to anchor the class I receptor molecule into the cell membrane, suggesting that the product of this gene is secreted and consequently not involved in classical class I antigen-presentation. The truncated and conserved character of Amcr-UA lead us to define it as a nonclassical gene that is related to the few available squamate class I sequences. However, phylogenetic analysis placed Amcr-UA in a basal position relative to other published classical MHC genes from squamates, suggesting that this gene diverged near the beginning of squamate diversification. PMID:18682845

  9. Expression, refolding and crystallization of murine MHC class I H-2Db in complex with human β2-microglobulin

    International Nuclear Information System (INIS)

    Sandalova, Tatyana; Michaëlsson, Jakob; Harris, Robert A.; Ljunggren, Hans-Gustaf; Kärre, Klas; Schneider, Gunter; Achour, Adnane

    2005-01-01

    Mouse MHC class I H-2Db in complex with human β2m and the LCMV-derived peptide gp33 has been produced and crystallized. Resolution of the structure of this complex combined with the structural comparison with the previously solved crystal structure of H-2Db/mβ2m/gp33 should lead to a better understanding of how the β2m subunit affects the overall conformation of MHC complexes as well as the stability of the presented peptides. β 2 -Microglobulin (β 2 m) is non-covalently linked to the major histocompatibility (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I can bind human β 2 m (hβ 2 m) and such hybrid molecules are often used in structural and functional studies. The replacement of mouse β 2 m (mβ 2 m) by hβ 2 m has important functional consequences for MHC class I complex stability and specificity, but the structural basis for this is unknown. To investigate the impact of species-specific β 2 m subunits on MHC class I conformation, murine MHC class I H-2D b in complex with hβ 2 m and the peptide gp33 derived from lymphocytic choriomeningitis virus (LCMV) has been expressed, refolded in vitro and crystallized. Crystals containing two complexes per asymmetric unit and belonging to the space group P2 1 , with unit-cell parameters a = 68.1, b = 65.2, c = 101.9 Å, β = 102.4°, were obtained

  10. BG1 has a major role in MHC-linked resistance to malignant lymphoma in the chicken.

    Science.gov (United States)

    Goto, Ronald M; Wang, Yujun; Taylor, Robert L; Wakenell, Patricia S; Hosomichi, Kazuyoshi; Shiina, Takashi; Blackmore, Craig S; Briles, W Elwood; Miller, Marcia M

    2009-09-29

    Pathogen selection is postulated to drive MHC allelic diversity at loci for antigen presentation. However, readily apparent MHC infectious disease associations are rare in most species. The strong link between MHC-B haplotype and the occurrence of virally induced tumors in the chicken provides a means for defining the relationship between pathogen selection and MHC polymorphism. Here, we verified a significant difference in resistance to gallid herpesvirus-2 (GaHV-2)-induced lymphomas (Marek's disease) conferred by two closely-related recombinant MHC-B haplotypes. We mapped the crossover breakpoints that distinguish these haplotypes to the highly polymorphic BG1 locus. BG1 encodes an Ig-superfamily type I transmembrane receptor-like protein that contains an immunoreceptor tyrosine-based inhibition motif (ITIM), which undergoes phosphorylation and is recognized by Src homology 2 domain-containing protein tyrosine phosphatase (SHP-2). The recombinant haplotypes are identical, except for differences within the BG1 3'-untranslated region (3'-UTR). The 3'-UTR of the BG1 allele associated with increased lymphoma contains a 225-bp insert of retroviral origin and showed greater inhibition of luciferase reporter gene translation compared to the other allele. These findings suggest that BG1 could affect the outcome of GaHV-2 infection through modulation of the lymphoid cell responsiveness to infection, a condition that is critical for GaHV-2 replication and in which the MHC-B haplotype has been previously implicated. This work provides a mechanism by which MHC-B region genetics contributes to the incidence of GaHV-2-induced malignant lymphoma in the chicken and invites consideration of the possibility that similar mechanisms might affect the incidence of lymphomas associated with other oncogenic viral infections.

  11. Characterization of a nonclassical class I MHC gene in a reptile, the Galápagos marine iguana (Amblyrhynchus cristatus.

    Directory of Open Access Journals (Sweden)

    Scott Glaberman

    Full Text Available Squamates are a diverse order of vertebrates, representing more than 7,000 species. Yet, descriptions of full-length major histocompatibility complex (MHC genes in this group are nearly absent from the literature, while the number of MHC studies continues to rise in other vertebrate taxa. The lack of basic information about MHC organization in squamates inhibits investigation into the relationship between MHC polymorphism and disease, and leaves a large taxonomic gap in our understanding of amniote MHC evolution. Here, we use both cDNA and genomic sequence data to characterize a class I MHC gene (Amcr-UA from the Galápagos marine iguana, a member of the squamate subfamily Iguaninae. Amcr-UA appears to be functional since it is expressed in the blood and contains many of the conserved peptide-binding residues that are found in classical class I genes of other vertebrates. In addition, comparison of Amcr-UA to homologous sequences from other iguanine species shows that the antigen-binding portion of this gene is under purifying selection, rather than balancing selection, and therefore may have a conserved function. A striking feature of Amcr-UA is that both the cDNA and genomic sequences lack the transmembrane and cytoplasmic domains that are necessary to anchor the class I receptor molecule into the cell membrane, suggesting that the product of this gene is secreted and consequently not involved in classical class I antigen-presentation. The truncated and conserved character of Amcr-UA lead us to define it as a nonclassical gene that is related to the few available squamate class I sequences. However, phylogenetic analysis placed Amcr-UA in a basal position relative to other published classical MHC genes from squamates, suggesting that this gene diverged near the beginning of squamate diversification.

  12. TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation

    NARCIS (Netherlands)

    Nair-Gupta, Priyanka; Baccarini, Alessia; Tung, Navpreet; Seyffer, Fabian; Florey, Oliver; Huang, Yunjie; Banerjee, Meenakshi; Overholtzer, Michael; Roche, Paul A.; Tampé, Robert; Brown, Brian D.; Amsen, Derk; Whiteheart, Sidney W.; Blander, J. Magarian

    2014-01-01

    Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly

  13. HUMAN NK CELLS: FROM SURFACE RECEPTORS TO THE THERAPY OF LEUKEMIAS AND SOLID TUMORS

    Directory of Open Access Journals (Sweden)

    LORENZO eMORETTA

    2014-03-01

    Full Text Available Natural Killer (NK cells are major effector cells of the innate immunity. The discovery, over two decades ago, of MHC-class I specific NK receptors and subsequently of activating receptors, recognizing ligands expressed by tumor or virus-infected cells, paved the way to our understanding of the mechanisms of selective recognition and killing of tumor cells. Although NK cells can efficiently kill tumor cells of different histotypes in vitro, their activity may be limited in vivo by their inefficient trafficking to tumor lesions and by the inhibition of their function induced by tumor cells themselves and by the tumor microenvironment. On the other hand, the important role of NK cells has been clearly demonstrated in the therapy of high risk leukemias in the haploidentical hematopoietic cell (HSC transplantation setting. NK cells derived from donor HSC kill leukemic cells residual after the conditioning regimen, thus preventing leukemia relapses. In addition, they also kill residual dendritic cells and T lymphocytes, thus preventing both GvHD and graft rejection.

  14. A comparative analysis of viral peptides presented by contemporary human and chimpanzee MHC class I molecules

    NARCIS (Netherlands)

    van Deutekom, Hanneke W. M.; Hoof, Ilka; Bontrop, Ronald E.; Keşmir, Can

    2011-01-01

    Genetic factors such as the MHC influence the immunocompetence of an individual. MHC genes are the most polymorphic genes in primates, which is often interpreted as an adaptation to establish good T cell responses to a wide range of (evolving) pathogens. Chimpanzee MHC (Patr) genes are less

  15. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  16. Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

    Directory of Open Access Journals (Sweden)

    Elaine F Kenny

    Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

  17. The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

    Science.gov (United States)

    Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

    2016-01-01

    The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for

  18. A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

    Science.gov (United States)

    Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

    1996-03-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

  19. B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway

    Science.gov (United States)

    Milillo, M. Ayelén; Velásquez, Lis N.; Trotta, Aldana; Delpino, M. Victoria; Balboa, Luciana; Vermeulen, Mónica; Espindola, Sonia L.; Rodriguez-Rodrigues, Nahuel; Fernández, Gabriela C.; Oliveira, Sergio Costa; Giambartolomei, Guillermo H.

    2017-01-01

    Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the

  20. B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway.

    Directory of Open Access Journals (Sweden)

    M Ayelén Milillo

    2017-08-01

    Full Text Available Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP. Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and

  1. Inhibition of MHC-I by Brucella abortus is an early event during infection and involves EGFR pathway.

    Science.gov (United States)

    Velásquez, Lis N; Milillo, M Ayelén; Delpino, M Victoria; Trotta, Aldana; Mercogliano, M Florencia; Pozner, Roberto G; Schillaci, Roxana; Elizalde, Patricia V; Giambartolomei, Guillermo H; Barrionuevo, Paula

    2017-04-01

    Brucella abortus is able to persist inside the host despite the development of potent CD8 + T-cell responses. We have recently reported the ability of B. abortus to inhibit the interferon-γ-induced major histocompatibility complex (MHC)-I cell surface expression on human monocytes. This phenomenon was due to the B. abortus-mediated retention of MHC-I molecules within the Golgi apparatus and was dependent on bacterial viability. However, the implications of bacterial virulence or replicative capacity and the signaling pathways remained unknown. Here we demonstrated that the B. abortus mutant strains RB51 and virB10 - are able to inhibit MHC-I expression in the same manner as wild-type B. abortus, even though they are unable to persist inside human monocytes for a long period of time. Consistent with this, the phenomenon was triggered early in time and could be observed at 8 h postinfection. At 24 and 48 h, it was even stronger. Regarding the signaling pathway, targeting epidermal growth factor (EGF) receptor (EGFR), ErbB2 (HER2) or inhibition of tumor necrosis factor-α-converting enzyme, one of the enzymes which generates soluble EGF-like ligands, resulted in partial recovery of MHC-I surface expression. Moreover, recombinant EGF and transforming growth factor-α as well as the combination of both were also able to reproduce the B. abortus-induced MHC-I downmodulation. Finally, when infection was performed in the presence of an extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, MHC-I surface expression was significantly recovered. Overall, these results describe how B. abortus evades CD8 + T-cell responses early during infection and exploits the EGFR-ERK signaling pathway to escape from the immune system and favor chronicity.

  2. Donor T cells primed on leukemia lysate-pulsed recipient APCs mediate strong graft-versus-leukemia effects across MHC barriers in full chimeras.

    Science.gov (United States)

    Ghosh, Arnab; Koestner, Wolfgang; Hapke, Martin; Schlaphoff, Verena; Länger, Florian; Baumann, Rolf; Koenecke, Christian; Cornberg, Markus; Welte, Karl; Blazar, Bruce R; Sauer, Martin G

    2009-04-30

    Antigen-presenting cells (APCs) of host origin drive graft-versus-leukemia (GVL) effects but can also trigger life-threatening graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) across major histocompatibility complex (MHC) barriers. We show that in vitro priming of donor lymphocytes can circumvent the need of recipient-derived APCs in vivo for mediating robust GVL effects and significantly diminishes the risk of severe GVHD. In vitro, generated and expanded T cells (ETCs) mediate anti-leukemia effects only when primed on recipient-derived APCs. Loading of APCs in vitro with leukemia cell lysate, chimerism status of the recipient, and timing of adoptive transfer after HCT are important factors determining the outcome. Delayed transfer of ETCs resulted in strong GVL effects in leukemia-bearing full chimera (FC) and mixed chimera (MC) recipients, which were comparable with the GVL/GVHD rates observed after the transfer of naive donor lymphocyte infusion (DLI). Upon early transfer, GVL effects were more pronounced with ETCs but at the expense of significant GVHD. The degree of GVHD was most severe in MCs after transfer of ETCs that had been in vitro primed either on nonpulsed recipient-derived APCs or with donor-derived APCs.

  3. Targeting of T Lymphocytes to Melanoma Cells Through Chimeric Anti-GD3 Immunoglobulin T-Cell Receptors

    Directory of Open Access Journals (Sweden)

    C.O. Yun

    2000-09-01

    Full Text Available Immunoglobulin T-cell receptors (IgTCRs combine the specificity of antibodies with the potency of cellular killing by grafting antibody recognition domains onto TCR signaling chains. IgTCR-modified T cells are thus redirected to kill tumor cells based on their expression of intact antigen on cell surfaces, bypassing the normal mechanism of activation through TCR—peptide—major histocompatibility complex (MHC recognition. Melanoma is one of the most immunoresponsive of human cancers and has served as a prototype for the development of a number of immunotherapies. The target antigen for this study is the ganglioside GD3, which is highly expressed on metastatic melanoma with only minor immunologic cross-reaction with normal tissues. To determine an optimal configuration for therapy, four combinations of IgTCRs were prepared and studied: sFv-ɛ, sFv-ζ, Fab-ɛ, Fab-ζ. These were expressed on the surface of human T cells by retroviral transduction. IgTCR successfully redirected T-cell effectors in an MHC-unrestricted manner, in this case against a non—T-dependent antigen, with specific binding, activation, and cytotoxicity against GD3+ melanoma cells. Soluble GD3 in concentrations up to 100 μg/ml did not interfere with recognition and binding of membrane-bound antigen. Based on the outcomes of these structural and functional tests, the sFv-ζ construct was selected for clinical development. These results demonstrate key features that emphasize the potential of anti-GD3 IgTCR-modified autologous T cells for melanoma therapies.

  4. Structural Simulation of MHC-peptide Interactions using T-cell Epitope in Iron-acquisition Protein of N. meningitides for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Namrata Mishra

    2010-12-01

    Full Text Available The present work uses a structural simulation approach to identify the potential target vaccine candidates or T cell epitopes (antigenic region that can activate T cell response in two iron acquisition proteins from Neisseria. An iron regulated outer membrane protein frpB: extracellular, [NMB1988], and a Major ferric Iron-binding protein fbpA: periplasmic, [NMB0634] critical for the survival of the pathogen in the host were used. Ten novel promiscuous epitopes from the two iron acquisition proteins were identified using bioinformatics interface. Of these epitopes, 630VQKAVGSIL638 present on frpB with high binding affinity for allele HLA*DR1 was identified with an anchor position at P2, an aliphatic residue at P4 and glycine at P6 making it thereby a potential quality choice for linking peptide-loaded MHC dynamics to T-cell activation and vaccine constructs. The feasibility and structural binding of predicted peptide to the respective HLA allele was investigated by molecular modeling and template-based structural simulation. The conformational properties of the linear peptide were investigated by molecular dynamics using GROMOS96 package and Swiss PDB viewer.

  5. MHC and Evolution in Teleosts

    OpenAIRE

    Grimholt, Unni

    2016-01-01

    Major histocompatibility complex (MHC) molecules are key players in initiating immune responses towards invading pathogens. Both MHC class I and class II genes are present in teleosts, and, using phylogenetic clustering, sequences from both classes have been classified into various lineages. The polymorphic and classical MHC class I and class II gene sequences belong to the U and A lineages, respectively. The remaining class I and class II lineages contain nonclassical gene sequences that, de...

  6. Structure-based, rational design of T cell receptors

    Directory of Open Access Journals (Sweden)

    Vincent eZoete

    2013-09-01

    Full Text Available Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce TCR modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction pMHC. Using the well-characterized 2C TCR/SIYR/H-2K(b structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. 54% of the designed sequence replacements exhibited improved pMHC-binding as compared to the native TCR, with up to 150 fold increase in affinity, while preserving specificity. Genetically-engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes. We have complemented the approach with a simplified rigid method to predict the TCR orientation over pMHC. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of

  7. Computational screening of Six Antigens for potential MHC class II restricted epitopes and evaluating its CD4+ T-Cell Responsiveness against Visceral Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Manas Ranjan

    2017-12-01

    Full Text Available Visceral leishmaniasis is one of the most neglected tropical diseases for which no vaccine exists. In spite of extensive efforts, no successful vaccine is available against this dreadful infectious disease. To support the vaccine development, immunoinformatics approach was applied to search for potential MHC-classII restricted epitopes that can activate the immune cells. Initially, a total of 37 epitopes derived from six, stage dependent over expressed antigens were predicted, which were presented by at least 26 diverse MHC class II alleles including: DRB10101, DRB10301, DRB10401, DRB10404, DRB10405, DRB10701, DRB10802, DRB10901, DRB11101, DRB11302, DRB11501, DRB30101, DRB40101, DRB50101, DPA10103-DPB10401, DPA10103-DPB10201, DPA10201-DPB10101, DPA10103-DPB10301_DPB10401, DPA10301-DPB10402, DPA10201-DPB105021, DQA10102-DQB10602, DQA10401-DQB10402, DQA10501-QB10201, DQA10501-DQB10301, DQA10301-DQB10302 and DQA10101-DQB10501. Based on the population coverage analysis and HLA cross presentation ability, six epitopes namely, FDLFLFSNGAVVWWG (P1, YPVYPFLASNAALLN (P2, VYPFLASNAALLNLI (P3, LALLIMLYALIATQF (P4, LIMLYALIATQFSDD (P5, IMLYALIATQFSDDA (P6 were selected for further analysis. Stimulation with synthetic peptide alone or as a cocktail triggered the intracellular IFN-γ production. Moreover, specific IgG class of antibodies was detected in the serum of active VL cases against P1, P4, P and P6 in order to evaluate peptide effect on humoral immune response. Additionally, most of the peptides, except P2, were found to be non-inducer of CD4+ IL-10 against both active VL as well as treated VL subjects. Peptide immunogenicity was validated in BALB/c mice immunized with cocktail of synthetic peptide emulsified in complete Freund’s adjuvant/incomplete Freund’s adjuvant. The immunized splenocytes induced strong spleen cell proliferation upon parasite re-stimulation. Furthermore, an increased IFN-γ, IL-12, IL-17 and IL-22 production augmented with

  8. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore.......05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...... studies of vitamin D's effect on TCC cells in vitro are necessary before the efficacy of treatment with vitamin D analogues in TCC can be evaluated in patients....

  9. NMDA Receptors in Glial Cells: Pending Questions.

    Science.gov (United States)

    Dzamba, David; Honsa, Pavel; Anderova, Miroslava

    2013-05-01

    Glutamate receptors of the N-methyl-D-aspartate (NMDA) type are involved in many cognitive processes, including behavior, learning and synaptic plasticity. For a long time NMDA receptors were thought to be the privileged domain of neurons; however, discoveries of the last 25 years have demonstrated their active role in glial cells as well. Despite the large number of studies in the field, there are many unresolved questions connected with NMDA receptors in glia that are still a matter of debate. The main objective of this review is to shed light on these controversies by summarizing results from all relevant works concerning astrocytes, oligodendrocytes and polydendrocytes (also known as NG2 glial cells) in experimental animals, further extended by studies performed on human glia. The results are divided according to the study approach to enable a better comparison of how findings obtained at the mRNA level correspond with protein expression or functionality. Furthermore, special attention is focused on the NMDA receptor subunits present in the particular glial cell types, which give them special characteristics different from those of neurons - for example, the absence of Mg(2+) block and decreased Ca(2+) permeability. Since glial cells are implicated in important physiological and pathophysiological roles in the central nervous system (CNS), the last part of this review provides an overview of glial NMDA receptors with respect to ischemic brain injury.

  10. Changing T cell specificity by retroviral T cell receptor display

    NARCIS (Netherlands)

    Kessels, H. W.; van den Boom, M. D.; Spits, H.; Hooijberg, E.; Schumacher, T. N.

    2000-01-01

    The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T

  11. Dopamine receptor repertoire of human granulosa cells

    Directory of Open Access Journals (Sweden)

    Kunz Lars

    2007-10-01

    Full Text Available Abstract Background High levels of dopamine (DA were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs derived from women undergoing in vitro fertilization (IVF are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality. Methods Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4 were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system. Results We found members of the two DA receptor families (D1- and D2 -like associated with different signaling pathways in human GCs, namely D1 (as expected and D5 (both are Gs coupled and linked to cAMP increase and D2, D4 (Gi/Gq coupled and linked to IP3/DAG. D3 was not found. The presence of the trophic hormone hCG (10 IU/ml in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR or protein levels (immunocytochemistry/Western blotting of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S. Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed

  12. Ia-restricted B-B cell interaction. I. The MHC haplotype of bone marrow cells present during B cell ontogeny dictates the self-recognition specificity of B cells in the polyclonal B cell activation by a B cell differentiation factor, B151-TRF2

    International Nuclear Information System (INIS)

    Ono, S.; Takahama, Y.; Hamaoka, T.

    1987-01-01

    We have demonstrated that B cell recognition of Ia molecules is involved in polyclonal B cell differentiation by B151-TRF2. The present study was undertaken to examine the Ia recognition specificity of B151-TRF2-responsive B cells in fully major histocompatibility complex (MHC)-allogeneic P1----P2, semiallogeneic P1----(P1 x P2)F1, and double donor (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 radiation bone marrow chimeras. The B cells from both P1----P2 and P1----(P1 x P2)F1 chimeras could give rise to in vitro immunoglobulin M-producing cells upon stimulation with B151-TRF2 comparable in magnitude to that of normal P1 B cells, and their responses were inhibited by anti-I-AP1 but not by anti-I-AP2 monoclonal antibody even in the presence of mitomycin C-treated T cell-depleted P2 spleen cells as auxiliary cells. In contrast, the B151-TRF2 responses of P1 B cells isolated from both (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 double bone marrow chimeras became sensitive to the inhibition of not only anti-I-AP1 but also anti-I-AP2 monoclonal antibody only when the culture was conducted in the presence of P2 auxiliary cells, demonstrating that they adaptively differentiate to recognize as self-structures allogeneic as well as syngeneic Ia molecules. Moreover, the experiments utilizing B cells from H-2-congenic mice and B cell hybridoma clones as auxiliary cells revealed that B151-TRF2-responsive B cells recognize Ia molecules expressed on B cells. Taken together, these results demonstrate that B151-TRF2-responsive B cells recognize Ia molecules expressed by B cells as self-structures and that their self-recognition specificity is dictated by the MHC haplotype of bone marrow cells present during the B cell ontogeny but not by the MHC haplotype of a radiation-resistant host environment

  13. Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

  14. Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor.

    Science.gov (United States)

    Ilchmann, Anne; Krause, Maren; Heilmann, Monika; Burgdorf, Sven; Vieths, Stefan; Toda, Masako

    2012-05-01

    The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove's modified Dulbecco's medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR) 2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly, BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4(+) T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. A strategy for bacterial production of a soluble functional human neonatal Fc receptor

    DEFF Research Database (Denmark)

    Andersen, Jan Terje; Justesen, Sune; Berntzen, Gøril

    2008-01-01

    The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus...

  16. Cloning the interleukin 1 receptor from human T cells

    International Nuclear Information System (INIS)

    Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

    1989-01-01

    cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

  17. MHC class II-associated invariant chain linkage of antigen dramatically improves cell-mediated immunity induced by adenovirus vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Mandrup Jensen, Camilla Maria; Orskov, Cathrine

    2008-01-01

    The ideal vaccine induces a potent protective immune response, which should be rapidly induced, long-standing, and of broad specificity. Recombinant adenoviral vectors induce potent Ab and CD8+ T cell responses against transgenic Ags within weeks of administration, and they are among the most...

  18. Sibling rivalry: competition between MHC class II family members inhibits immunity.

    Science.gov (United States)

    Denzin, Lisa K; Cresswell, Peter

    2013-01-01

    Peptide loading of major histocompatibility complex (MHC) class II molecules in the endosomes and lysosomes of antigen-presenting cells is catalyzed by human leukocyte antigen-DM (HLA-DM) and modulated by HLA-DO. In a structural study in this issue, Guce et al. show that HLA-DO is an MHC class II mimic and functions as a competitive and essentially irreversible inhibitor of HLA-DM activity, thereby inhibiting MHC class II antigen presentation.

  19. MHC class II molecules and tumour immunotherapy

    International Nuclear Information System (INIS)

    Oven, I.

    2005-01-01

    Background. Tumour immunotherapy attempts to use the specificity and capability of the immune system to kill malignant cells with a minimum damage to normal tissue. Increasing knowledge of the identity of tumour antigens should help us design more effective therapeutic vaccines. Increasing evidence has demonstrated that MHC class II molecules and CD4+ T cells play important roles in generating and maintaining antitumour immune responses in animal models. These data suggest that it may be necessary to involve both CD4+ and CD8+ T cells for more effective antitumour therapy. Novel strategies have been developed for enhancing T cell responses against cancer by prolonging antigen presentation of dendritic cells to T cells, by the inclusion of MHC class II-restricted tumour antigens and by genetically modifying tumour cells to present antigen to T lymphocytes directly. Conclusions. Vaccines against cancers aim to induce tumour-specific effector T cells that can reduce tumour mass and induce development of tumour-specific T cell memory, that can control tumour relapse. (author)

  20. NK cell receptor/H2-Dk-dependent host resistance to viral infection is quantitatively modulated by H2q inhibitory signals.

    Science.gov (United States)

    Fodil-Cornu, Nassima; Loredo-Osti, J Concepción; Vidal, Silvia M

    2011-04-01

    The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2(k), we generated double congenic mice between MA/My and BALB.K mice and an F(2) cross between FVB/N (H-2(q)) and BALB.K (H2(k)) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2(k) in conjunction with Cmv3(MA/My) or Cmv3(FVB) were resistant to MCMV infection. Subsequently, an F(3) cross was carried out between transgenic FVB/H2-D(k) and MHC-I deficient mice in which only the progeny expressing Cmv3(FVB) and a single H2-D(k) class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell-dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2(q) alleles influence the expression level of H2(q) molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2(q) alleles. Our results support a model in which H-2(q) molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-D(k) on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell-mediated control of viral load.

  1. Ligation of MHC class I molecules on peripheral blood T lymphocytes induces new phenotypes and functions

    DEFF Research Database (Denmark)

    Bregenholt, S; Röpke, M; Skov, S

    1996-01-01

    of T cell-mediated cytotoxicity. Immediately following MHC-I ligation, the T cells responded with increased protein tyrosine phosphorylation, with new bands appearing in the SDS-PAGE. Exposure of T cells to immobilized anti-MHC-I Ab for 24 h induced an increased surface expression of the TCR/CD3 and CD......28 molecules. MHC-I-induced proliferation of purified T cells was dependent on cellular interactions with non-T cells. Under certain conditions, in which MHC-I was ligated by picogram concentrations of immobilized anti-MHC-I Ab, anti-TCR/CD3 Ab-induced proliferation of T cells was strongly inhibited....... These data clearly demonstrate that ligation of the MHC-I complex on T cells may induce both positive and negative signals. Since the physiologic ligands for MHC-I molecules are TCR and the CD8 molecules, our data may suggest that MHC-I molecules are instrumental in cellular interactions between T cells....

  2. Prevention of MHC-alloimmunization by UV-B irradiation in a murine model: effects of UV dose and number of transfused cells

    International Nuclear Information System (INIS)

    Grijzenhout, M.A.; Claas, F.H.J.

    1994-01-01

    The optimal dose of UV-B radiation for prevention of in vivo alloimmunization (AI) against major histocompatibility complex (MHC) antigens was investigated in a murine transfusion model. Two groups with five C57BL/6 mice (H-2 b ) each were transfused at weekly intervals with 1 x 10 5 or 1 x 10 6 DBA/2 (H-2 d ) leucocytes. Both suspensions induced anti-H-2 d antibodies in all mice after the second transfusion. The minimal UV-B dose required for abolition of alloreactivity in the mixed leucocyte reaction (MLR) was 0.6 J/cm 2 . This dose completely prevented the onset of MHC-AI in all five mice transfused with six suspensions containing 1 x 10 5 leucocytes. In contrast, suspensions with 1 x 10 6 leucocytes and exposed to 0.6 J/cm 2 induced immunization in 4/5 mice. Further increase of the dose to 1.8 or 5.4 J/cm 2 did not prevent the onset of MHC-AI. We conclude that the number of leucocytes per transfusion determines the efficacy of UV irradiation for the prevention of MHC-AI. For UV irradiation of human platelet concentrates (PCs) we propose to reduce the number of leucocytes by centrifugation prior to UV exposure. UV-B irradiation of PCs with high numbers of leucocytes may not be effective for prevention of alloimmunization. (Author)

  3. Acute inhibition of selected membrane-proximal mouse T cell receptor signaling by mitochondrial antagonists.

    Directory of Open Access Journals (Sweden)

    Kwangmi Kim

    2009-11-01

    Full Text Available T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR with cognate peptide/major histocompatibility complex (MHC plus lymphocyte function-associated antigen 1 (LFA-1 with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin, resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s. Thus, activation of Akt and PLC-gamma1 and entry of extracellular Ca(2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

  4. Complex assembly, crystallization and preliminary X-ray crystallographic studies of duck MHC class I molecule

    International Nuclear Information System (INIS)

    Zhang, Jianhua; Chen, Yong; Gao, Feng; Chen, Weihong; Qi, Jianxun; Xia, Chun

    2009-01-01

    Using a peptide derived from H5N1, a complex of duck MHC class I molecule (DuMHC I) with duck β 2 -microglobulin (Duβ 2 m) was assembled and crystallized. Initial structure analysis indicated that the crystals did not contain the complete DuMHC I complex but instead contained DuMHC I α3-domain and Duβ 2 m subunits. In order to understand the biological properties of the immune systems of waterfowl and to establish a system for structural studies of duck class I major histocompatibility complex (DuMHC I), a complex of DuMHC I with duck β 2 -microglobulin (Duβ 2 m) and the peptide AEIEDLIF (AF8) derived from H5N1 NP residues 251–258 was assembled. The complex was crystallized; the crystals belonged to space group C222 1 , with unit-cell parameters a = 54.7, b = 72.4, c = 102.2 Å, and diffracted to 2.3 Å resolution. Matthews coefficient calculation and initial structure determination by molecular replacement showed that the crystals did not contain the whole DuMHC I complex, but instead contained the DuMHC I α3 domain and a Duβ2m molecule (DuMHC I α3+β2m). Another complex of DuMHC I with the peptide IDWFDGKE derived from a chicken fusion protein also generated the same results. The stable structure of DuMHC I α3+β2m may reflect some unique characteristics of DuMHC I and pave the way for novel MHC structure-related studies in the future

  5. Circumvention of MHC class II restriction by genetic immunization.

    Science.gov (United States)

    Schuler, K; Lu, C; Chang, H D; Croft, M; Zanetti, M; Gerloni, M

    2001-11-12

    The fate of T cell responses to peptide-based vaccination is subject to constraints by the major histocompatibility complex (MHC), MHC restriction. Using as a model system of T and B cell epitopes from the circumsporozoite protein of Plasmodium falciparum malaria parasite, we show that vaccination by somatic transgene immunization readily primes Balb/c mice (H-2(d)) a strain previously reported to be non-responder to immunization with a synthetic peptide vaccine encompassing these epitopes. Following genetic vaccination Balb/c mice developed a primary T cell response comparable to that of the responder strain C57Bl/6 (H-2(b)). Following booster immunization on day 45 Balb/c mice responded with a typical T cell memory response. Priming induced the formation of specific antibodies, which rose sharply after booster immunization. These findings suggests that genetic immunization can circumvent MHC class II restriction.

  6. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  7. B lymphocytes as natural antigen-presenting cells (APC) of their own Ig receptor determinants

    International Nuclear Information System (INIS)

    Yurin, V.L.; Rudensky, A.Yu.; Rabinovich, O.R.; Kulakova, O.G.; Bobreneva, R.A.

    1986-01-01

    The authors use Igk-lb allotype-specific rat T cell proliferation(Pr) in vitro as a model of natural Ig determinants B cell presentation in Ig-specific T-B cell interactions. As shown before Igk-lb-specific responsiveness of AUG(RT-l/sup c/, Igk-la) and WAG (RT-l, Igk-la) rats is controlled by dominant Ir gene, linked to RT-l/sup c/. Only IgG(Igk-lb)-pulsed splenic APC of AUG(responder) but not WAG(non-responder) origin induce specific F 1 (WAGxAUG) T cell Pr. The same restriction was observed if purified B cells from Igk-l congeneic AUG-lb and WAG-lb rats were used as APC. B cell presentation was found to be sensitive to high irradiation dose(2000 rad). Anti-RT-l monoclonal antibody inhibition studies suggested RT-lB(I-A) molecule as a main restricting element of Igk-lb T cell recognition. B cell and splenic APC presentation of Igk-lb allotype was not inhibited by poly- and monoclonal anti-Igk-lb antibodies. Allelic exclusion of Igk-lb presentation by B cells from heterozygous F 1 (WAG-lbx AUG) rats was demonstrated by panning with antiallotypic reagents. Important, that irradiated anti-Igk-lb T cells induce specific Pr of normal Igk-lb-positive B cells. The data demonstrate MHC-restricted B cell presentation of their own receptor determinants, distinct from serologically-defined epitopes. T cell recognition of these determinants induce specific Pr of Ig-recognizing T cells and Ig-presenting B lymphocytes

  8. Bacterial superantigens promote acute nasopharyngeal infection by Streptococcus pyogenes in a human MHC Class II-dependent manner.

    Directory of Open Access Journals (Sweden)

    Katherine J Kasper

    2014-05-01

    Full Text Available Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs as 'trademark' virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS, how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6 mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC -II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms.

  9. T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis.

    Science.gov (United States)

    Held, Kathrin; Beltrán, Eduardo; Moser, Markus; Hohlfeld, Reinhard; Dornmair, Klaus

    2015-09-01

    Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161(hi)TRAV1-2(+) T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161(hi)TRAV1-2(+) TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  10. Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael

    2011-01-01

    a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data......In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC...

  11. NK Cell Receptor/H2-Dk–Dependent Host Resistance to Viral Infection Is Quantitatively Modulated by H2 q Inhibitory Signals

    Science.gov (United States)

    Fodil-Cornu, Nassima; Loredo-Osti, J. Concepción; Vidal, Silvia M.

    2011-01-01

    The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and H2k, we generated double congenic mice between MA/My and BALB.K mice and an F2 cross between FVB/N (H-2q) and BALB.K (H2k) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2k in conjunction with Cmv3MA/My or Cmv3FVB were resistant to MCMV infection. Subsequently, an F3 cross was carried out between transgenic FVB/H2-Dk and MHC-I deficient mice in which only the progeny expressing Cmv3FVB and a single H2-Dk class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell–dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2q alleles influence the expression level of H2q molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2q alleles. Our results support a model in which H-2q molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-Dk on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell–mediated control of viral load. PMID:21533075

  12. CSF-1 Receptor Signaling in Myeloid Cells

    Science.gov (United States)

    Stanley, E. Richard; Chitu, Violeta

    2014-01-01

    The CSF-1 receptor (CSF-1R) is activated by the homodimeric growth factors colony-stimulating factor-1 (CSF-1) and interleukin-34 (IL-34). It plays important roles in development and in innate immunity by regulating the development of most tissue macrophages and osteoclasts, of Langerhans cells of the skin, of Paneth cells of the small intestine, and of brain microglia. It also regulates the differentiation of neural progenitor cells and controls functions of oocytes and trophoblastic cells in the female reproductive tract. Owing to this broad tissue expression pattern, it plays a central role in neoplastic, inflammatory, and neurological diseases. In this review we summarize the evolution, structure, and regulation of expression of the CSF-1R gene. We review, the structures of CSF-1, IL-34, and the CSF-1R and the mechanism of ligand binding to and activation of the receptor. We further describe the pathways regulating macrophage survival, proliferation, differentiation, and chemotaxis downstream from the CSF-1R. PMID:24890514

  13. Induction of protective immunity against MHC class I-deficient, HPV16-associated tumours with peptide and dendritic cell-based vaccines

    Czech Academy of Sciences Publication Activity Database

    Reiniš, Milan; Štěpánek, Ivan; Šímová, Jana; Bieblová, Jana; Přibylová, Hana; Indrová, Marie; Bubeník, Jan

    2010-01-01

    Roč. 36, č. 3 (2010), s. 545-551 ISSN 1019-6439 R&D Projects: GA AV ČR IAA500520605; GA AV ČR IAA500520807 EU Projects: European Commission(XE) 18933 - CLINIGENE Institutional research plan: CEZ:AV0Z50520514 Keywords : MHC class I-deficient tumours * CpG oligodeoxynucleotides * human papilloma virus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.571, year: 2010

  14. Exosomal cancer immunotherapy is independent of MHC molecules on exosomes.

    Science.gov (United States)

    Hiltbrunner, Stefanie; Larssen, Pia; Eldh, Maria; Martinez-Bravo, Maria-Jose; Wagner, Arnika K; Karlsson, Mikael C I; Gabrielsson, Susanne

    2016-06-21

    Peptide-loaded exosomes are promising cancer treatment vehicles; however, moderate T cell responses in human clinical trials indicate a need to further understand exosome-induced immunity. We previously demonstrated that antigen-loaded exosomes carry whole protein antigens and require B cells for inducing antigen-specific T cells. Therefore, we investigated the relative importance of exosomal major histocompatibility complex (MHC) class I for the induction of antigen-specific T cell responses and tumour protection. We show that ovalbumin-loaded dendritic cell-derived exosomes from MHCI-/- mice induce antigen-specific T cells at the same magnitude as wild type exosomes. Furthermore, exosomes lacking MHC class I, as well as exosomes with both MHC class I and II mismatch, induced tumour infiltrating T cells and increased overall survival to the same extent as syngeneic exosomes in B16 melanoma. In conclusion, T cell responses are independent of exosomal MHC/peptide complexes if whole antigen is present. This establishes the prospective of using impersonalised exosomes, and will greatly increase the feasibility of designing exosome-based vaccines or therapeutic approaches in humans.

  15. Kupffer cell complement receptor clearance function and host defense.

    Science.gov (United States)

    Loegering, D J

    1986-01-01

    Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.

  16. Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

    Science.gov (United States)

    Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

    2017-01-01

    Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

  17. The prognostic role of classical and nonclassical MHC class I expression in endometrial cancer

    NARCIS (Netherlands)

    Bijen, C.B.; Bantema-Joppe, E.J.; de Jong, Renske; Leffers, N.; Mourits, M.J.; Eggink, Henk F.; van der Zee, A.G.; Hollema, H.; de Bock, G.H.; Nijman, H.W.

    2010-01-01

    The aim of this study was to investigate classical MHC class I and nonclassical MHC (human leukocyte antigen-G [HLA-GJ) expression in a large cohort of patients with endometrial cancer, to determine the prognostic value of these cell surface markers and their relation with clinicopathological

  18. Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.; Morozov, Giora I.; Mage, Michael G.; Margulies, David H. (NIH); (Hebrew)

    2017-10-12

    Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

  19. Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A-431) cells

    Science.gov (United States)

    Tan, Y; Chiow, KH; Huang, D; Wong, SH

    2010-01-01

    Background and purpose: Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. Experimental approach: We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. Key results: Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. Conclusion and implications: This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death. PMID:20233216

  20. Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

    Directory of Open Access Journals (Sweden)

    Channakeshava Sokke Umeshappa

    Full Text Available Involvement of CD4(+ helper T (Th cells is crucial for CD8(+ cytotoxic T lymphocyte (CTL-mediated immunity. However, CD4(+ Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA-pulsed dendritic cell (DCova. CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I complex signaling, CD4(+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2 and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7 molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

  1. Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

    Science.gov (United States)

    Umeshappa, Channakeshava Sokke; Xie, Yufeng; Xu, Shulin; Nanjundappa, Roopa Hebbandi; Freywald, Andrew; Deng, Yulin; Ma, Hong; Xiang, Jim

    2013-01-01

    Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

  2. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    International Nuclear Information System (INIS)

    Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J.

    2011-01-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  3. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Iri-Sofla, Farnoush Jafari [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O (Denmark); Rasaee, Mohammad J. [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  4. Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

    OpenAIRE

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERR...

  5. A Novel Therapeutic Vaccine for Metastatic Mammary Carcinoma: Focusing MHC/Peptide Complexes to Lipid Rafts

    National Research Council Canada - National Science Library

    Dolan, Brian

    2004-01-01

    Genetic engineering of tumor cells to express MHC class and subsequent use of said cells for treatment of established and metastatic tumors has yielded promising results in animal models for treatment of breast cancer...

  6. Tropomyosin Receptor Kinase A Expression on Merkel Cell Carcinoma Cells.

    Science.gov (United States)

    Wehkamp, Ulrike; Stern, Sophie; Krüger, Sandra; Hauschild, Axel; Röcken, Christoph; Egberts, Friederike

    2017-11-01

    Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor frequently associated with the Merkel cell polyomavirus. Immune checkpoint therapy showed remarkable results, although not all patients are responsive to this therapy. Anti-tropomyosin receptor kinase A (TrkA)-targeted treatment has shown promising results in several tumor entities. To determine TrkA expression in MCC as a rationale for potential targeted therapy. This case series study investigated the MCC specimens of 55 patients treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany, from January 1, 2005, through December 31, 2015. Thirty-nine of the 55 samples were suitable for further histopathologic examination. Expression of TrkA was explored by immunohistochemical analysis. Diagnosis of MCC was confirmed by staining positive for cytokeratin 20 (CK20) and synaptophysin. Expression of TrkA on the tumor cells. Specimens of 39 patients (21 women and 18 men; mean [SD] age, 75.0 [7.8] years) underwent immunohistochemical investigation. Thirty-eight of 38 specimens expressed CK20 and synaptophysin on the MCC tumor cells (100% expression). Merkel cell polyomavirus was detected in 32 of 38 specimens (84%). Tropomyosin receptor kinase A was found in all 36 evaluable specimens on the tumor cells; 34 (94%) showed a weak and 2 (6%) showed a strong cytoplasmic expression. In addition, strongly positive perinuclear dots were observed in 30 of 36 specimens (83%). Tropomyosin receptor kinase A was expressed on MCC tumor cells in 100% of evaluable specimens. This result may lead to the exploration of new targeted treatment options in MCC, especially for patients who do not respond to anti-programmed cell death protein 1 treatment.

  7. 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.

  8. NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells

    International Nuclear Information System (INIS)

    Bhat, Rauf; Rommelaere, Jean

    2013-01-01

    Investigating how the immune system functions during malignancies is crucial to developing novel therapeutic strategies. Natural killer (NK) cells, an important component of the innate immune system, play a vital role in immune defense against tumors and virus-infected cells. The poor survival rate in colon cancer makes it particularly important to develop novel therapeutic strategies. Oncolytic viruses, in addition to lysing tumor cells, may have the potential to augment antitumor immune responses. In the present study, we investigate the role of NK cells and how parvovirus H-1PV can modulate NK-cell mediated immune responses against colon carcinoma. Human NK cells were isolated from the blood of healthy donors. The cytotoxicity and antibody-mediated inhibition of NK cells were measured in chromium release assays. Phenotypic assessment of colon cancer and dendritic cells was done by FACS. The statistical significance of the results was calculated with Student’s t test (*p <0.05; **, p < 0.01; ***, p < 0.001). We show that IL-2-activated human NK cells can effectively kill colon carcinoma cells. Killing of colon carcinoma cells by NK cells was further enhanced upon infection of the former cells with parvovirus H-1PV. H-1PV has potent oncolytic activity against various tumors, yet its direct killing effect on colon carcinoma cells is limited. The cytotoxicity of NK cells towards colon carcinoma cells, both mock- and H-1PV-infected, was found to be mostly mediated by a combination of natural cytotoxicity receptors (NCRs), namely NKp30, 44, and 46. Colon carcinoma cells displayed low to moderate expression of NK cell ligands, and this expression was modulated upon H-1PV infection. Lysates of H-1PV-infected colon carcinoma cells were found to increase MHC class II expression on dendritic cells. Altogether, these data suggest that IL-2-activated NK cells actively kill colon carcinoma cells and that this killing is mediated by several natural cytotoxicity receptors

  9. MHC Intratumoral Heterogeneity May Predict Cancer Progression and Response to Immunotherapy

    Directory of Open Access Journals (Sweden)

    Irene Romero

    2018-01-01

    Full Text Available An individual tumor can present intratumoral phenotypic heterogeneity, containing tumor cells with different phenotypes that do not present irreversible genetic alterations. We have developed a mouse cancer model, named GR9, derived from a methylcholanthrene-induced fibrosarcoma that was adapted to tissue culture and cloned into different tumor cell lines. The clones showed diverse MHC-I phenotypes, ranging from highly positive to weakly positive MHC-I expression. These MHC-I alterations are due to reversible molecular mechanisms, because surface MHC-I could be recovered by IFN-γ treatment. Cell clones with high MHC-I expression demonstrated low local oncogenicity and high spontaneous metastatic capacity, whereas MHC-I-low clones showed high local oncogenicity and no spontaneous metastatic capacity. Although MHC-I-low clones did not metastasize, they produced MHC-I-positive dormant micrometastases controlled by the host immune system, i.e., in a state of immunodormancy. The metastatic capacity of each clone was directly correlated with the host T-cell subpopulations; thus, a strong decrease in cytotoxic and helper T lymphocytes was observed in mice with numerous metastases derived from MHC-I positive tumor clones but a strong increase was observed in those with dormant micrometastases. Immunotherapy was administered to the hosts after excision of the primary tumor, producing a recovery in their immune status and leading to the complete eradication of overt spontaneous metastases or their decrease. According to these findings, the combination of MHC-I surface expression in primary tumor and metastases with host T-cell subsets may be a decisive indicator of the clinical outcome and response to immunotherapy in metastatic disease, allowing the identification of responders to this approach.

  10. ER stress affects processing of MHC class I-associated peptides

    Directory of Open Access Journals (Sweden)

    Meloche Sylvain

    2009-02-01

    Full Text Available Abstract Background Viral infection and neoplastic transformation trigger endoplasmic reticulum (ER stress. Thus, a large proportion of the cells that must be recognized by the immune system are stressed cells. Cells respond to ER stress by launching the unfolded protein response (UPR. The UPR regulates the two key processes that control major histocompatibility complex class I (MHC I-peptide presentation: protein synthesis and degradation. We therefore asked whether and how the UPR impinges on MHC I-peptide presentation. Results We evaluated the impact of the UPR on global MHC I expression and on presentation of the H2Kb-associated SIINFEKL peptide. EL4 cells stably transfected with vectors coding hen egg lysozyme (HEL-SIINFEKL protein variants were stressed with palmitate or exposed to glucose deprivation. UPR decreased surface expression of MHC I but did not affect MHC I mRNA level nor the total amount of intracellular MHC I proteins. Impaired MHC I-peptide presentation was due mainly to reduced supply of peptides owing to an inhibition of overall protein synthesis. Consequently, generation of H2Kb-SIINFEKL complexes was curtailed during ER stress, illustrating how generation of MHC I peptide ligands is tightly coupled to ongoing protein synthesis. Notably, the UPR-induced decline of MHC I-peptide presentation was more severe when the protein source of peptides was localized in the cytosol than in the ER. This difference was not due to changes in the translation rates of the precursor proteins but to increased stability of the cytosolic protein during ER stress. Conclusion Our results demonstrate that ER stress impairs MHC I-peptide presentation, and that it differentially regulates expression of ER- vs. cytosol-derived peptides. Furthermore, this work illustrates how ER stress, a typical feature of infected and malignant cells, can impinge on cues for adaptive immune recognition.

  11. Patterns of MHC-G-Like and MHC-B Diversification in New World Monkeys.

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    Juan S Lugo

    Full Text Available The MHC class I (MHC-I region in New World monkeys (Platyrrhini has remained relatively understudied. To evaluate the diversification patterns and transcription behavior of MHC-I in Platyrrhini, we first analyzed public genomic sequences from the MHC-G-like subregion in Saimiri boliviensis, Ateles geoffroyi and Callicebus moloch, and from the MHC-B subregion in Saimiri boliviensis. While S. boliviensis showed multiple copies of both MHC-G-like (10 and -B (15 loci, A. geoffroyi and C. moloch had only three and four MHC-G-like genes, respectively, indicating that not all Platyrrhini species have expanded their MHC-I loci. We then sequenced MHC-G-like and -B cDNAs from nine Platyrrhini species, recovering two to five unique cDNAs per individual for both loci classes. In two Saguinus species, however, no MHC-B cDNAs were found. In phylogenetic trees, MHC-G-like cDNAs formed genus-specific clusters whereas the MHC-B cDNAs grouped by Platyrrhini families, suggesting a more rapid diversification of the former. Furthermore, cDNA sequencing in 12 capuchin monkeys showed that they transcribe at least four MHC-G-like and five MHC-B polymorphic genes, showing haplotypic diversity for gene copy number and signatures of positive natural selection at the peptide binding region. Finally, a quantitative index for MHC:KIR affinity was proposed and tested to predict putative interacting pairs. Altogether, our data indicate that i MHC-I genes has expanded differentially among Platyrrhini species, ii Callitrichinae (tamarins and marmosets MHC-B loci have limited or tissue-specific expression, iii MHC-G-like genes have diversified more rapidly than MHC-B genes, and iv the MHC-I diversity is generated mainly by genetic polymorphism and gene copy number variation, likely promoted by natural selection for ligand binding.

  12. Genetic engineering with T cell receptors.

    Science.gov (United States)

    Zhang, Ling; Morgan, Richard A

    2012-06-01

    In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers. Published by Elsevier B.V.

  13. Paternal HLA-C and Maternal Killer-Cell Immunoglobulin-Like Receptor Genotypes in the Development of Autism.

    Science.gov (United States)

    Gamliel, Moriya; Anderson, Karen L; Ebstein, Richard P; Yirmiya, Nurit; Mankuta, David

    2016-01-01

    Killer-cell immunoglobulin-like receptors (KIRs) are a family of cell surface proteins found on natural killer cells, which are components of the innate immune system. KIRs recognize MHC class I proteins, mainly HLA-C and are further divided into two groups: short-tailed 2/3DS activating receptors and long-tailed 2/3DL inhibitory receptors. Based on the Barker Hypothesis, the origins of illness can be traced back to embryonic development in the uterus, and since KIR:HLA interaction figures prominently in the maternal-fetal interface, we investigated whether specific KIR:HLA combinations may be found in autism spectrum disorders (ASD) children compared with their healthy parents. This study enrolled 49 ASD children from different Israeli families, and their healthy parents. Among the parents, a higher frequency of HLA-C2 allotypes was found in the fathers, while its corresponding ligand 2DS1 was found in higher percentage in the maternal group. However, such skewing in KIR:HLA frequencies did not appear in the ASD children. Additionally, analysis of "overall activation" indicated higher activation in maternal than in paternal cohorts.

  14. Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

    Directory of Open Access Journals (Sweden)

    Karasuyama Hajime

    2011-04-01

    Full Text Available Abstract Background There have been few reports on the role of Fc receptors (FcRs and immunoglobulin G (IgG in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa. Methods In FcγRIIB deficient (KO and C57BL/6 (WT mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA. Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL. Results In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously. Conclusion Antigen-specific IgG ameliorates

  15. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  16. MHC Class II epitope predictive algorithms

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lund, Ole; Buus, S

    2010-01-01

    Major histocompatibility complex class II (MHC-II) molecules sample peptides from the extracellular space, allowing the immune system to detect the presence of foreign microbes from this compartment. To be able to predict the immune response to given pathogens, a number of methods have been...... developed to predict peptide-MHC binding. However, few methods other than the pioneering TEPITOPE/ProPred method have been developed for MHC-II. Despite recent progress in method development, the predictive performance for MHC-II remains significantly lower than what can be obtained for MHC-I. One reason...

  17. Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition.

    Directory of Open Access Journals (Sweden)

    Maud Condomines

    Full Text Available Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1 costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

  18. Forming a complex with MHC class I molecules interferes with mouse CD1d functional expression.

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    Renukaradhya J Gourapura

    Full Text Available CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of mouse CD1d on the surface of cells. Low pH (3.0 acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.

  19. Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

    Science.gov (United States)

    Peñas-Cazorla, Raúl; Vilaró, M Teresa

    2015-11-01

    Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists.

  20. Mouse NK cell-mediated rejection of bone marrow allografts exhibits patterns consistent with Ly49 subset licensing.

    Science.gov (United States)

    Sun, Kai; Alvarez, Maite; Ames, Erik; Barao, Isabel; Chen, Mingyi; Longo, Dan L; Redelman, Doug; Murphy, William J

    2012-02-09

    Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49(+) subsets in BMC rejection by using β2-microglobulin deficient (β2m(-/-)) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind "self-MHC" were found to be the dominant subset responsible for β2m(-/-) BMC rejection. This provides in vivo evidence for host MHC class I-dependent functional education. Interestingly, all H2(d) strain mice regardless of background were able to resist significantly greater amounts of β2m(-/-), but not wild-type BMC than H2(b) mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.

  1. Functional recombinant MHC class II molecules and high-throughput peptide-binding assays

    DEFF Research Database (Denmark)

    Justesen, Sune; Harndahl, Mikkel; Lamberth, Kasper

    2009-01-01

    BACKGROUND: Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle......-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately....... CONCLUSION: We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools....

  2. Peripheral nerve injury causes transient expression of MHC class I antigens in rat motor neurons and skeletal muscles

    DEFF Research Database (Denmark)

    Maehlen, J; Nennesmo, I; Olsson, A B

    1989-01-01

    After a peripheral nerve lesion (rat facial and sciatic) an induction of major histocompatibility complex (MHC) antigens class I was detected immunohistochemically in skeletal muscle fibers and motor neurons. This MHC expression was transient after a nerve crush, when regeneration occurred......, but persisted after a nerve cut, when regeneration was prevented. Since the time course of MHC class I expression correlates to that of regeneration a role for this cell surface molecule in regeneration may be considered....

  3. Role of laminin receptor in tumor cell migration

    DEFF Research Database (Denmark)

    Wewer, U M; Taraboletti, G; Sobel, M E

    1987-01-01

    Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986......). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor...... antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin...

  4. Nonequivalence of classical MHC class I loci in ability to direct effective antiviral immunity.

    Directory of Open Access Journals (Sweden)

    Kevin D Pavelko

    2012-02-01

    Full Text Available Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b but encoding the peptide binding domain of D(b, develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(bα1α2D(b gene (low and D(b (high in the CNS of infected mice mirror expression levels of their endogenous H-2(q counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

  5. NetMHCpan 4.0: Improved peptide-MHC class I interaction predictions integrating eluted ligand and peptide binding affinity data

    OpenAIRE

    Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten

    2017-01-01

    Cytotoxic T cells are of central importance in the immune systems response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC (major histocompatibility complex) class I molecules. Peptide binding to MHC molecules is the single most selective step in the antigen presentation pathway. On the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has therefore attracted large attention. In the past, predictors of peptide-...

  6. Orf virus interferes with MHC class I surface expression by targeting vesicular transport and Golgi

    Directory of Open Access Journals (Sweden)

    Rohde Jörg

    2012-07-01

    Full Text Available Abstract Background The Orf virus (ORFV, a zoonotic Parapoxvirus, causes pustular skin lesions in small ruminants (goat and sheep. Intriguingly, ORFV can repeatedly infect its host, despite the induction of a specific immunity. These immune modulating and immune evading properties are still unexplained. Results Here, we describe that ORFV infection of permissive cells impairs the intracellular transport of MHC class I molecules (MHC I as a result of structural disruption and fragmentation of the Golgi apparatus. Depending on the duration of infection, we observed a pronounced co-localization of MHC I and COP-I vesicular structures as well as a reduction of MHC I surface expression of up to 50%. These subversion processes are associated with early ORFV gene expression and are accompanied by disturbed carbohydrate trimming of post-ER MHC I. The MHC I population remaining on the cell surface shows an extended half-life, an effect that might be partially controlled also by late ORFV genes. Conclusions The presented data demonstrate that ORFV down-regulates MHC I surface expression in infected cells by targeting the late vesicular export machinery and the structure and function of the Golgi apparatus, which might aid to escape cellular immune recognition.

  7. Non-MHC genes influence virus clearance through regulation of the antiviral T-cell response: correlation between virus clearance and Tc and Td activity in segregating backcross progeny

    DEFF Research Database (Denmark)

    Christensen, Jan Pravsgaard; Marker, O; Thomsen, Allan Randrup

    1994-01-01

    ) was followed by measurement of footpad swelling. Ten days after virus inoculation, the animals were sacrificed and spleen virus titer together with splenic Tc activity was measured. With regard to all three parameters a continuous distribution was observed in this backcross population. However, using cutoff...... values based on parental and F1 animals tested in parallel, 11/30 animals were assigned Tc responders, 23/30 DTH responders and 10/30 cleared virus with maximal efficiency. Comparison of responder status with regard to the different parameters revealed a strong correlation between Tc responsiveness...... and the ability to clear virus. Amongst Tc low responders a correlation between DTH reactivity and virus clearance was observed. Taken together, these results indicate that non-MHC genes affect virus clearance through regulation of the antiviral T-cell response, especially the virus-specific Tc response. However...

  8. Rice Bran Feruloylated Oligosaccharides Activate Dendritic Cells via Toll-Like Receptor 2 and 4 Signaling

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    Chi Chen Lin

    2014-04-01

    Full Text Available This work presents the effects of feruloylated oligosaccharides (FOs of rice bran on murine bone marrow-derived dendritic cells (BMDCs and the potential pathway through which the effects are mediated. We found that FOs induced phenotypic maturation of DCs, as shown by the increased expression of CD40, CD80/CD86 and MHC-I/II molecules. FOs efficiently induced maturation of DCs generated from C3H/HeN or C57BL/6 mice with normal toll-like receptor 4 (TLR-4 or TLR-2 but not DCs from mice with mutated TLR4 or TLR2. The mechanism of action of FOs may be mediated by increased phosphorylation of ERK, p38 and JNK mitogen-activated protein kinase (MAPKs and increased NF-kB activity, which are important signaling molecules downstream of TLR-4 and TLR-2. These data suggest that FOs induce DCs maturation through TLR-4 and/or TLR-2 and that FOs might have potential efficacy against tumor or virus infection or represent a candidate-adjuvant approach for application in immunotherapy and vaccination.

  9. Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands.

    Science.gov (United States)

    Das, Dibyendu Kumar; Mallis, Robert J; Duke-Cohan, Jonathan S; Hussey, Rebecca E; Tetteh, Paul W; Hilton, Mark; Wagner, Gerhard; Lang, Matthew J; Reinherz, Ellis L

    2016-12-02

    The pre-T cell receptor (pre-TCR) is a pTα-β heterodimer functioning in early αβ T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αβTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cβ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαβ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vβ patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of β chains with self-reactivity but is occluded by α subunit replacement of pTα upon αβTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αβTCR repertoire tuning via the pre-TCR. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

    NARCIS (Netherlands)

    C.H.J. Lamers (Cor); S. van Steenbergen-Langeveld (Sabine); M. van Brakel (Mandy); C.M. Groot-van Ruijven (Corrien); P.M.M.L. van Elzakker (Pascal); B.A. van Krimpen (Brigitte); S. Sleijfer (Stefan); J.E.M.A. Debets (Reno)

    2014-01-01

    textabstractTherapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T

  11. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...... whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays...

  12. Constitutive MHC class I molecules negatively regulate TLR-triggered inflammatory responses via the Fps-SHP-2 pathway.

    Science.gov (United States)

    Xu, Sheng; Liu, Xingguang; Bao, Yan; Zhu, Xuhui; Han, Chaofeng; Zhang, Peng; Zhang, Xuemin; Li, Weihua; Cao, Xuetao

    2012-04-22

    The molecular mechanisms that fine-tune Toll-like receptor (TLR)-triggered innate inflammatory responses remain to be fully elucidated. Major histocompatibility complex (MHC) molecules can mediate reverse signaling and have nonclassical functions. Here we found that constitutively expressed membrane MHC class I molecules attenuated TLR-triggered innate inflammatory responses via reverse signaling, which protected mice from sepsis. The intracellular domain of MHC class I molecules was phosphorylated by the kinase Src after TLR activation, then the tyrosine kinase Fps was recruited via its Src homology 2 domain to phosphorylated MHC class I molecules. This led to enhanced Fps activity and recruitment of the phosphatase SHP-2, which interfered with TLR signaling mediated by the signaling molecule TRAF6. Thus, constitutive MHC class I molecules engage in crosstalk with TLR signaling via the Fps-SHP-2 pathway and control TLR-triggered innate inflammatory responses.

  13. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  14. Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

    Directory of Open Access Journals (Sweden)

    Jimenez-Perez Miriam I

    2012-02-01

    Full Text Available Abstract Background Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity. Results We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT. Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells. Conclusions Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

  15. Internalisation of gonadotrophin-receptor complex in ovarian luteal cells

    International Nuclear Information System (INIS)

    Conn, P.M.; Conti, M.; Harwood, J.P.; Dufau, M.L.; Catt, K.J.

    1978-01-01

    Following evidence that certain protein hormones can enter target cells the present investigation was undertaken which shows that gonadotrophin-induced receptor loss may occur by a process of internalisation of the hormone-receptor complex following the initial interaction of gonadotrophin with the cell surface. Localisation studies were carried out in 33-d old female rats previously treated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin (hCG) to induce ovarian luteinisation. Animals were injected with 125 I-hCG to label the ovarian receptors for luteinising hormone in vivo. Microscope autoradiographs demonstrating distribution of 125 I-hCG in ovaries at various times following injection are shown. The combined results from the autoradiographs and from solubilisation experiments were used to determine the location and nature of the hCG-receptor complex following occupancy and loss of receptors from the plasma membrane of luteinised ovarian cells. (U.K.)

  16. Functional somatostatin receptors on a rat pancreatic acinar cell line

    International Nuclear Information System (INIS)

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A.

    1988-01-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125 I-[Tyr 11 ]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/10 6 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125 I-[Tyr 11 ]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N i to inhibit adenylate cyclase

  17. The Role of the Sweet Taste Receptor in Enteroendocrine Cells and Pancreatic β-Cells

    Directory of Open Access Journals (Sweden)

    Itaru Kojima

    2011-10-01

    Full Text Available The sweet taste receptor is expressed in taste cells located in taste buds of the tongue. This receptor senses sweet substances in the oral cavity, activates taste cells, and transmits the taste signals to adjacent neurons. The sweet taste receptor is a heterodimer of two G protein-coupled receptors, T1R2 and T1R3. Recent studies have shown that this receptor is also expressed in the extragustatory system, including the gastrointestinal tract, pancreatic β-cells, and glucose-responsive neurons in the brain. In the intestine, the sweet taste receptor regulates secretion of incretin hormones and glucose uptake from the lumen. In β-cells, activation of the sweet taste receptor leads to stimulation of insulin secretion. Collectively, the sweet taste receptor plays an important role in recognition and metabolism of energy sources in the body.

  18. pH dependence of MHC class I-restricted peptide presentation

    DEFF Research Database (Denmark)

    Stryhn, A; Pedersen, L O; Romme, T

    1996-01-01

    The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited....... Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset...

  19. Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Worning, Peder

    2004-01-01

    Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus......, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates...

  20. Chimeric antigen receptor T-cell therapy for solid tumors

    Directory of Open Access Journals (Sweden)

    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.

  1. MHC variability in heritage breeds of chickens.

    Science.gov (United States)

    Fulton, J E; Lund, A R; McCarron, A M; Pinegar, K N; Korver, D R; Classen, H L; Aggrey, S; Utterbach, C; Anthony, N B; Berres, M E

    2016-02-01

    The chicken Major Histocompatibility Complex (MHC) is very strongly associated with disease resistance and thus is a very important region of the chicken genome. Historically, MHC (B locus) has been identified by the use of serology with haplotype specific alloantisera. These antisera can be difficult to produce and frequently cross-react with multiple haplotypes and hence their application is generally limited to inbred and MHC-defined lines. As a consequence, very little information about MHC variability in heritage chicken breeds is available. DNA-based methods are now available for examining MHC variability in these previously uncharacterized populations. A high density SNP panel consisting of 101 SNP that span a 230,000 bp region of the chicken MHC was used to examine MHC variability in 17 heritage populations of chickens from five universities from Canada and the United States. The breeds included 6 heritage broiler lines, 3 Barred Plymouth Rock, 2 New Hampshire and one each of Rhode Island Red, Light Sussex, White Leghorn, Dark Brown Leghorn, and 2 synthetic lines. These heritage breeds contained from one to 11 haplotypes per line. A total of 52 unique MHC haplotypes were found with only 10 of them identical to serologically defined haplotypes. Furthermore, nine MHC recombinants with their respective parental haplotypes were identified. This survey confirms the value of these non-commercially utilized lines in maintaining genetic diversity. The identification of multiple MHC haplotypes and novel MHC recombinants indicates that diversity is being generated and maintained within these heritage populations. © 2016 Poultry Science Association Inc.

  2. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

    2015-01-01

    The presence of AT2 receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and the role in the induction...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  3. Perspectives on the Trypanosoma cruzi–host cell receptor interactions

    Science.gov (United States)

    Villalta, Fernando; Scharfstein, Julio; Ashton, Anthony W.; Tyler, Kevin M.; Guan, Fangxia; Mukherjee, Shankar; Lima, Maria F.; Alvarez, Sandra; Weiss, Louis M.; Huang, Huan; Machado, Fabiana S.

    2009-01-01

    Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets. PMID:19283409

  4. Prospects and limitations of T cell receptor gene therapy

    NARCIS (Netherlands)

    Jorritsma, Annelies; Schotte, Remko; Coccoris, Miriam; de Witte, Moniek A.; Schumacher, Ton N. M.

    2011-01-01

    Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining

  5. Protective influences on experimental autoimmune encephalomyelitis by MHC class I and class II alleles

    DEFF Research Database (Denmark)

    Mustafa, M; Vingsbo, C; Olsson, T

    1994-01-01

    are resistant. Interestingly, rats with the MHC u haplotype develop an immune response to the MBP 63-88, but do not get EAE. In this study we have used intra-MHC recombinant rat strains to compare the influences of the MHC u with the a haplotype. We discovered the following: 1) The class II region of the MHC...... a haplotype permits EAE and a Th1 type of immune response as measured by IFN-gamma production after in vitro challenge of in vivo-primed T cells with MBP 63-88. 2) The class II region of the u haplotype is associated with a disease-protective immune response characterized by production of not only IFN......Experimental autoimmune encephalomyelitis (EAE) is influenced by polymorphism of the MHC. We have previously found that Lewis rats with certain MHC haplotypes are susceptible to disease induced with the myelin basic protein (MBP) peptide 63-88, whereas Lewis rats with other MHC haplotypes...

  6. Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

    Science.gov (United States)

    Kahaleh, M B; Fan, P S; Otsuka, T

    1999-05-01

    In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc. Copyright 1999 Academic Press.

  7. ZAP-70 and p72syk are signaling response elements through MHC class II molecules

    DEFF Research Database (Denmark)

    Kanner, S B; Grosmaire, L S; Blake, J

    1995-01-01

    Ligation of major histocompatibility complex (MHC) class II antigens expressed on antigen-activated human CD4+ T-lymphocytes induces early signal transduction events including the activation of tyrosine kinases, the tyrosine phosphorylation of phospholipase-C gamma 1 and the mobilization...... of intracellular calcium. Similar responses have been observed in B-cells following stimulation of MHC class II molecules, including the increased production of intracellular cAMP. In this report, we demonstrate that the ZAP-70 tyrosine kinase is a responsive signaling element following cross-linking of HLA...... by herbimycin A. MHC class II ligation on B-lymphocytes resulted in cell death, which was both qualitatively distinct from Fas-induced apoptosis and partially protected by herbimycin A pretreatment. Thus, ligation of MHC class II molecules expressed on human lymphocytes stimulates the ZAP-70/p72syk family...

  8. Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

    Science.gov (United States)

    Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

    2009-01-01

    High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

  9. IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation.

    Directory of Open Access Journals (Sweden)

    Qiliang Cai

    2013-10-01

    Full Text Available Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters. This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

  10. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    International Nuclear Information System (INIS)

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-01-01

    Highlights: ► Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. ► EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. ► EGCG-treated DCs inhibited MAPKs activation and NF-κB p65 translocation via 67LR. ► EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  11. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Eui-Baek [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Choi, Han-Gyu [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Sung, Nak-Yun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong, E-mail: ehbyun80@gmail.com [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of)

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  12. Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

    DEFF Research Database (Denmark)

    Siddle, Hannah V; Kreiss, Alexandre; Tovar, Cesar

    2013-01-01

    Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should...... be rejected by the host-immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo......, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can...

  13. 5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

    DEFF Research Database (Denmark)

    Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

    2006-01-01

    39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events in the ce......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial...

  14. Correlated receptor transport processes buffer single-cell heterogeneity.

    Directory of Open Access Journals (Sweden)

    Stefan M Kallenberger

    2017-09-01

    Full Text Available Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

  15. Aminobisphosphonates and Toll-like receptor ligands: recruiting Vγ9Vδ2 T cells for the treatment of hematologic malignancy.

    Science.gov (United States)

    Kalyan, S; Wesch, D; Kabelitz, D

    2011-01-01

    Gamma delta (γδ) T cells are intrinsically important for preventing the development and progression of hematologic cancers. These innate T cells are particularly suited for the application of cancer therapy due to the fact they: 1) recognize transformed cells independent of antigen processing or presentation by classical MHC molecules, and 2) embody the anti-tumour effector functions of both NK cells and cytotoxic T cells. It was serendipitously discovered that aminobisphosphonates (ABP), a class of drugs used as adjuvant cancer therapy for the treatment of malignant osteolytic bone disease, have the unexpected side-effect of potently activating the antitumour effector functions of human peripheral γδ T cells. Such beneficial therapeutic synergisms are rare, and no time has been wasted to determine how to best harness the anti-cancer potential of γδ T cells and ABP. Despite promising experimental results, the full clinical potential of this immunotherapeutic strategy has been hampered by the subversive strategies employed by cancer cells to obstruct activation of anti-tumour immune responses. These include the promotion of regulatory T cells (Tregs) that maintain tumour tolerance and the corruption of dendritic cell (DC) function and maturation. Toll-like receptor (TLR) agonists have a long history of breaking free of tumour-induced immune-suppression by resetting DC function and abrogating Treg induced tolerance. This review presents data to support the notion that TLR signalling may perfectly complement the anti-tumour synergy of ABP and activated γδ T cells, and this combined innate artillery could provide the necessary ammunition to topple malignancy's stronghold on the immune system.

  16. Cellular expression or binding of desLys58-beta2 microglobulin is not dependent on the presence of the tri-molecular MHC class I complex

    DEFF Research Database (Denmark)

    Wang, M; Corlin, D B; Heegaard, N H H

    2008-01-01

    -exposure to dbeta2m. Binding of 332-01 antibody could not be displaced by addition of high concentrations of native beta2m. In conclusion, our data indicate that dbeta2m - in contrast to native beta2m - binds to a hitherto unknown cell surface receptor independent of classical MHC class I molecules. As beta2m has...... previously been shown to display biological activities such as the induction of both growth promotion and apoptosis, C1 complement activity, shown to mediate cleavage of beta2m, could be involved in these processes....

  17. Dynamics of Corticosteroid Receptors: Lessons from Live Cell Imaging

    International Nuclear Information System (INIS)

    Nishi, Mayumi

    2011-01-01

    Adrenal corticosteroids (cortisol in humans or corticosterone in rodents) exert numerous effects on the central nervous system that regulates the stress response, mood, learning and memory, and various neuroendocrine functions. Corticosterone (CORT) actions in the brain are mediated via two receptor systems: the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). It has been shown that GR and MR are highly colocalized in the hippocampus. These receptors are mainly distributed in the cytoplasm without hormones and translocated into the nucleus after treatment with hormones to act as transcriptional factors. Thus the subcellular dynamics of both receptors are one of the most important issues. Given the differential action of MR and GR in the central nervous system, it is of great consequence to clarify how these receptors are trafficked between cytoplasm and nucleus and their interactions are regulated by hormones and/or other molecules to exert their transcriptional activity. In this review, we focus on the nucleocytoplasmic and subnuclear trafficking of GR and MR in neural cells and non-neural cells analyzed by using molecular imaging techniques with green fluorescent protein (GFP) including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET), and discuss various factors affecting the dynamics of these receptors. Furthermore, we discuss the future directions of in vivo molecular imaging of corticosteroid receptors at the whole brain level

  18. An MHC class I immune evasion gene of Marek׳s disease virus.

    Science.gov (United States)

    Hearn, Cari; Preeyanon, Likit; Hunt, Henry D; York, Ian A

    2015-01-15

    Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198-205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  20. The E5 protein of human papillomavirus type 16 perturbs MHC class II antigen maturation in human foreskin keratinocytes treated with interferon-γ

    International Nuclear Information System (INIS)

    Zhang Benyue; Li Ping; Wang Exing; Brahmi, Zacharie; Dunn, Kenneth W.; Blum, Janice S.; Roman, Ann

    2003-01-01

    Major histocompatibility complex (MHC) class II antigens are expressed on human foreskin keratinocytes (HFKs) following exposure to interferon gamma. The expression of MHC class II proteins on the cell surface may allow keratinocytes to function as antigen-presenting cells and induce a subsequent immune response to virus infection. Invariant chain (Ii) is a chaperone protein which plays an important role in the maturation of MHC class II molecules. The sequential degradation of Ii within acidic endocytic compartments is a key process required for the successful loading of antigenic peptide onto MHC class II molecules. Since human papillomavirus (HPV) 16 E5 can inhibit the acidification of late endosomes in HFKs, the E5 protein may be able to affect proper peptide loading onto the MHC class II molecule. To test this hypothesis, HFKs were infected with either control virus or a recombinant virus expressing HPV16 E5 and the infected cells were subsequently treated with interferon-γ. ELISAs revealed a decrease of MHC class II expression on the surface of E5-expressing cells compared with control virus-infected cells after interferon treatment. Western blot analysis showed that, in cells treated with interferon gamma, E5 could prevent the breakdown of Ii and block the formation of peptide-loaded, SDS-stable mature MHC class II dimers, correlating with diminished surface MHC class II expression. These data suggest that HPV16 E5 may be able to decrease immune recognition of infected keratinocytes via disruption of MHC class II protein function

  1. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction

    Directory of Open Access Journals (Sweden)

    Lund Ole

    2009-09-01

    Full Text Available Abstract Background The major histocompatibility complex (MHC molecule plays a central role in controlling the adaptive immune response to infections. MHC class I molecules present peptides derived from intracellular proteins to cytotoxic T cells, whereas MHC class II molecules stimulate cellular and humoral immunity through presentation of extracellularly derived peptides to helper T cells. Identification of which peptides will bind a given MHC molecule is thus of great importance for the understanding of host-pathogen interactions, and large efforts have been placed in developing algorithms capable of predicting this binding event. Results Here, we present a novel artificial neural network-based method, NN-align that allows for simultaneous identification of the MHC class II binding core and binding affinity. NN-align is trained using a novel training algorithm that allows for correction of bias in the training data due to redundant binding core representation. Incorporation of information about the residues flanking the peptide-binding core is shown to significantly improve the prediction accuracy. The method is evaluated on a large-scale benchmark consisting of six independent data sets covering 14 human MHC class II alleles, and is demonstrated to outperform other state-of-the-art MHC class II prediction methods. Conclusion The NN-align method is competitive with the state-of-the-art MHC class II peptide binding prediction algorithms. The method is publicly available at http://www.cbs.dtu.dk/services/NetMHCII-2.0.

  2. Adiponectin mediated MHC class II mismatched cardiac graft rejection in mice is IL-4 dependent.

    Directory of Open Access Journals (Sweden)

    Daxu Li

    Full Text Available BACKGROUND: Adiponectin regulates glucose and fatty-acid metabolism but its role in chronic graft rejection mediated by Th2 cytokines remains ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: Wild type and adiponectin-null mice were used as graft recipients in mouse MHC class II disparate cardiac transplantation (bm12 toB6 and the graft rejection was monitored. In adiponectin-null mice we observed that the cellular infiltrate of eosinophils, CD4(+ and CD8(+ T cells was reduced in grafts compared to the controls as was collagen deposition and vessel occlusion. A similar outcome was observed for skin transplants except that neutrophil infiltration was increased. Low levels of IL-4 were detected in the grafts and serum. The effect of adiponectin signaling on IL-4 expression was further investigated. Treatment with AMPK and p38 MAPK inhibitors blocked adiponectin enhanced T cell proliferation in mixed lymphocyte reactions. Inhibition of AMPK reduced eosinophil infiltration in skin grafts in wild type recipients and in contrast AMPK activation increased eosinophils in adiponectin-null recipients. The addition of adiponectin increased IL-4 production by the T cell line EL4 with augmented nuclear GATA-3 and phospho-STAT6 expression which were suppressed by knockdown of adiponectin receptor 1 and 2. CONCLUSIONS: Our results demonstrate a direct effect of adiponectin on IL-4 expression which contributes to Th2 cytokine mediated rejection in mouse MHC class II histoincompatible transplants. These results add to our understanding of the interrelationship of metabolism and immune regulation and raise the possibility that AMPK inhibitors may be beneficial in selected types of rejection.

  3. Structural requirements for the interaction between class II MHC molecules and peptide antigens

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Appella, E

    1990-01-01

    of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity (IA(d) or IE(d)); 3) IA(d) and IE(d) molecules recognize different and independent structures on the antigen molecule; 4) only about 10% of the single...... IA(d) and IE(d) molecules and their peptide ligands, we found that some structural characteristics apply to both antigen-MHC interactions. In particular, we found: 1) each MHC molecule is capable of binding many unrelated peptides through the same peptide-binding site; 2) despite this permissiveness...... amino acid substitutions tested on two IA(d)- and IE(d)-binding peptides had significant effect on their MHC-binding capacities, while over 80% of these substitutions significantly impaired T cell recognition of the Ia-peptide complex; 5) based on the segregation between residues that are crucial for T...

  4. A Novel Therapeutic Vaccine for Metastatic Mammary Carcinoma: Focusing MHC/Peptide Complexes to Lipid Rafts

    Science.gov (United States)

    2006-11-01

    reportable outcomes). Briefly, the T cell lymphoma EL4 and the immortalized fibroblast cell line DAP (both expressing ova) were used to measure...and Use Committee. Cells , transfections, and antibodies B16.BL6 8.2, A20, EL4 and EL4 /ova were cultured as described (20-22). NIH3T3 cells were...types can donate MHC class I molecules to DC. To determine if the levels of MHC class I on the donor cell affected the efficiency of transfer, EL4 /ova

  5. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N

    1997-01-01

    by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U...

  6. The role of purinergic receptors in stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Constanze Kaebisch

    2015-01-01

    Full Text Available A major challenge modern society has to face is the increasing need for tissue regeneration due to degenerative diseases or tumors, but also accidents or warlike conflicts. There is great hope that stem cell-based therapies might improve current treatments of cardiovascular diseases, osteochondral defects or nerve injury due to the unique properties of stem cells such as their self-renewal and differentiation potential. Since embryonic stem cells raise severe ethical concerns and are prone to teratoma formation, adult stem cells are still in the focus of research. Emphasis is placed on cellular signaling within these cells and in between them for a better understanding of the complex processes regulating stem cell fate. One of the oldest signaling systems is based on nucleotides as ligands for purinergic receptors playing an important role in a huge variety of cellular processes such as proliferation, migration and differentiation. Besides their natural ligands, several artificial agonists and antagonists have been identified for P1 and P2 receptors and are already used as drugs. This review outlines purinergic receptor expression and signaling in stem cells metabolism. We will briefly describe current findings in embryonic and induced pluripotent stem cells as well as in cancer-, hematopoietic-, and neural crest-derived stem cells. The major focus will be placed on recent findings of purinergic signaling in mesenchymal stem cells addressed in in vitro and in vivo studies, since stem cell fate might be manipulated by this system guiding differentiation towards the desired lineage in the future.

  7. Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

    Science.gov (United States)

    Taupin, J L; Anderson, P

    1994-12-01

    The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

  8. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    International Nuclear Information System (INIS)

    Levy, J.R.; Olefsky, J.M.

    1988-01-01

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 0 C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 0 C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

  9. Investigations on the insulin receptor of isolated fat cells

    International Nuclear Information System (INIS)

    Eichler, W.

    1980-01-01

    Fat cells, isolated from the epididymal adipose tissue of rats, were incubed with iodine 125 insulin after previous incubation with various antagonists. By varying the antagonist concentration, it was possible to determine the effect these substances have on the insulin receptor, i.e. the insulin similarity. By varying the preincubation time, toxicity of the test substances could be detected, which pretended repression effects; and by finally verying the incubation time the effects on the receptor via the membrane could be distinguished from direct receptor bindings of the antagonist. (orig./MG) [de

  10. Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay

    DEFF Research Database (Denmark)

    Harndahl, Mikkel; Rasmussen, Michael; Røder, Gustav Andreas

    2011-01-01

    and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed the rates of dissociation that ranged from 0.1h to 46h depending on the peptide used.......Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely...... timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal...

  11. NMDA Receptors in Glial Cells: Pending Questions

    Czech Academy of Sciences Publication Activity Database

    Džamba, Dávid; Honsa, Pavel; Anděrová, Miroslava

    2013-01-01

    Roč. 11, č. 3 (2013), s. 250-262 ISSN 1570-159X R&D Projects: GA ČR GA309/08/1381; GA ČR(CZ) GBP304/12/G069 Grant - others:GA UK(CZ) 604212 Institutional support: RVO:68378041 Keywords : astrocytes * ischemia * NMDA receptors Subject RIV: FH - Neurology Impact factor: 2.347, year: 2013

  12. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    Science.gov (United States)

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation.

    Science.gov (United States)

    Landais, Elise; Romagnoli, Pablo A; Corper, Adam L; Shires, John; Altman, John D; Wilson, Ian A; Garcia, K Christopher; Teyton, Luc

    2009-12-15

    Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

  14. 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 ...

  15. Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC and its application for DNA methylation analysis

    Directory of Open Access Journals (Sweden)

    Ottaviani Diego

    2008-05-01

    Full Text Available Abstract Background The major histocompatibility complex (MHC is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome. Methods To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP, methylated DNA immunoprecipitation (MeDIP, array comparative genomic hybridization (aCGH and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls. Results Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs. Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation. Conclusion A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics.

  16. Specialities of the differentiation conditions for the memory cells - either initial or enriched secondary cytotoxic T lymphocyte precursors (pCTL-2) specific to the MHC molecule class 1

    International Nuclear Information System (INIS)

    Brondz, B.D.; Osipova, T.V.; Aptikaeva, G.F.; Kronin, V.V.

    1989-01-01

    The in vivo induced pCTL-2 with phenotype L3T4 - Lyt2 + , specific to the H-2K b molecule, turn into effector CTLs during 4 days in the mixed lymphocyte culture (with heat-treated donor stimulators) much more efficiency when donor and recipient are different from one another not only in MHC class 1 (anti-BIO, MBR BIO.AKM) but in 1+2 (K b +1 b ) anti-C57BL/6 BIAD2(R1O1). The initial pCTL-2 differentiation in enhanced as a result of synergistic effect between the K b alloantigen and rIL2. The anti-K b pCTL-2, being separated from helper T cells by means of absorption onto the macrophage donor monolayer and elution from it, give rise to pronounced differentiation in simplified conditions, irrespective of the stimulator presence and without external rIL2. It is supposed that these phenomena are roused to secrection of the CTL differentiation factor by the eluted pCTL-2 themselves, and besides, rIL2 may promote for secretion of this factor additionally

  17. Labeling of lectin receptors during the cell cycle.

    Science.gov (United States)

    Garrido, J

    1976-12-01

    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

  18. Clonorchis sinensis adult-derived proteins elicit Th2 immune responses by regulating dendritic cells via mannose receptor.

    Directory of Open Access Journals (Sweden)

    Lu Zhao

    2018-03-01

    Full Text Available Clonorchis sinensis (C. sinensis is the most widespread human liver fluke in East Asia including China and Korea. Clonorchiasis as a neglected tropical zoonosis, leads to serious economic and public health burden in China. There are considerable evidences for an etiological relation between chronic clonorchiasis and liver fibrosis in human beings. Liver fibrosis is a highly conserved and over-protected response to hepatic tissue injury. Immune cells including CD4+ T cell as well as dendritic cell (DC, and pro-fibrogenic cytokines like interleukin 4 (IL-4, IL-13 have been identified as vital manipulators in liver fibrogenesis. Our previous studies had a mere glimpse of T helper type 2 (Th2 dominant immune responses as key players in liver fibrosis induced by C. sinensis infection, but little is known about the involved mechanisms in this pathological process.By flow cytometry (FACS, adult-derived total proteins of C. sinensis (CsTPs down-regulated the expression of surface markers CD80, CD86 and major histocompatibility complex class II (MHC-II on lipopolysaccharide (LPS induced DC. ELISA results demonstrated that CsTPs inhibited IL-12p70 release from LPS-treated bone marrow-derived dendritic cells (BMDC. IL-10 level increased in a time-dependent manner in LPS-treated BMDCs after incubation with CsTPs. CD4+ T cells incubated with LPS-treated BMDCs plus CsTPs could significantly elevate IL-4 level by ELISA. Meanwhile, elevated expression of pro-fibrogenic mediators including IL-13 and IL-4 were detected in a co-culture system of LPS-activated BMDCs and naive T cells containing CsTPs. In vivo, CsTPs-immunized mice enhanced expression of type 2 cytokines IL-13, IL-10 and IL-4 in both splenocytes and hepatic tissue. Exposure of BMDCs to CsTPs activated expression of mannose receptor (MR but not toll like receptor 2 (TLR2, TLR4, C-type lectin receptor DC-SIGN and Dectin-2 on the cell surface by RT-PCR and FACS. Blockade of MR almost completely

  19. Quantifying the importance of pMHC valency, total pMHC dose and frequency on nanoparticle therapeutic efficacy.

    Science.gov (United States)

    Sugarman, Jordan; Tsai, Sue; Santamaria, Pere; Khadra, Anmar

    2013-05-01

    Nanoparticles (NPs) coated with β-cell-specific peptide major histocompatibility complex (pMHC) class I molecules can effectively restore normoglycemia in spontaneously diabetic nonobese diabetic mice. They do so by expanding pools of cognate memory autoreactive regulatory CD8+ T cells that arise from naive low-avidity T-cell precursors to therapeutic levels. Here we develop our previously constructed mathematical model to explore the effects of compound design parameters (NP dose and pMHC valency) on therapeutic efficacy with the underlying hypothesis that the functional correlates of the therapeutic response (expansion of autoregulatory T cells and deletion of autoantigen-loaded antigen-presenting cells by these T cells) are biphasic. We show, using bifurcation analysis, that the model exhibits a 'resonance'-like behavior for a given range of NP dose in which bistability between the healthy state (possessing zero level of effector T-cell population) and autoimmune state (possessing elevated level of the same population) disappears. A heterogeneous population of model mice subjected to several treatment protocols under these new conditions is conducted to quantify both the average percentage of autoregulatory T cells in responsive and nonresponsive model mice, and the average valency-dependent minimal optimal dose needed for effective therapy. Our results reveal that a moderate increase (≥1.6-fold) in the NP-dependent expansion rate of autoregulatory T-cell population leads to a significant increase in the efficacy and the area corresponding to the effective treatment regimen, provided that NP dose ≥8 μg. We expect the model developed here to generalize to other autoimmune diseases and serve as a computational tool to understand and optimize pMHC-NP-based therapies.

  20. A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer

    Directory of Open Access Journals (Sweden)

    Kandalaft Lana E

    2012-08-01

    Full Text Available Abstract Purpose In spite of increased rates of complete response to initial chemotherapy, most patients with advanced ovarian cancer relapse and succumb to progressive disease. Rationale Genetically reprogrammed, patient-derived chimeric antigen receptor (CAR-T lymphocytes with the ability to recognize predefined surface antigens with high specificity in a non-MHC restricted manner have shown increasing anti-tumor efficacy in preclinical and clinical studies. Folate receptor-α (FRα is an ovarian cancer-specific tumor target; however, it is expressed at low levels in certain organs with risk for toxicity. Design Here we propose a phase I study testing the feasibility, safety and preliminary activity of FRα-redirected CAR-T cells bearing the CD137 (4-1BB costimulatory domain, administered after lymphodepletion for the treatment of recurrent ovarian cancer. A novel trial design is proposed that maximizes safety features. Innovation This design involves an initial accelerated dose escalation phase of FR-α CAR-T cells followed by a standard 3 + 3 escalation phase. A split-dose approach is proposed to mitigate acute adverse events. Furthermore, infusion of bulk untransduced autologous peripheral blood lymphocytes (PBL is proposed two days after CAR-T cell infusion at the lower dose levels of CAR-T cells, to suppress excessive expansion of CAR-T cells in vivo and mitigate toxicity.

  1. Entry of Francisella tularensis into Murine B Cells: The Role of B Cell Receptors and Complement Receptors.

    Directory of Open Access Journals (Sweden)

    Lenka Plzakova

    Full Text Available Francisella tularensis, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell-pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of F. tularensis into B cells within in vivo and in vitro infection models. Here, we present data showing that Francisella tularensis subsp. holarctica strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of Francisella into B cells is mediated by B cell receptors (BCRs with or without complement receptor CR1/2. However, F. tularensis strain FSC200 ΔiglC and ΔftdsbA deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, F. tularensis LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate F. tularensis engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria's internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to F. tularensis.

  2. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

    International Nuclear Information System (INIS)

    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D'Avis, J.C.; Wartofsky, L.

    1991-01-01

    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding [125I] ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound [125I]ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized [125I]ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity

  3. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

  4. Secretin receptor involvement in prion-infected cells and animals.

    Science.gov (United States)

    Kimura, Tomohiro; Nishizawa, Keiko; Oguma, Ayumi; Nishimura, Yuki; Sakasegawa, Yuji; Teruya, Kenta; Nishijima, Ichiko; Doh-ura, Katsumi

    2015-07-08

    The cellular mechanisms behind prion biosynthesis and metabolism remain unclear. Here we show that secretin signaling via the secretin receptor regulates abnormal prion protein formation in prion-infected cells. Animal studies demonstrate that secretin receptor deficiency slightly, but significantly, prolongs incubation time in female but not male mice. This gender-specificity is consistent with our finding that prion-infected cells are derived from females. Therefore, our results provide initial insights into the reasons why age of disease onset in certain prion diseases is reported to occur slightly earlier in females than males. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Lipid rafts and B cell signaling.

    Science.gov (United States)

    Gupta, Neetu; DeFranco, Anthony L

    2007-10-01

    B cells comprise an essential component of the humoral immune system. They are equipped with the unique ability to synthesize and secrete pathogen-neutralizing antibodies, and share with professional antigen presenting cells the ability to internalize foreign antigens, and process them for presentation to helper T cells. Recent evidence indicates that specialized cholesterol- and glycosphingolipid-rich microdomains in the plasma membrane commonly referred to as lipid rafts, serve to compartmentalize key signaling molecules during the different stages of B cell activation including B cell antigen receptor (BCR)-initiated signal transduction, endocytosis of BCR-antigen complexes, loading of antigenic peptides onto MHC class II molecules, MHC-II associated antigen presentation to helper T cells, and receipt of helper signals via the CD40 receptor. Here we review the recent literature arguing for a role of lipid rafts in the spatial organization of B cell function.

  6. Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

    Directory of Open Access Journals (Sweden)

    Drew Michael GB

    2006-03-01

    Full Text Available Abstract Background MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results A large dataset comprising MHC-peptide structural complexes was created by re-modelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion The QSAR techniques of Genetic Function Approximation (GFA and Genetic Partial Least Squares (G/PLS algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

  7. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    Science.gov (United States)

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  8. NK cells and T cells: mirror images?

    NARCIS (Netherlands)

    Versteeg, R.

    1992-01-01

    The expression of MHC class I molecules protects cells against lysis by natural killer (NK) cells. It is possible that NK cells are 'educated' to recognize self MHC class I molecules and that the combination of self peptide and MHC class I molecule blocks NK-mediated lysis. Here, Rogier Versteeg

  9. Killer Cell Immunoglobulin-like Receptors and their Ligands

    Directory of Open Access Journals (Sweden)

    Tajik N.

    2010-09-01

    Full Text Available The Natural killer (NK cells are a subset of lymphocytes comprising around 10% of total lymphocytes in peripheral blood. Due to their role in the innate response, NK cells provide a ‘first line of defense’ against infectious agents and cancer and are also thought to play a role in autoimmunity. The killer cell immunoglobulin-like receptors (KIR are regulatory surface molecules, found on NK cells and on a subset of T lymphocytes. The genes for KIR are present on chromosome 19 in the leukocyte receptor complex and show a major difference for both the type and number of KIR genes present among different ethnic groups. They have been divided into two groups of 2D or 3D, depending on the number of external immunoglobulin domains. The presence of a long cytoplasmic tail with two immune tyrosine-based inhibitory motifs (ITIM allows the transduction of inhibitory signals and characterizes the inhibitory KIRs (2DL and 3DL, whereas the presence of short cytoplasmic tails corresponds to the activating KIR receptors (2DS and 3DS.These polymorphic receptors interact with specific motifs on human leukocyte antigen (HLA class I molecules, modulate NK cytolytic activity. Some KIRs are known to interact with HLA-C molecules of target cells, HLA-Bw4 molecules and HLA-A3/11. For some KIRs the corresponding ligands are still unknown.

  10. Structural properties of MHC class II ligands, implications for the prediction of MHC class II epitopes.

    Directory of Open Access Journals (Sweden)

    Kasper Winther Jørgensen

    2010-12-01

    Full Text Available Major Histocompatibility class II (MHC-II molecules sample peptides from the extracellular space allowing the immune system to detect the presence of foreign microbes from this compartment. Prediction of MHC class II ligands is complicated by the open binding cleft of the MHC class II molecule, allowing binding of peptides extending out of the binding groove. Furthermore, only a few HLA-DR alleles have been characterized with a sufficient number of peptides (100-200 peptides per allele to derive accurate description of their binding motif. Little work has been performed characterizing structural properties of MHC class II ligands. Here, we perform one such large-scale analysis. A large set of SYFPEITHI MHC class II ligands covering more than 20 different HLA-DR molecules was analyzed in terms of their secondary structure and surface exposure characteristics in the context of the native structure of the corresponding source protein. We demonstrated that MHC class II ligands are significantly more exposed and have significantly more coil content than other peptides in the same protein with similar predicted binding affinity. We next exploited this observation to derive an improved prediction method for MHC class II ligands by integrating prediction of MHC- peptide binding with prediction of surface exposure and protein secondary structure. This combined prediction method was shown to significantly outperform the state-of-the-art MHC class II peptide binding prediction method when used to identify MHC class II ligands. We also tried to integrate N- and O-glycosylation in our prediction methods but this additional information was found not to improve prediction performance. In summary, these findings strongly suggest that local structural properties influence antigen processing and/or the accessibility of peptides to the MHC class II molecule.

  11. Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules.

    Science.gov (United States)

    Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G; Mandelboim, Ofer

    2017-11-15

    NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus

  12. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2013-10-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  13. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  14. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    International Nuclear Information System (INIS)

    Matsuda, Satoru; Kitagishi, Yasuko

    2013-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

  15. T cell clones which share T cell receptor epitopes differ in phenotype, function and specificity

    NARCIS (Netherlands)

    Yssel, H.; Blanchard, D.; Boylston, A.; de Vries, J. E.; Spits, H.

    1986-01-01

    Recently, we described a monoclonal antibody (3D6) that reacts with the T cell receptor (Ti) of the T leukemic cell line HPB-ALL and that cross-reacts with 2-10% of the T cells of normal healthy individuals. In this study we report the establishment of T cell clones that are 3D6+ but that differ in

  16. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    Science.gov (United States)

    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...

  17. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell......Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...

  18. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  19. Nonimmune cells equipped with T-cell-receptor-like signaling for cancer cell ablation.

    Science.gov (United States)

    Kojima, Ryosuke; Scheller, Leo; Fussenegger, Martin

    2018-01-01

    The ability to engineer custom cell-contact-sensing output devices into human nonimmune cells would be useful for extending the applicability of cell-based cancer therapies and for avoiding risks associated with engineered immune cells. Here we have developed a new class of synthetic T-cell receptor-like signal-transduction device that functions efficiently in human nonimmune cells and triggers release of output molecules specifically upon sensing contact with a target cell. This device employs an interleukin signaling cascade, whose OFF/ON switching is controlled by biophysical segregation of a transmembrane signal-inhibitory protein from the sensor cell-target cell interface. We further show that designer nonimmune cells equipped with this device driving expression of a membrane-penetrator/prodrug-activating enzyme construct could specifically kill target cells in the presence of the prodrug, indicating its potential usefulness for target-cell-specific, cell-based enzyme-prodrug cancer therapy. Our study also contributes to the advancement of synthetic biology by extending available design principles to transmit extracellular information to cells.

  20. The MHC motif viewer: a visualization tool for MHC binding motifs

    DEFF Research Database (Denmark)

    Rapin, Nicolas; Hoof, Ilka; Lund, Ole

    2010-01-01

    is hampered by the lack of tools for browsing and comparing specificity of these molecules. We have developed a Web server, MHC Motif Viewer, which allows the display of the binding motif for MHC class I proteins for human, chimpanzee, rhesus monkey, mouse, and swine, as well as HLA-DR protein sequences...

  1. Regulation of HIV receptor expression in cervical epithelial cells by ...

    African Journals Online (AJOL)

    Background. Sexually transmitted infections (STIs) caused by the Gram-negative bacteria Chlamydia trachomatis and Neisseria gonorrhoeae are associated with an increased risk of HIV acquisition in South African women. HIV infection involves binding of the virus to CD4+ receptors on host cells and subsequent binding to ...

  2. Enhanced vaccine-induced CD8+ T cell responses to malaria antigen ME-TRAP by fusion to MHC class ii invariant chain.

    Directory of Open Access Journals (Sweden)

    Alexandra J Spencer

    Full Text Available The orthodox role of the invariant chain (CD74; Ii is in antigen presentation to CD4+ T cells, but enhanced CD8+ T cells responses have been reported after vaccination with vectored viral vaccines encoding a fusion of Ii to the antigen of interest. In this study we assessed whether fusion of the malarial antigen, ME-TRAP, to Ii could increase the vaccine-induced CD8+ T cell response. Following single or heterologous prime-boost vaccination of mice with a recombinant chimpanzee adenovirus vector, ChAd63, or recombinant modified vaccinia virus Ankara (MVA, higher frequencies of antigen-specific CD4+ and CD8+ T cells were observed, with the largest increases observed following a ChAd63-MVA heterologous prime-boost regimen. Studies in non-human primates confirmed the ability of Ii-fusion to augment the T cell response, where a 4-fold increase was maintained up to 11 weeks after the MVA boost. Of the numerous different approaches explored to increase vectored vaccine induced immunogenicity over the years, fusion to the invariant chain showed a consistent enhancement in CD8+ T cell responses across different animal species and may therefore find application in the development of vaccines against human malaria and other diseases where high levels of cell-mediated immunity are required.

  3. MHC and non-MHC genes regulate elimination of lymphocytic choriomeningitis virus and antiviral cytotoxic T lymphocyte and delayed-type hypersensitivity mediating T lymphocyte activity in parallel

    DEFF Research Database (Denmark)

    Thomsen, Allan Randrup; Marker, O

    1989-01-01

    with regard to all three parameters was recessive, indicating that natural tolerance is not the mechanism explaining non-MHC dependent low responsiveness in this system. The implications of these findings are discussed with specific reference to the role of MHC genes in controlling resistance to infectious......, indicating that both H-2 and non-H-2 genes may influence the elimination of this virus. Differences in virus spread prior to appearance of the immune response could not explain the observed differences in clearance rate. On the other hand, inefficient clearance always correlated with low T cell...

  4. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data

    DEFF Research Database (Denmark)

    Jurtz, Vanessa Isabell; Paul, Sinu; Andreatta, Massimo

    2017-01-01

    by mass spectrometry have been reported containing information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging......Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I molecules. Peptide binding to MHC molecules is the single most selective step in the Ag-presentation pathway....... Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing number of MHC-presented peptides identified...

  5. NLRC5: a newly discovered MHC class I transactivator (CITA)

    OpenAIRE

    Meissner, Torsten B.; Li, Amy; Kobayashi, Koichi S.

    2011-01-01

    Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class II gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NLR protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as “CITA” (MHC class I transactivator)...

  6. Plant cell wall signalling and receptor-like kinases.

    Science.gov (United States)

    Wolf, Sebastian

    2017-02-15

    Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  7. Rapid internalization of the insulin receptor in rat hepatoma cells

    International Nuclear Information System (INIS)

    Backer, J.M.; White, M.F.; Kahn, C.R.

    1987-01-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4 0 C, stimulated with insulin at 37 0 C, and then cooled rapidly, trypsinized at 4 0 C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37 0 C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4 0 C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific [ 125 I]insulin binding measured at 4 0 C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways

  8. Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice.

    Directory of Open Access Journals (Sweden)

    Mark T Orr

    2005-09-01

    Full Text Available Herpes simplex virus (HSV has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis.

  9. Receptor-mediated endocytosis of trichosanthin in choriocarcinoma cells

    International Nuclear Information System (INIS)

    Chan, W.Y.; Huang Hai; Tam, S.-C.

    2003-01-01

    Trichosanthin (TCS) is a ribosome inactivating protein (RIP). It is generally believed that its many biological activities act through inhibition of ribosomes resulting in a decrease in protein synthesis. It has been hypothesized that the rate of entry of TCS into cells to reach ribosomes is an important factor in determining its biological activity. To prove this hypothesis, we have mapped out and compared the intracellular routing of TCS in two cell lines, namely the choriocarcinoma JAR cell line, which is known to be highly sensitive to the toxic effects of TCS, and the hepatoma H35 cell line, to which TCS shows minimal toxicity. Results from laser scanning confocal microscopy indicated that fluorescein isothiocyanate labeled TCS quickly accumulated inside JAR cells within 4 h of incubation while only a low level of fluorescent signals was detected in H35 cells during the same period of time. When TCS was conjugated with gold particles (Au) and its intracellular locations were traced with a transmission electron microscope, it was found that most of TCS were bound to coated pits on the JAR cell surface and were rapidly internalized within an hour. By 4 h, TCS reached almost every cytoplasmic region including ribosomes, and the JAR cell began to degenerate. In H35 cells, however, the binding of TCS to coated pits was not observed, but instead, a small amount of TCS was found to penetrate the cell non-specifically by direct diffusion across the cell membrane. Our observations suggest that most of TCS enter JAR cells via a specific receptor mediated pathway, which allows a swift transport of TCS across the membrane and a rapid accumulation of intracellular TCS, while in H35 cells, TCS takes a slow and non-specific route. The receptor-mediated uptake together with the specific intracellular routing of TCS may partly account for the differential vulnerability of the choriocarcinoma cell line towards the toxicity of TCS

  10. New insights into how trafficking regulates T cell receptor signaling

    Directory of Open Access Journals (Sweden)

    Jieqiong Lou

    2016-07-01

    Full Text Available AbstractThere is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR signaling. The trafficking molecules involved in lytic granule (LG secretion in cytotoxic T lymphocytes (CTL have been well studied due to the immune disorder known as familial hemophagocytic lymphohisiocytosis (FHLH. However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions.

  11. Receptor control in mesenchymal stem cell engineering

    Science.gov (United States)

    Dalby, Matthew J.; García, Andrés J.; Salmeron-Sanchez, Manuel

    2018-03-01

    Materials science offers a powerful tool to control mesenchymal stem cell (MSC) growth and differentiation into functional phenotypes. A complex interplay between the extracellular matrix and growth factors guides MSC phenotypes in vivo. In this Review, we discuss materials-based bioengineering approaches to direct MSC fate in vitro and in vivo, mimicking cell-matrix-growth factor crosstalk. We first scrutinize MSC-matrix interactions and how the properties of a material can be tailored to support MSC growth and differentiation in vitro, with an emphasis on MSC self-renewal mechanisms. We then highlight important growth factor signalling pathways and investigate various materials-based strategies for growth factor presentation and delivery. Integrin-growth factor crosstalk in the context of MSC engineering is introduced, and bioinspired material designs with the potential to control the MSC niche phenotype are considered. Finally, we summarize important milestones on the road to MSC engineering for regenerative medicine.

  12. MHC-I-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by T cells 30 years after vaccination

    DEFF Research Database (Denmark)

    Tang, Sheila Tuyet; Wang, M.; Lamberth, K.

    2008-01-01

    It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T...... lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-gamma release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity...... of K(D) less than or equal to 5 nM, whereas no T cell responses were detected against 60 peptides with an HLA affinity of K(D) more than 5 nM. All epitopes were fully conserved in seven variola, vaccinia and cowpox strains. Knowledge of the long-term response to smallpox vaccination may lead...

  13. Therapy of MHC Class I+ and Class I- HPV 16-Associated Tumours with Cytokines, Genetically Modified Tumour Vaccines and Dendritic Cells

    Czech Academy of Sciences Publication Activity Database

    Bubeník, Jan; Šímová, Jana; Mikyšková, Romana; Vonka, V.

    2004-01-01

    Roč. 27, č. 4 (2004), s. 161D ISSN 0147-958X. [International Congress of Immunology /12./ and Annual Conference of FOCIS /4./. Montreal, 18.07.2004-23.07.2004] Institutional research plan: CEZ:AV0Z5052915 Keywords : HPV 16 * tumour vaccines * dendritic cells Subject RIV: EC - Immunology Impact factor: 1.051, year: 2004

  14. Glucose transporters are expressed in taste receptor cells.

    Science.gov (United States)

    Merigo, Flavia; Benati, Donatella; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

    2011-08-01

    In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C β type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism. © 2011 The Authors. Journal of Anatomy © 2011

  15. AN MHC class I immune evasion gene of Marek's disease virus

    Science.gov (United States)

    Marek's disease virus (MDV) is a widespread a-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198–205 (2001)), but the gene(s) involved have not been identified. Here...

  16. Specific deletion of LDL receptor-related protein on macrophages has skewed in vivo effects on cytokine production by invariant natural killer T cells.

    Directory of Open Access Journals (Sweden)

    Roman Covarrubias

    Full Text Available Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr on antigen presenting cells (APCs has been shown to enhance invariant natural killer T (iNKT cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP, plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ. We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO. LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.

  17. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Yi, Ka Hee; Kim, Chang Min

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves' patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves' disease will be elucidated. (author). 25 refs

  18. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  19. One-pot, mix-and-read peptide-MHC tetramers

    DEFF Research Database (Denmark)

    Leisner, Christian Valdemar Vinge; Loeth, Nina; Lamberth, Kasper

    2008-01-01

    BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development...... molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation...

  20. DMPD: Signals and receptors involved in recruitment of inflammatory cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 7744810 Signals and receptors involved in recruitment of inflammatory cells. Ben-Ba...ow Signals and receptors involved in recruitment of inflammatory cells. PubmedID 7744810 Title Signals and receptors involved in recr...uitment of inflammatory cells. Authors Ben-Baruch A, Mic

  1. TAM receptors in apoptotic cell clearance, autoimmunity, and cancer.

    Science.gov (United States)

    Nguyen, Khanh-Quynh; Tsou, Wen-I; Kotenko, Sergei; Birge, Raymond B

    2013-08-01

    Receptor tyrosine kinases, Tyro-3, Axl and Mer, collectively designated as TAM, are involved in the clearance of apoptotic cells. TAM ligands, Gas6 and Protein S, bind to the surfaces of apoptotic cells, and at the same time, interact directly with TAM expressed on phagocytes, impacting the engulfment and clearance of apoptotic cells and debris. The well-tuned and balanced actions of TAM may affect a variety of human pathologies including autoimmunity, retinal degeneration, and cancer. This article emphasizes some of the emerging findings and mechanistic insights into TAM functions that are clinically relevant and possibly therapeutically targeted.

  2. Glucocorticoid receptor beta increases migration of human bladder cancer cells.

    Science.gov (United States)

    McBeth, Lucien; Nwaneri, Assumpta C; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D

    2016-05-10

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease.

  3. A Comprehensive Nuclear Receptor Network for Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ralf Kittler

    2013-02-01

    Full Text Available In breast cancer, nuclear receptors (NRs play a prominent role in governing gene expression, have prognostic utility, and are therapeutic targets. We built a regulatory map for 24 NRs, six chromatin state markers, and 14 breast-cancer-associated transcription factors (TFs that are expressed in the breast cancer cell line MCF-7. The resulting network reveals a highly interconnected regulatory matrix where extensive crosstalk occurs among NRs and other breast -cancer-associated TFs. We show that large numbers of factors are coordinately bound to highly occupied target regions throughout the genome, and these regions are associated with active chromatin state and hormone-responsive gene expression. This network also provides a framework for stratifying and predicting patient outcomes, and we use it to show that the peroxisome proliferator-activated receptor delta binds to a set of genes also regulated by the retinoic acid receptors and whose expression is associated with poor prognosis in breast cancer.

  4. P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells.

    NARCIS (Netherlands)

    Constantinescu, P.; Wang, B.; Kovacevic, K.; Jalilian, I.; Bosman, G.J.C.G.M.; Wiley, J.S.; Sluyter, R.

    2010-01-01

    Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR,

  5. Premalignant quiescent melanocytic nevi do not express the MHC class I chain-related protein A

    Directory of Open Access Journals (Sweden)

    Mercedes B. Fuertes

    2011-08-01

    Full Text Available The MHC class I chain-related protein A (MICA is an inducible molecule almost not expressed by normal cells but strongly up-regulated in tumor cells. MICA-expressing cells are recognized by natural killer (NK cells, CD8+ aßTCR and ?dTCR T lymphocytes through the NKG2D receptor. Engagement of NKG2D by MICA triggers IFN-? secretion and cytotoxicity against malignant cells. Although most solid tumors express MICA and this molecule is a target during immune surveillance against tumors, it has been observed that high grade tumors from different histotypes express low amounts of cell surface MICA due to a metalloprotease- induced shedding. Also, melanomas develop after a complex process of neotransformation of normal melanocytes. However, the expression of MICA in premalignant stages (primary human quiescent melanocytic nevi remains unknown. Here, we assessed expression of MICA by flow cytometry using cell suspensions from 15 primary nevi isolated from 11 patients. When collected material was abundant, cell lysates were prepared and MICA expression was also analyzed by Western blot. We observed that MICA was undetectable in the 15 primary nevi (intradermic, junction, mixed, lentigo and congenital samples as well as in normal skin, benign lesions (seborrheic keratosis, premalignant lesions (actinic keratosis and benign basocellular cancer. Conversely, a primary recently diagnosed melanoma showed intense cell surface MICA. We conclude that the onset of MICA expression is a tightly regulated process that occurs after melanocytes trespass the stage of malignant transformation. Thus, analysis of MICA expression in tissue sections of skin samples may constitute a useful marker to differentiate between benign and malignant nevi.

  6. The Role of Estrogen Related Receptor in Modulating Estrogen Receptor Mediated Transcription in Breast Cancer Cells

    Science.gov (United States)

    2005-04-01

    tumors correlates with an unfavorable prognosis (Ariazi 2002; Lu 2001; Suzuki 2004; Vanacker 1999). The transcriptional activity of ERRa is not inhibited...SA. 101:6570-5. Needham, M ., S. Raines, J. McPheat, C. Stacey, J. Ellston, S. Hoare, and M . Parker. 2000. Differential interaction of steroid hormone...R. Graves, M . Wright, and B.M. Spiegelman. 1998. A cold- inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell. 92:829- 39

  7. Vaccination against lymphocytic choriomeningitis virus infection in MHC class II-deficient mice

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2011-01-01

    response could be elicited in MHC class II-deficient mice by vaccination with adenovirus encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein tethered to MHC class II-associated invariant chain. Moreover, the response induced conferred significant cytolytic CD8(+) T cell-mediated protection...... against challenge with a high dose of the invasive clone 13 strain of LCMV. In contrast, vaccination with adenovirus encoding unlinked LCMV glycoprotein induced weak virus control in the absence of CD4(+) T cells, and mice may die of increased immunopathology associated with incomplete protection. Acute...... mortality was not observed in any vaccinated mice following infection with the less-invasive Traub strain. However, LCMV Traub infection caused accelerated late mortality in unvaccinated MHC class II-deficient mice; in this case, we observed a strong trend toward delayed mortality in vaccinated mice...

  8. TLR4-HMGB1 signaling pathway affects the inflammatory reaction of autoimmune myositis by regulating MHC-I.

    Science.gov (United States)

    Wan, Zemin; Zhang, Xiujuan; Peng, Anping; He, Min; Lei, Zhenhua; Wang, Yunxiu

    2016-12-01

    To analyze the effects of TLR4 on the expression of the HMGB1, MHC-I and downstream cytokines IL-6 and TNF-α, and to investigate the biological role of the TLR4-HMGB1 signaling pathway in the development of the autoimmune myositis. We built mice models with experimental autoimmune myositis (EAM) and used the inverted screen experiment to measure their muscle endurance; we also examined inflammatory infiltration of muscle tissues after HE staining; and we assessed the expression of MHC-I using immunohistochemistry. In addition, peripheral blood mononuclear cells (PBMC) were extracted and flow cytometry was utilized to detect the effect of IFN-γ on the expression of MHC-I. Furthermore, PBMCs were treated with IFN-γ, anti-TLR4, anti-HMGB1 and anti-MHC-I. Real-time PCR and western blotting were employed to examine the expressions of TLR4, HMGB1 and MHC-I in different groups. The ELISA method was also utilized to detect the expression of the downstream cytokines TNF-α and IL-6. The expressions of TLR4, HMGB1 and MHC-I in muscle tissues from mice with EAM were significantly higher than those in the control group (all Pmyositis inflammation by regulating the expression of MHC-I and other pro-inflammatory cytokines. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Kornbluth Richard S

    2009-08-01

    Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

  10. Extensive shared polymorphism at non-MHC immune genes in recently diverged North American prairie grouse

    Science.gov (United States)

    Minias, Piotr; Bateson, Zachary W.; Whittingham, Linda A.; Johnson, Jeff A.; Oyler-McCance, Sara J.; Dunn, Peter O.

    2018-01-01

    Gene polymorphisms shared between recently diverged species are thought to be widespread and most commonly reflect introgression from hybridization or retention of ancestral polymorphism through incomplete lineage sorting. Shared genetic diversity resulting from incomplete lineage sorting is usually maintained for a relatively short period of time, but under strong balancing selection it may persist for millions of years beyond species divergence (balanced trans-species polymorphism), as in the case of the major histocompatibility complex (MHC) genes. However, balancing selection is much less likely to act on non-MHC immune genes. The aim of this study was to investigate the patterns of shared polymorphism and selection at non-MHC immune genes in five grouse species from Centrocercus and Tympanuchus genera. For this purpose, we genotyped five non-MHC immune genes that do not interact directly with pathogens, but are involved in signaling and regulate immune cell growth. In contrast to previous studies with MHC, we found no evidence for balancing selection or balanced trans-species polymorphism among the non-MHC immune genes. No haplotypes were shared between genera and in most cases more similar allelic variants sorted by genus. Between species within genera, however, we found extensive shared polymorphism, which was most likely attributable to introgression or incomplete lineage sorting following recent divergence and large ancestral effective population size (i.e., weak genetic drift). Our study suggests that North American prairie grouse may have attained relatively low degree of reciprocal monophyly at nuclear loci and reinforces the rarity of balancing selection in non-MHC immune genes.

  11. Endothelium in brain: Receptors, mitogenesis, and biosynthesis in glial cells

    International Nuclear Information System (INIS)

    MacCumber, M.W.; Ross, C.A.; Snyder, S.H.

    1990-01-01

    The authors have explored the cellular loci of endothelin (ET) actions and formation in the brain, using cerebellar mutant mice was well as primary and continuous cell cultures. A glial role is favored by several observations: (1) mutant mice lacking neuronal Purkinje cells display normal ET receptor binding and enhanced stimulation by ET of inositolphospholipid turnover; (ii) in weaver mice lacking neuronal granule cells, ET stimulation of inositolphospholipid turnover is not significantly diminished; (iii) C 6 glioma cells and primary cultures of cerebellar astroglia exhibit substantial ET receptor binding and ET-induced stimulation of inositolphospholipid turnover; (iv) ET promotes mitogenesis of C 6 glioma cells and primary cerebellar astroglia; and (v) primary cultures of cerebellar astroglia contain ET mRNA. ET also appears to have a neuronal role, since it stimulates inositolphospholipid turnover in primary cultures of cerebellar granule cells, and ET binding declines in granule cell-deficient mice. Thus, ET can be produced by glia and act upon both glia and neurons in a paracrine fashion

  12. Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

    International Nuclear Information System (INIS)

    Androlewicz, M.J.; Straus, D.S.; Brandenburg, D.F.

    1989-01-01

    Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125 I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degree C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation

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

    Science.gov (United States)

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

    2017-10-12

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

  14. Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Rieber, E P; Linke, R P; Riethmueller, G [Tuebingen Univ. (Germany, F.R.). Abt. fuer Experimentelle Chirurgie und Immunologie; Heyden, H.W. von; Waller, H D [Tuebingen Univ. (Germany, F.R.). Abt. Innere Medizin 2

    1976-01-01

    Using /sup 125/I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab')/sub 2/-fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of ..mu..-chains was detected. ..gamma..-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria.

  15. Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

    International Nuclear Information System (INIS)

    Rieber, E.P.; Linke, R.P.; Riethmueller, G.; Heyden, H.W. von; Waller, H.D.

    1976-01-01

    Using 125 I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab') 2 -fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of μ-chains was detected. γ-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria. (orig.) [de

  16. [Functional properties of taste bud cells. Mechanisms of afferent neurotransmission in Type II taste receptor cells].

    Science.gov (United States)

    Romanov, R A

    2013-01-01

    Taste Bud cells are heterogeneous in their morphology and functionality. These cells are responsible for sensing a wide variety of substances and for associating detected compounds with a different taste: bitter, sweet, salty, sour and umami. Today we know that each of the five basic tastes corresponds to distinct cell populations organized into three basic morpho-functional cell types. In addition, some receptor cells of the taste bud demonstrate glia-related functions. In this article we expand on some properties of these three morphological receptor cell types. Main focus is devoted to the Type II cells and unusual mechanism for afferent neurotransmission in these cells. Taste cells of the Type II consist of three populations detecting bitter, sweet and umami tastes, and, thus, evoke a serious scientific interest.

  17. Brucella abortus Inhibits Major Histocompatibility Complex Class II Expression and Antigen Processing through Interleukin-6 Secretion via Toll-Like Receptor 2▿

    Science.gov (United States)

    Barrionuevo, Paula; Cassataro, Juliana; Delpino, M. Victoria; Zwerdling, Astrid; Pasquevich, Karina A.; Samartino, Clara García; Wallach, Jorge C.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2008-01-01

    The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-γ)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-γ production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection. PMID:17984211

  18. Renal cells activate the platelet receptor CLEC-2 through podoplanin

    Science.gov (United States)

    Christou, Charita M.; Pearce, Andrew C.; Watson, Aleksandra A.; Mistry, Anita R.; Pollitt, Alice Y.; Fenton-May, Angharad E.; Johnson, Louise A.; Jackson, David G.; Watson, Steve P.; O'Callaghan, Chris A.

    2009-01-01

    We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin, rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing human immunodeficiency virus type 1 (HIV-1). This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of this study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to 293T cells in which the HIV can be grown. Further, 293T cells activate both platelets and CLEC-2-transfected DT-40 B cells. The transmembrane protein podoplanin was identified on 293T cells and demonstrated to mediate both binding of 293T cells to CLEC-2 and 293T cell activation of CLEC-2-transfected DT-40 B cells. Podoplanin is expressed on renal cells (podocytes). Further, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5 ± 3.7μM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells. PMID:18215137

  19. Staphylococcal enterotoxin-A directly stimulates signal transduction and interferon-gamma production in psoriatic T-cell lines

    DEFF Research Database (Denmark)

    Nielsen, M B; Odum, N; Gerwien, J

    1998-01-01

    class II. Here we address the question of whether SEA can directly activate psoriatic T cells in the absence of professional antigen-presenting cells. We show that SEA induces i) tyrosine phosphorylation of several proteins, ii) downregulation of the T-cell receptor (TCR), and iii) production......-mediated proliferation. In contrast, SEA with a mutation in the MHC class II alpha-binding site induces IFN-gamma and a qualitatively changed tyrosine phosphorylation profile. Both mutations delete the co-stimulatory effect on cytokine-mediated proliferation. This suggests that both MHC class II binding sites...

  20. Ultraviolet irradiation modulates MHC-alloreactive cytotoxic T-cell precursors involved in the onset of graft-versus-host disease

    International Nuclear Information System (INIS)

    Prooijen, H.C. Van; Aarts-Riemens, M.I.; Weelden, H. Van; Grijzenhout, M.A.

    1992-01-01

    Ultraviolet B (UVB) irradiation of cellular blood components has been proposed as a new technology to prevent HLA sensitization in recipients. Earlier studies have shown that a dose of 2 J/cm 2 abrogates the ability of lymphocytes to serve as stimulators in mixed lymphocyte cultures (MLC). In this study the authors evaluate the effect of UV energy on T-lymphocytes for the prevention of transfusion-associated graft-versus-host disease (TA-GvHD). The response of cytotoxic T-lymphocyte precursors against host alloantigens was almost undetectable at a dose of 0.5 J/cm 2 . T-cell proliferation in MLC or in response to phytohaemagglutinin was inhibited by more than 95% at doses of 1 J/cm 2 or higher. The data suggest that UV irradiation can be used to prevent both HLA sensitization and TA-GvHD in recipients. (Author)

  1. Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

    National Research Council Canada - National Science Library

    Worsham, Maria J

    2007-01-01

    .... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

  2. Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

    National Research Council Canada - National Science Library

    Worsham, Maria

    2001-01-01

    .... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

  3. Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

    National Research Council Canada - National Science Library

    Worsham, Maria J; Raju, Usha; Abrams, Judith

    2005-01-01

    .... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

  4. Population Based Assessment of MHC Class 1 Antigens Down Regulation as Marker in Increased Risk for Development and Progression of Breast Cancer From Benign Breast Lesions

    National Research Council Canada - National Science Library

    Worsham, Maria J

    2006-01-01

    .... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

  5. Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer From Benign Breast Lesions

    National Research Council Canada - National Science Library

    Worsham, Maria

    2004-01-01

    .... The major histocompatibility complex (MHC) class I molecules are found on the cell membrane of all cells in the body and are involved in intercellular communications and in complex interactions with the immune...

  6. A hybrid approach for predicting promiscuous MHC class I restricted ...

    Indian Academy of Sciences (India)

    Prakash

    2006-09-15

    Sep 15, 2006 ... with existing MHC binder prediction methods for alleles studied by both ... in locating the promiscuous MHC binding regions from antigen sequence. ... Artificial neural network; MHC class I alleles; promiscuous binders; ... this problem by developing methods for prediction for ... In case equal number of.

  7. Selective receptor expression restricts Nipah virus infection of endothelial cells

    Directory of Open Access Journals (Sweden)

    Diederich Sandra

    2008-11-01

    Full Text Available Abstract Nipah virus (NiV is a highly pathogenic paramyxovirus that causes severe diseases in animals and humans. Endothelial cell (EC infection is an established hallmark of NiV infection in vivo. Despite systemic virus spread via the vascular system, EC in brain and lung are preferentially infected whereas EC in other organs are less affected. As in vivo, we found differences in the infection of EC in cell culture. Only brain-derived primary or immortalized EC were found to be permissive to NiV infection. Using a replication-independent fusion assay, we could show that the lack of infection in non-brain EC was due to a lack of receptor expression. The NiV entry receptors ephrinB2 (EB2 or ephrinB3 were only expressed in brain endothelia. The finding that EB2 expression in previously non-permissive aortic EC rendered the cells permissive to infection then demonstrated that EB2 is not only necessary but also sufficient to allow the establishment of a productive NiV infection. This strongly suggests that limitations in receptor expression restrict virus entry in certain EC subsets in vivo, and are thus responsible for the differences in EC tropism observed in human and animal NiV infections.

  8. Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

    Science.gov (United States)

    Laurin, David; Marin, Virna; Biagi, Ettore; Pizzitola, Irene; Agostoni, Valentina; Gallot, Géraldine; Vié, Henri; Jacob, Marie Christine; Chaperot, Laurence; Aspord, Caroline; Plumas, Joël

    2013-04-01

    T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

  9. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  10. Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Nørgaard, P; Abrahamsen, N

    1999-01-01

    Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung...

  11. Gastrin receptor characterization: affinity cross-linking of the gastrin receptor on canine gastric parietal cells

    International Nuclear Information System (INIS)

    Matsumoto, M.; Park, J.; Yamada, T.

    1987-01-01

    The authors applied affinity cross-linking methods to label the gastrin receptor on isolated canine gastric parietal cells in order to elucidate the nature of its chemical structure. 125 I-labeled Leu 15 -gastrin and 125 I-labeled gastrin/sub 2-17/ bound to intact parietal cells and their membranes with equal affinity, and half-maximal inhibition of binding was obtained at an incubation concentration of 3.2 x 10 -10 M unlabeled gastrin. 125 I-gastrin/sub 2-17/ was cross-linked to plasma membranes or intact parietal cells by incubation in disuccinimidyl suberate. The membrane pellets were solubilized with or without dithiothreitol and applied to electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels. Autoradiograms revealed a band of labeling at M/sub r/ 76,000 and labeling of this band was inhibited in a dose-dependent fashion by addition of unlabeled gastrin to the incubation mixture. Dithiothreitol in concentrations as high as 100 mM did not later the electrophoretic mobility of the labeled band. After taking into account the molecular weight of 125 I-gastrin/sub 2-17/, the results suggest that the gastrin receptor on parietal cells is a single protein of M/sub r/ 74,000 without disulfide-linked subunits

  12. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Directory of Open Access Journals (Sweden)

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  13. Tolerance to MHC class II disparate allografts through genetic modification of bone marrow

    Science.gov (United States)

    Jindra, Peter T.; Tripathi, Sudipta; Tian, Chaorui; Iacomini, John; Bagley, Jessamyn

    2012-01-01

    Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-Ab (I-Ab) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction. PMID:22833118

  14. MHC classⅠ gene in two duck lines

    Indian Academy of Sciences (India)

    Navya

    Weishan Ma duck (WS) is the eugenic endemic breed, one of the four famous ducks in China, as well Cherry Valley duck (CV) is the largest number of breeding variety. WS is egg strain and CV is meat type. The two duck lines mainly support the. Chinese waterfowl industry. In this study, MHC class Ⅰ genes of WS and CV ...

  15. MHC Region and Its Related Disease Study

    DEFF Research Database (Denmark)

    Cao, Hongzhi

    The major histocompatibility complex (MHC) is one of the most gene dense regions in the human genome and many disorders, including primary immune deficiencies, autoimmune conditions, infections, cancers and mental disorder have been found to be associated with this region. However, due to a high ...

  16. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  17. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    International Nuclear Information System (INIS)

    Wang, Feng; Yang, Yong

    2014-01-01

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers

  18. Stimulation of cell proliferation by histamine H2 receptors in dimethylhdrazine-induced adenocarcinomata.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1978-03-01

    Cell proliferation in dimethylhydrazine-induced colonic carcinomata was stimulated by histamine and by the histamine H2 receptor agonist dimaprit and inhibited by the histamine H2 receptor antagonists Metiamide and Cimetidine but not by the histamine H1 receptor antagonist Mepyramine. In contrast histamine had no effect on colonic crypt cell proliferation in normal or dimethylhydrazine-treated rats.

  19. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    Kolko, Miriam; de Turco, Elena B; Diemer, Nils Henrik

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H...

  20. Aborted germinal center reactions and B cell memory by follicular T cells specific for a B cell receptor V region peptide.

    Science.gov (United States)

    Heiser, Ryan A; Snyder, Christopher M; St Clair, James; Wysocki, Lawrence J

    2011-07-01

    A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.

  1. MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo.

    Science.gov (United States)

    Cavalli, Giulio; Hayashi, Masahiro; Jin, Ying; Yorgov, Daniel; Santorico, Stephanie A; Holcomb, Cherie; Rastrou, Melinda; Erlich, Henry; Tengesdal, Isak W; Dagna, Lorenzo; Neff, C Preston; Palmer, Brent E; Spritz, Richard A; Dinarello, Charles A

    2016-02-02

    Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an "adjuvant" during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity.

  2. The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer.

    Science.gov (United States)

    Zimmermann, Kerstin; Eells, Rebecca; Heinrich, Frank; Rintoul, Stefanie; Josey, Brian; Shekhar, Prabhanshu; Lösche, Mathias; Stern, Lawrence J

    2017-10-27

    Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ cyt ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ cyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ cyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ cyt to the bilayers in dynamic equilibrium: one in which ζ cyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ cyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

  3. CD54/intercellular adhesion molecule 1 and major histocompatibility complex II signaling induces B cells to express interleukin 2 receptors and complements help provided through CD40 ligation

    DEFF Research Database (Denmark)

    Poudrier, J; Owens, T

    1994-01-01

    We have examined signaling roles for CD54 intercellular adhesion molecule 1 and major histocompatibility complex (MHC) II as contact ligands during T help for B cell activation. We used a T helper 1 (Th1)-dependent helper system that was previously shown to be contact as well as interleukin 2 (IL-2......) dependent to demonstrate the relative roles of CD54, MHC II, and CD40 signaling in the events leading to the induction of B cell proliferation and responsiveness to IL-2. Paraformaldehyde-fixed activated Th1-induced expression of IL-2R alpha, IL-2R beta, and B7, and upregulated MHC II and CD54 on B cells...... resulted in the upregulated expression of MHC II and of CD54 and B7, respectively, analogous to the effect of fixed activated Th1 cells. B7 expression was further enhanced by co-cross-linking CD54 and MHC II. Cross-linking of CD40 achieved comparable effects. Strikingly, cross-linking ligation of CD54...

  4. T-cell receptor gamma delta bearing cells in normal human skin

    NARCIS (Netherlands)

    Bos, J. D.; Teunissen, M. B.; Cairo, I.; Krieg, S. R.; Kapsenberg, M. L.; Das, P. K.; Borst, J.

    1990-01-01

    T-cell antigen receptors (TCR) are divided into common alpha beta and less common gamma delta types. In the murine skin, TCR gamma delta+ cells have been reported to form the great majority of epidermal T lymphocytes. We have examined the relative contribution of TCR alpha beta+ and TCR gamma delta+

  5. Towards the generation of B-cell receptor retrogenic mice.

    Directory of Open Access Journals (Sweden)

    Jenny Freitag

    Full Text Available Transgenic expression of B- and T-cell receptors (BCRs and TCRs, respectively has been a standard tool to study lymphocyte development and function in vivo. The generation of transgenic mice is time-consuming and, therefore, a faster method to study the biology of defined lymphocyte receptors in vivo would be highly welcome. Using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells, TCRs can be expressed rapidly in mice of any background. We aimed at adopting this retrogenic technology to the in vivo expression of BCRs. Using a well characterised BCR specific for hen egg lysozyme (HEL, we achieved surface expression of the retrogenically encoded BCR in a Rag-deficient pro B-cell line in vitro. In vivo, retrogenic BCRs were detectable only intracellularly but not on the surface of B cells from wild type or Rag2-deficient mice. This data, together with the fact that no BCR retrogenic mouse model has been published in the 7 years since the method was originally published for TCRs, strongly suggests that achieving BCR-expression in vivo with retrogenic technology is highly challenging if not impossible.

  6. MHC class I epitope binding prediction trained on small data sets

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Nielsen, Morten; Lamberth, K.

    2004-01-01

    The identification of potential T-cell epitopes is important for development of new human or vetenary vaccines, both considering single protein/subunit vaccines, and for epitope/peptide vaccines as such. The highly diverse MHC class I alleles bind very different peptides, and accurate binding pre...... in situations where only very limited data are available for training....

  7. Improved methods for predicting peptide binding affinity to MHC class II molecules.

    Science.gov (United States)

    Jensen, Kamilla Kjaergaard; Andreatta, Massimo; Marcatili, Paolo; Buus, Søren; Greenbaum, Jason A; Yan, Zhen; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

    2018-01-06

    Major histocompatibility complex class II (MHC-II) molecules are expressed on the surface of professional antigen-presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC-II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T-cell epitopes. We here present updated versions of two MHC-II-peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC-peptide binding affinity data obtained from the Immune Epitope Database covering HLA-DR, HLA-DQ, HLA-DP and H-2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2. © 2018 John Wiley & Sons Ltd.

  8. Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection.

    Science.gov (United States)

    Boardman, D A; Philippeos, C; Fruhwirth, G O; Ibrahim, M A A; Hannen, R F; Cooper, D; Marelli-Berg, F M; Watt, F M; Lechler, R I; Maher, J; Smyth, L A; Lombardi, G

    2017-04-01

    Regulatory T cell (Treg) therapy using recipient-derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune-mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  9. MHC Expression on Spleen Lymphocyte Subsets in Genetically Resistant and Susceptible Chickens Infected with Marek's Disease Virus

    DEFF Research Database (Denmark)

    Dalgaard, Tina; Bøving, Mette K.; Handberg, Kurt

    2009-01-01

    cytometry for MHC surface expression. In the spleen, constitutive MHC class I surface expression was found to be highest in homozygous B19, lowest in homozygous B21, and intermediate in heterozygous B19/B21 animals. This was observed on CD4(+), CD8(+), and Bu-1(+) splenic lymphocytes. Chickens of all three...... genotypes were subjected to infection with MD virus (GA strain) and spleen samples from infected as well as MHC-matched negative controls were analyzed at 1, 4, and 8 wk post-infection (p.i.). It was observed that MDV induced an increase in MHC class I expression late in the infection. Thus, MHC class I...... was increased on the surface of CD4(+) cells from infected chickens of all genotypes at 4 and 8 wk p.i. compared with negative controls. Also, MHC class I expression was increased on CD8(+) cells from infected chickens of all genotypes at 4 and 8 wk p.i., except for the homozygous B19 animals, that showed...

  10. TCR tuning of T cell subsets.

    Science.gov (United States)

    Cho, Jae-Ho; Sprent, Jonathan

    2018-05-01

    After selection in the thymus, the post-thymic T cell compartments comprise heterogenous subsets of naive and memory T cells that make continuous T cell receptor (TCR) contact with self-ligands bound to major histocompatibility complex (MHC) molecules. T cell recognition of self-MHC ligands elicits covert TCR signaling and is particularly important for controlling survival of naive T cells. Such tonic TCR signaling is tightly controlled and maintains the cells in a quiescent state to avoid autoimmunity. Here, we review how naive and memory T cells are differentially tuned and wired for TCR sensitivity to self and foreign ligands. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Palindromic nucleotide analysis in human T cell receptor rearrangements.

    Directory of Open Access Journals (Sweden)

    Santosh K Srivastava

    Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

  12. Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

    Science.gov (United States)

    Smirnov, Anna; Pohlmann, Stephanie; Nehring, Melanie; Ali, Shafaqat; Mann-Nüttel, Ritu; Scheu, Stefanie; Antoni, Anne-Charlotte; Hansen, Wiebke; Büettner, Manuela; Gardiasch, Miriam J.; Westendorf, Astrid M.; Wirsdörfer, Florian; Pastille, Eva; Dudda, Marcel; Flohé, Stefanie B.

    2017-01-01

    Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs) secrete enhanced levels of interleukin (IL) 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP), a model for human polymicrobial sepsis. Bone marrow cells (BMC) were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs) were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR) 2, the receptor for C-C chemokine ligand (CCL) 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs) that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are activated

  13. Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

    Directory of Open Access Journals (Sweden)

    Anna Smirnov

    2017-11-01

    Full Text Available Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs secrete enhanced levels of interleukin (IL 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP, a model for human polymicrobial sepsis. Bone marrow cells (BMC were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR 2, the receptor for C-C chemokine ligand (CCL 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are

  14. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    Science.gov (United States)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  15. Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

    Directory of Open Access Journals (Sweden)

    Mohanan Valiya Veettil

    2014-10-01

    Full Text Available Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

  16. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

  17. Involvement of the Soluble Urokinase Receptor in Chondrosarcoma Cell Mobilization

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    Katia Bifulco

    2011-01-01

    Full Text Available High levels of urokinase receptor (uPAR in tissue and serum of patients with chondrosarcoma correlate with poor prognosis. First, we analyzed the uPAR levels in tissues and plasma of five patients affected by chondrosarcoma. Interestingly, very high levels of uPAR and its soluble forms (SuPAR were found on tumor cell surfaces and plasma, respectively, of two patients with lung metastases. Therefore, to investigate the role of SuPAR in chondrosaromas, we generated a primary cell culture from a chondrosarcoma tissue overexpressing uPAR on cell surfaces. We found that chondrosarcoma-like primary culture cells release a large amount of SuPAR in the medium. In vitro, SuPAR elicits chondrosarcoma cell migration likely through its uPAR88-92 sequence, since the DII88-183 or DIIDIIR88-284 uPAR domains retain motogen effect whereas DI1-87 or DIII184-284 domains, both lacking the uPAR88-92 sequence, are ineffective. Chondrosarcoma cells cross matrigel in response to SuPAR, and their invasion capability is abrogated by RERF peptide which inhibits uPAR88-92 signalling. These findings assign a role to uPAR in mobilizing chondrosarcoma cells and suggest that RERF peptide may be regarded as a prototype to generate new therapeutics for the chondrosarcoma treatment.

  18. Activation of postnatal neural stem cells requires nuclear receptor TLX.

    Science.gov (United States)

    Niu, Wenze; Zou, Yuhua; Shen, Chengcheng; Zhang, Chun-Li

    2011-09-28

    Neural stem cells (NSCs) continually produce new neurons in postnatal brains. However, the majority of these cells stay in a nondividing, inactive state. The molecular mechanism that is required for these cells to enter proliferation still remains largely unknown. Here, we show that nuclear receptor TLX (NR2E1) controls the activation status of postnatal NSCs in mice. Lineage tracing indicates that TLX-expressing cells give rise to both activated and inactive postnatal NSCs. Surprisingly, loss of TLX function does not result in spontaneous glial differentiation, but rather leads to a precipitous age-dependent increase of inactive cells with marker expression and radial morphology for NSCs. These inactive cells are mispositioned throughout the granular cell layer of the dentate gyrus during development and can proliferate again after reintroduction of ectopic TLX. RNA-seq analysis of sorted NSCs revealed a TLX-dependent global expression signature, which includes the p53 signaling pathway. TLX regulates p21 expression in a p53-dependent manner, and acute removal of p53 can rescue the proliferation defect of TLX-null NSCs in culture. Together, these findings suggest that TLX acts as an essential regulator that ensures the proliferative ability of postnatal NSCs by controlling their activation through genetic interaction with p53 and other signaling pathways.

  19. The combination of major histocompatibility complex (MHC) and non-MHC genes influences murine lymphocytic choriomeningitis virus pathogenesis

    DEFF Research Database (Denmark)

    Eyler, Y L; Pfau, C J; Broomhall, K S

    1989-01-01

    with the recessive disease phenotype. In all cases, susceptibility was dominant. In backcross progeny obtained from matings of parental strains differing in both major histocompatibility complex (MHC) and non-MHC (SWR; C3H), 90% of the challenged mice died, indicating that at least three loci controlled...... susceptibility to the disease. When the parental strains carried similar MHC haplotypes but dissimilar background genes (B10.BR; CBA), 78% of the backcross mice succumbed, indicating that at least two non-MHC loci influenced disease susceptibility. It is unlikely, however, that the same two non-MHC loci...... are critical in all genetic combinations, since F1 produced from two H-2 identical, resistant strains (B10.BR; C3H) were found to be fully susceptible. When congenic mice, differing only in the D-end of the MHC region, were analysed, 50% of the backcross animals died, indicating that one gene in the MHC region...

  20. Role of nuclear receptors in breast cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    Alessio; Papi; Marina; Orlandi

    2016-01-01

    The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells,capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells(CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs(BCSCs) are likely to sustain the growth of the primary tumour mass, as wellas to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and proinflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the antiinflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.

  1. Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors

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    Yi Zhao

    2017-04-01

    Full Text Available IntroductionMany antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells.MethodsCultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS in the presence of dead and dying cells, their supernatants (SNs, or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo.ResultsThe stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment.ConclusionInosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

  2. Haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of MHC I expression.

    Science.gov (United States)

    Bell, Charlotte R; MacHugh, Niall D; Connelley, Timothy K; Degnan, Kathryn; Morrison, W Ivan

    2015-07-09

    Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells

  3. VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires.

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    Mikhail Shugay

    2015-11-01

    Full Text Available Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools.

  4. Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

    International Nuclear Information System (INIS)

    Cameron, W.; Doyle, K.; Rocklin, R.E.

    1986-01-01

    A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

  5. Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

    Science.gov (United States)

    Velásquez, Lis N; Milillo, M Ayelén; Delpino, M Victoria; Trotta, Aldana; Fernández, Pablo; Pozner, Roberto G; Lang, Roland; Balboa, Luciana; Giambartolomei, Guillermo H; Barrionuevo, Paula

    2017-03-01

    Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4 + T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection. © Society for Leukocyte Biology.

  6. S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; van Loenen, Pieter B.; Hajji, Najat; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2009-01-01

    Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells

  7. The Androgen Receptor Bridges Stem Cell-Associated Signaling Nodes in Prostate Stem Cells

    Directory of Open Access Journals (Sweden)

    Alastair H. Davies

    2016-01-01

    Full Text Available The therapeutic potential of stem cells relies on dissecting the complex signaling networks that are thought to regulate their pluripotency and self-renewal. Until recently, attention has focused almost exclusively on a small set of “core” transcription factors for maintaining the stem cell state. It is now clear that stem cell regulatory networks are far more complex. In this review, we examine the role of the androgen receptor (AR in coordinating interactions between signaling nodes that govern the balance of cell fate decisions in prostate stem cells.

  8. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  9. Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

    Science.gov (United States)

    Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

    2018-05-01

    The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

  10. Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways

    International Nuclear Information System (INIS)

    Heizmann, Beate; Sellars, MacLean; Macias-Garcia, Alejandra; Chan, Susan; Kastner, Philippe

    2016-01-01

    The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

  11. Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Heizmann, Beate [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Sellars, MacLean [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Macias-Garcia, Alejandra [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Institute for Medical Engineering and Science at MIT, Cambridge, MA 02139 (United States); Chan, Susan, E-mail: scpk@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Kastner, Philippe, E-mail: scpk@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR 7104, Université de Strasbourg, 67404 Illkirch (France); Faculté de Médecine, Université de Strasbourg, Strasbourg (France)

    2016-02-12

    The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

  12. Rat hepatic β2-adrenergic receptor: structural similarities to the rat fat cell β1-adrenergic receptor

    International Nuclear Information System (INIS)

    Graziano, M.P.

    1984-01-01

    The mammalian β 2 -adrenergic receptor from rat liver has been purified by sequential cycles of affinity chromatography followed by steric-exclusion high performance liquid chromatography. Electrophoresis of highly purified receptor preparations on polyacrylamide gels in the presence of sodium dodecyl sulfate under reducing conditions reveals a single peptide M/sub r/ = 67,000, as judged by silver staining. Purified β 2 -adrenergic receptor migrates on steric-exclusion high performance liquid chromatography in two peaks, with M/sub r/ = 140,000 and 67,000. Specific binding of the high affinity, β-adrenergic receptor antagonists (-)[ 3 H]dihydroalprenolol and (-)[ 125 I]iodocyanopindolol to purified rat liver β-adrenergic receptor preparations displays stereoselectivity for (-)isomers of agonists and a rank order of potencies for agonists characteristics of a β 2 -adrenergic receptor. Radioiodinated, β 1 -adrenergic receptors from rat fat cells and β 2 -adrenergic receptors from rat liver purified in the presence of protease inhibitors comigrate in electrophoretic separations on polyacrylamide gels in the presence of sodium dodecyl sulfate as 67,000-M/sub r/ peptides. Autoradiograms of two dimensional partial proteolytic digests of the purified, radioiodinated rat liver β 2 -adrenergic receptor, generated with α-chymotrypsin, S. aureus V8 protease and elastase reveal a pattern of peptide fragments essentially identical to those generated by partial proteolytic digests of the purified, radioiodinated β 1 -adrenergic receptor from rat fat cells, by these same proteases. These data indicate that a high degree of homology exists between these two pharmacologically distinct mammalian β-adrenergic receptor proteins

  13. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells

    Directory of Open Access Journals (Sweden)

    Gin-Wen Chang

    2016-05-01

    Full Text Available Natural killer (NK cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

  14. Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

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    Qiang Zhang

    Full Text Available The Nkrp1 (Klrb1-Clr (Clec2 genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b, Nkrp1c (Klrb1c, and Clr-c (Clec2f genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d and Clr-g (Clec2i showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells, as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

  15. Reaction and Aggregation Dynamics of Cell Surface Receptors

    Science.gov (United States)

    Wang, Michelle Dong

    This dissertation is composed of both theoretical and experimental studies of cell surface receptor reaction and aggregation. Project I studies the reaction rate enhancement due to surface diffusion of a bulk dissolved ligand with its membrane embedded target, using numerical calculations. The results show that the reaction rate enhancement is determined by ligand surface adsorption and desorption kinetic rates, surface and bulk diffusion coefficients, and geometry. In particular, we demonstrate that the ligand surface adsorption and desorption kinetic rates, rather than their ratio (the equilibrium constant), are important in rate enhancement. The second and third projects are studies of acetylcholine receptor clusters on cultured rat myotubes using fluorescence techniques after labeling the receptors with tetramethylrhodamine -alpha-bungarotoxin. The second project studies when and where the clusters form by making time-lapse movies. The movies are made from overlay of the pseudocolored total internal reflection fluorescence (TIRF) images of the cluster, and the schlieren images of the cell cultures. These movies are the first movies made using TIRF, and they clearly show the cluster formation from the myoblast fusion, the first appearance of clusters, and the eventual disappearance of clusters. The third project studies the fine structural features of individual clusters observed under TIRF. The features were characterized with six parameters by developing a novel fluorescence technique: spatial fluorescence autocorrelation. These parameters were then used to study the feature variations with age, and with treatments of drugs (oligomycin and carbachol). The results show little variation with age. However, drug treatment induced significant changes in some parameters. These changes were different for oligomycin and carbachol, which indicates that the two drugs may eliminate clusters through different mechanisms.

  16. Desensitization of parathyroid hormone receptors on cultured bone cells

    International Nuclear Information System (INIS)

    Pun, K.K.; Ho, P.W.; Nissenson, R.A.; Arnaud, C.D.

    1990-01-01

    Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1-34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. [Nle8,Nle18,Tyr34]bPTH-(3-34)amide significantly impaired the desensitization process by PTH-(1-34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 microM), prostaglandin E1 (50 microM), or prostaglandin E2 (50 microM) for 2 h. Binding experiments with [125I]PLP-(1-36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1-34) but not by cAMP generation itself

  17. Role of CD3 gamma in T cell receptor assembly

    DEFF Research Database (Denmark)

    Dietrich, J; Neisig, A; Hou, X

    1996-01-01

    . In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta......The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC...... predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression...

  18. Signal peptide-dependent inhibition of MHC class I heavy chain translation by rhesus cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Colin J Powers

    2008-10-01

    Full Text Available The US2-11 region of human and rhesus cytomegalovirus encodes a conserved family of glycoproteins that inhibit MHC-I assembly with viral peptides, thus preventing cytotoxic T cell recognition. Since HCMV lacking US2-11 is no longer able to block assembly and transport of MHC-I, we examined whether this is also observed for RhCMV lacking the corresponding region. Unexpectedly, recombinant RhCMV lacking US2-11 was still able to inhibit MHC-I expression in infected fibroblasts, suggesting the presence of an additional MHC-I evasion mechanism. Progressive deletion analysis of RhCMV-specific genomic regions revealed that MHC-I expression is fully restored upon additional deletion of rh178. The protein encoded by this RhCMV-specific open reading frame is anchored in the endoplasmic reticulum membrane. In the presence of rh178, RhCMV prevented MHC-I heavy chain (HC expression, but did not inhibit mRNA transcription or association of HC mRNA with translating ribosomes. Proteasome inhibitors stabilized a HC degradation intermediate in the absence of rh178, but not in its presence, suggesting that rh178 prevents completion of HC translation. This interference was signal sequence-dependent since replacing the signal peptide with that of CD4 or murine HC rendered human HCs resistant to rh178. We have identified an inhibitor of antigen presentation encoded by rhesus cytomegalovirus unique in both its lack of homology to any other known protein and in its mechanism of action. By preventing signal sequence-dependent HC translocation, rh178 acts prior to US2, US3 and US11 which attack MHC-I proteins after protein synthesis is completed. Rh178 is the first viral protein known to interfere at this step of the MHC-I pathway, thus taking advantage of the conserved nature of HC leader peptides, and represents a new mechanism of translational interference.

  19. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method.

    Science.gov (United States)

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2007-07-04

    Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. The SMM-align method was shown to outperform other

  20. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    Directory of Open Access Journals (Sweden)

    Lund Ole

    2007-07-01

    Full Text Available Abstract Background Antigen presenting cells (APCs sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR, we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion

  1. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    Science.gov (United States)

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Regulation of Innate Lymphoid Cells by Aryl Hydrocarbon Receptor

    Science.gov (United States)

    Li, Shiyang; Bostick, John W.; Zhou, Liang

    2018-01-01

    With striking similarity to their adaptive T helper cell counterparts, innate lymphoid cells (ILCs) represent an emerging family of cell types that express signature transcription factors, including T-bet+ Eomes+ natural killer cells, T-bet+ Eomes− group 1 ILCs, GATA3+ group 2 ILCs, RORγt+ group 3 ILCs, and newly identified Id3+ regulatory ILC. ILCs are abundantly present in barrier tissues of the host (e.g., the lung, gut, and skin) at the interface of host–environment interactions. Active research has been conducted to elucidate molecular mechanisms underlying the development and function of ILCs. The aryl hydrocarbon receptor (Ahr) is a ligand-dependent transcription factor, best known to mediate the effects of xenobiotic environmental toxins and endogenous microbial and dietary metabolites. Here, we review recent progresses regarding Ahr function in ILCs. We focus on the Ahr-mediated cross talk between ILCs and other immune/non-immune cells in host tissues especially in the gut. We discuss the molecular mechanisms of the action of Ahr expression and activity in regulation of ILCs in immunity and inflammation, and the interaction between Ahr and other pathways/transcription factors in ILC development and function with their implication in disease. PMID:29354125

  3. Regulation of Innate Lymphoid Cells by Aryl Hydrocarbon Receptor

    Directory of Open Access Journals (Sweden)

    Shiyang Li

    2018-01-01

    Full Text Available With striking similarity to their adaptive T helper cell counterparts, innate lymphoid cells (ILCs represent an emerging family of cell types that express signature transcription factors, including T-bet+ Eomes+ natural killer cells, T-bet+ Eomes− group 1 ILCs, GATA3+ group 2 ILCs, RORγt+ group 3 ILCs, and newly identified Id3+ regulatory ILC. ILCs are abundantly present in barrier tissues of the host (e.g., the lung, gut, and skin at the interface of host–environment interactions. Active research has been conducted to elucidate molecular mechanisms underlying the development and function of ILCs. The aryl hydrocarbon receptor (Ahr is a ligand-dependent transcription factor, best known to mediate the effects of xenobiotic environmental toxins and endogenous microbial and dietary metabolites. Here, we review recent progresses regarding Ahr function in ILCs. We focus on the Ahr-mediated cross talk between ILCs and other immune/non-immune cells in host tissues especially in the gut. We discuss the molecular mechanisms of the action of Ahr expression and activity in regulation of ILCs in immunity and inflammation, and the interaction between Ahr and other pathways/transcription factors in ILC development and function with their implication in disease.

  4. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    International Nuclear Information System (INIS)

    Wang, Jing; Yang, Qifeng; Haffty, Bruce G.; Li, Xiaoyan; Moran, Meena S.

    2013-01-01

    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

  5. Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

    International Nuclear Information System (INIS)

    Scott, C.D.; Baxter, R.C.

    1987-01-01

    Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

  6. Cell-specific cre recombinase expression allows selective ablation of glutamate receptors from mouse horizontal cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Ströh

    Full Text Available In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57, a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99% and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl. In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50% in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less

  7. Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

    Science.gov (United States)

    Janssen-Bienhold, Ulrike; Schultz, Konrad; Cimiotti, Kerstin; Weiler, Reto; Willecke, Klaus; Dedek, Karin

    2013-01-01

    In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input

  8. DNA sequence of the Peromyscus leucopus MHC class II gene Aa (MhcPeleAa)

    Energy Technology Data Exchange (ETDEWEB)

    Crew, M.D.; Bates, L.M. [Univ. of Arkansas for Medical Sciences, Little Rock, AR (United States)

    1996-09-01

    The genus Peromyscus has been extensively studied by populations biologists and ecologists for over eighty years, with P. leucopus (the white-footed mouse) being one of the most intensively investigated species. Polymorphic major histocompatibility complex (MHC) genes have proven useful in population genetic studies and might be helpful in understanding the population dynamics of Peromyscus species which are ubiquitously distributed over North and Central America. Polymorphism of P. leucopus MHC (MhcPele) class II genes was evident by restriction fragment length polymorphism (RFLP) analyses using human and mouse probes and Pele class II loci exhibited degrees of polymorphism similar to H2 class II genes (A-like>E-like). 8 refs., 2 figs.

  9. A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

    2014-10-31

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Characterization of estrogen receptors alpha and beta in uterine leiomyoma cells.

    Science.gov (United States)

    Valladares, Francisco; Frías, Ignacio; Báez, Delia; García, Candelaria; López, Francisco J; Fraser, James D; Rodríguez, Yurena; Reyes, Ricardo; Díaz-Flores, Lucio; Bello, Aixa R

    2006-12-01

    Cellular and subcellular localization of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in uterine leiomyomas. Retrospective study. University of La Laguna (ULL) and Canary University Hospital (HUC). Premenopausal and postmenopausal women with uterine leiomyomas. Hysterectomy and myomectomy. Estrogen receptor alpha was only present in smooth muscle cells with variation in the subcellular location in different leiomyomas. Estrogen receptor beta was widely distributed in smooth muscle, endothelial, and connective tissue cells with nuclear location in all cases studied; variations were only found in the muscle cells for this receptor. Estrogens operate in leiomyoma smooth muscle cells through different receptors, alpha and beta. However they only act through the ERbeta in endothelial and connective cells.

  11. The quantum chemical causality of pMHC-TCR biological avidity: Peptide atomic coordination data and the electronic state of agonist N termini

    Directory of Open Access Journals (Sweden)

    Georgios S.E. Antipas

    2015-06-01

    Full Text Available The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC and a T cell receptor (TCR is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1 peptide along with three artificially altered variants, all of which were presented by the (Class I HLA-A201 protein in complexation with the human (CD8+ A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist and peptide pair distribution function (PDF. In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3+ terminal group while antagonist peptides are not.

  12. RAE-1 is expressed in the adult subventricular zone and controls cell proliferation of neurospheres

    DEFF Research Database (Denmark)

    Popa, Natalia; Cédile, Oriane; Pollet-Villard, Xavier

    2011-01-01

    playing either immune or nonimmune function. Among the latter, MHC functions in the central nervous system has started to receive recent interest. Here, our first goal was to investigate the potential relationship between MHC class I molecules and neurogenesis. For the first time, we report the expression......, and we demonstrate they persist in one of the main area of adult neurogenesis, the subventricular zone (SVZ). So far, RAE-1 is only known for its immune functions as a ligand of the activating receptor NKG2D expressed by natural killer (NK) cells, natural killer T, Tγδ, and some T CD8 lymphocytes. Here...

  13. Functional importance of GLP-1 receptor species and expression levels in cell lines.

    Science.gov (United States)

    Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

    2012-04-10

    Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

    Science.gov (United States)

    Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

    2014-05-01

    Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

  15. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  16. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    International Nuclear Information System (INIS)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-01-01

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  17. Microfluidic biofunctionalisation protocols to form multi-valent interactions for cell rolling and phenotype modification investigations

    KAUST Repository

    Perozziello, Gerardo

    2013-07-01

    In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro-fluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi-valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC-I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Directory of Open Access Journals (Sweden)

    Kuszynski Charles

    2011-07-01

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

  19. Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

    OpenAIRE

    Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

    2007-01-01

    TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal ...

  20. Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

    Science.gov (United States)

    Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

    2015-11-19

    Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

  1. 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.

  2. Memory control by the B cell antigen receptor.

    Science.gov (United States)

    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.

  3. Targeting Gallium to Cancer Cells through the Folate Receptor

    Directory of Open Access Journals (Sweden)

    Nerissa Viola-Villegas

    2008-01-01

    Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG 'spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N, N′, N′, N′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

  4. The vitamin D receptor and T cell function

    Directory of Open Access Journals (Sweden)

    Martin eKongsbak

    2013-06-01

    Full Text Available The vitamin D receptor (VDR is a nuclear, ligand-dependent transcription factor that in complex with hormonally active vitamin D, 1,25(OH2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions. The impact of 1,25(OH2D3-VDR signaling on immune function has been the focus of many recent studies as a link between 1,25(OH2D3 and sus-ceptibility to various infections and to development of a variety of inflammatory diseases has been suggested. It is also becoming increasingly clear that microbes slow down immune reactivity by dysregulating the VDR ultimately to increase their chance of survival. Immune modulatory therapies that enhance VDR expression and activity are therefore considered in the clinic today to a greater extent. As T cells are of great importance for both protective immunity and development of inflammatory diseases a variety of studies have been engaged investigating the impact of VDR ex-pression in T cells and found that VDR expression and activity plays an important role in both T cell development, differentiation and effector function. In this review we will analyze current know-ledge of VDR regulation and function in T cells and discuss its importance for immune activity.

  5. Targeting Gallium to Cancer Cells through the Folate Receptor

    Directory of Open Access Journals (Sweden)

    Nerissa Viola-Villegas

    2008-01-01

    Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG ‘spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

  6. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells

    DEFF Research Database (Denmark)

    Engelstoft, Maja S; Park, Won-Mee; Sakata, Ichiro

    2013-01-01

    The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro......, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi....../o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi...

  7. The Determinations of Estrogen and Progesterone Receptor in Breast Cancer Cell by Radioimmunoassay Method

    International Nuclear Information System (INIS)

    Kim, Chi Yeul

    1981-01-01

    The estrogen and progesterone receptors which are bound to the cytoplasmic protein of cancer cells were measured in 20 patients with the early breast cancer by means of radioimmunoassay using charcoal. 1) The patients with estrogen receptor positive were 13 (65%) of 20 cases and with progestrone receptor positive were 7 cases (35%) in the early breast cancer. 2) Coexistence of estrogen and progesterone receptor positive was noted in 7 cases (35%). The cases of estrogen receptor positive and progesterone receptor negative were 6 cases (33.3%), while there were no cases of estrogen receptor negative with progesterone receptor positive. 3) Coincidence of estrogen and progesterone negative was noticed in 7 cases (35%). Conclusively it is considered that the measurement of estrogen and progesterone receptors has relevance as predictive value, in the response to hormonal manipulations and chemotherapy for breast cancer patients.

  8. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1 into Diverse Memory T-Cell Populations.

    Directory of Open Access Journals (Sweden)

    Drew C Deniger

    Full Text Available T cells modified with chimeric antigen receptors (CARs targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1 is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28 or CD137 (designated ROR1RCD137 and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC, which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

  9. Primordial linkage of β2-microglobulin to the MHC.

    Science.gov (United States)

    Ohta, Yuko; Shiina, Takashi; Lohr, Rebecca L; Hosomichi, Kazuyoshi; Pollin, Toni I; Heist, Edward J; Suzuki, Shingo; Inoko, Hidetoshi; Flajnik, Martin F

    2011-03-15