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Sample records for cell antigen recognition

  1. CD1-Restricted T Cell Recognition of Microbial Lipoglycan Antigens

    NASA Astrophysics Data System (ADS)

    Sieling, P. A.; Chatterjee, D.; Porcelli, S. A.; Prigozy, T. I.; Mazzaccaro, R. J.; Soriano, T.; Bloom, B. R.; Brenner, M. B.; Kronenberg, M.; Brennan, P. J.; Modlin, R. L.

    1995-07-01

    It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to αβ T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with α(1-->2) linkages and a phosphatidylinositol unit. T cells activated by LAM produced interferon γ and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.

  2. Essential flexibility in the T-cell recognition of antigen

    NASA Astrophysics Data System (ADS)

    Kersh, Gilbert J.; Allen, Paul M.

    1996-04-01

    αβ T cells specifically recognize a ligand composed of a peptide bound to a self-major-histocompatibility-complex molecule, but the recognition of slightly altered ligands by T cells can lead to a partial activation. This flexibility is crucial for T-cell development and can have both beneficial and harmful effects on peripheral T cells.

  3. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

    PubMed

    Roybal, Kole T; Rupp, Levi J; Morsut, Leonardo; Walker, Whitney J; McNally, Krista A; Park, Jason S; Lim, Wendell A

    2016-02-11

    T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.

  4. Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

    PubMed

    Roybal, Kole T; Rupp, Levi J; Morsut, Leonardo; Walker, Whitney J; McNally, Krista A; Park, Jason S; Lim, Wendell A

    2016-02-11

    T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT. PMID:26830879

  5. Atypical natural killer T-cell receptor recognition of CD1d–lipid antigens

    PubMed Central

    Le Nours, Jérôme; Praveena, T.; Pellicci, Daniel G.; Gherardin, Nicholas A.; Ross, Fiona J.; Lim, Ricky T.; Besra, Gurdyal S.; Keshipeddy, Santosh; Richardson, Stewart K.; Howell, Amy R.; Gras, Stephanie; Godfrey, Dale I.; Rossjohn, Jamie; Uldrich, Adam P.

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  6. A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey

    SciTech Connect

    Deng, Lu; Velikovsky, C. Alejandro; Xu, Gang; Iyer, Lakshminarayan M.; Tasumi, Satoshi; Kerzic, Melissa C.; Flajnik, Martin F.; Aravind, L.; Pancer, Zeev; Mariuzza, Roy A.

    2010-10-28

    Adaptive immunity in jawless vertebrates is mediated by leucine-rich repeat proteins called 'variable lymphocyte receptors' (VLRs). Two types of VLR (A and B) are expressed by mutually exclusive lymphocyte populations in lamprey. VLRB lymphocytes resemble the B cells of jawed vertebrates; VLRA lymphocytes are similar to T cells. We determined the structure of a high-affinity VLRA isolated from lamprey immunized with hen egg white lysozyme (HEL) in unbound and antigen-bound forms. The VLRA-HEL complex demonstrates that certain VLRAs, like {gamma}{delta} T-cell receptors (TCRs) but unlike {alpha}{beta} TCRs, can recognize antigens directly, without a requirement for processing or antigen-presenting molecules. Thus, these VLRAs feature the nanomolar affinities of antibodies, the direct recognition of unprocessed antigens of both antibodies and {gamma}{delta} TCRs, and the exclusive expression on the lymphocyte surface that is unique to {alpha}{beta} and {gamma}{delta} TCRs.

  7. Recognition of Major Histocompatibility Complex Antigens on Cultured Human Biliary Epithelial Cells by Alloreactive Lymphocytes

    PubMed Central

    Saidman, Susan L.; Duquesnoy, Rene J.; Zeevi, Adriana; Fung, John J.; Starzl, Thomas E.; Demetris, A. Jake

    2010-01-01

    We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-γ-interferon monoclonal antibody. In addition, recombinant human γ-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell–induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody–blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID:1704868

  8. On the logic of restrictive recognition of peptide by the T-cell antigen receptor

    PubMed Central

    2011-01-01

    This essay provides an analysis of the inadequacy of the current view of restrictive recognition of peptide by the T-cell antigen receptor. A competing model is developed, and the experimental evidence for the prevailing model is reinterpreted in the new framework. The goal is to contrast the two models with respect to their consistency, coverage of the data, explanatory power, and predictability. PMID:20931295

  9. Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

    PubMed Central

    Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

    2014-01-01

    In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

  10. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    PubMed

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation.

  11. Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

    PubMed

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2014-12-18

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates.

  12. Antigen recognition and presentation in periapical tissues: a role for TLR expressing cells?

    PubMed

    Desai, S V; Love, R M; Rich, A M; Seymour, G J

    2011-02-01

    Bacteria are the prime cause of periapical diseases and root canal microbiology is a well-researched area of endodontics. Antigen-presenting cells (APCs) are present in periapical lesions of endodontic origin and play a substantial role in recognizing, processing and presenting pathogenic antigens to the adaptive immune system such as an effective and long-lasting immune response is generated against the specific pathogens. Toll-like receptors (TLRs) are germ-line encoded pathogen recognition receptors (PRR) expressed by various APCs which induce their maturation, lead to gene transcription in the nucleus and the production of several pro- and anti-inflammatory cytokines. Thirteen TLRs have been discovered, 10 of which have been identified in humans so far. Preliminary studies of dental pulp tissue have demonstrated various cell types expressing different TLRs in response to commonly encountered microorganisms. However, there is little information available regarding the expression and function of the various TLRs in human periapical lesions. This review discusses the interactions of various APCs in periapical lesions and the possible roles of different TLRs and APCs in pulp/periapical pathogen recognition and presentation to the adaptive immune system in the initiation and sustaining of periapical diseases. PMID:21083574

  13. Molecular basis of mycobacterial lipid antigen presentation by CD1c and its recognition by αβ T cells

    PubMed Central

    Roy, Sobhan; Ly, Dalam; Li, Nan-Sheng; Altman, John D.; Piccirilli, Joseph A.; Moody, D. Branch; Adams, Erin J.

    2014-01-01

    CD1c is a member of the group 1 CD1 family of proteins that are specialized for lipid antigen presentation. Despite high cell surface expression of CD1c on key antigen-presenting cells and the discovery of its mycobacterial lipid antigen presentation capability, the molecular basis of CD1c recognition by T cells is unknown. Here we present a comprehensive functional and molecular analysis of αβ T-cell receptor (TCR) recognition of CD1c presenting mycobacterial phosphomycoketide antigens. Our structure of CD1c with the mycobacterial phosphomycoketide (PM) shows similarities to that of CD1c-mannosyl-β1-phosphomycoketide in that the A' pocket accommodates the mycoketide alkyl chain; however, the phosphate head-group of PM is shifted ∼6 Å in relation to that of mannosyl-β1-PM. We also demonstrate a bona fide interaction between six human TCRs and CD1c-mycoketide complexes, measuring high to moderate affinities. The crystal structure of the DN6 TCR and mutagenic studies reveal a requirement of five complementarity determining region (CDR) loops for CD1c recognition. Furthermore, mutagenesis of CD1c reveals residues in both the α1 and α2 helices involved in TCR recognition, yet not entirely overlapping among the examined TCRs. Unlike patterns for MHC I, no archetypical binding footprint is predicted to be shared by CD1c-reactive TCRs, even when recognizing the same or similar antigens. PMID:25298532

  14. Immune recognition of protein antigens

    SciTech Connect

    Laver, W.G.; Air, G.M.

    1985-01-01

    This book contains 33 papers. Some of the titles are: Antigenic Structure of Influenze Virus Hemagglutinin; Germ-line and Somatic Diversity in the Antibody Response to the Influenza Virus A/PR/8/34 Hemagglutinin; Recognition of Cloned Influenza A Virus Gene Products by Cytotoxic T Lymphocytes; Antigenic Structure of the Influenza Virus N2 Neuraminidase; and The Molecular and Genetic Basis of Antigenic Variation in Gonococcal Pillin.

  15. Co-potentiation of antigen recognition: A mechanism to boost weak T cell responses and provide immunotherapy in vivo

    PubMed Central

    Hoffmann, Michele M.; Molina-Mendiola, Carlos; Nelson, Alfreda D.; Parks, Christopher A.; Reyes, Edwin E.; Hansen, Michael J.; Rajagopalan, Govindarajan; Pease, Larry R.; Schrum, Adam G.; Gil, Diana

    2015-01-01

    Adaptive immunity is mediated by antigen receptors that can induce weak or strong immune responses depending on the nature of the antigen that is bound. In T lymphocytes, antigen recognition triggers signal transduction by clustering T cell receptor (TCR)/CD3 multiprotein complexes. In addition, it hypothesized that biophysical changes induced in TCR/CD3 that accompany receptor engagement may contribute to signal intensity. Nonclustering monovalent TCR/CD3 engagement is functionally inert despite the fact that it may induce changes in conformational arrangement or in the flexibility of receptor subunits. We report that the intrinsically inert monovalent engagement of TCR/CD3 can specifically enhance physiologic T cell responses to weak antigens in vitro and in vivo without stimulating antigen-unengaged T cells and without interrupting T cell responses to strong antigens, an effect that we term as “co-potentiation.” We identified Mono-7D6-Fab, which biophysically altered TCR/CD3 when bound and functionally enhanced immune reactivity to several weak antigens in vitro, including a gp100-derived peptide associated with melanoma. In vivo, Mono-7D6-Fab induced T cell antigen–dependent therapeutic responses against melanoma lung metastases, an effect that synergized with other anti-melanoma immunotherapies to significantly improve outcome and survival. We conclude that Mono-7D6-Fab directly co-potentiated TCR/CD3 engagement by weak antigens and that such concept can be translated into an immunotherapeutic design. The co-potentiation principle may be applicable to other receptors that could be regulated by otherwise inert compounds whose latent potency is only invoked in concert with specific physiologic ligands. PMID:26601285

  16. Recognition of minor histocompatibility antigens on lymphocytic and myeloid leukemic cells by cytotoxic T-cell clones.

    PubMed

    van der Harst, D; Goulmy, E; Falkenburg, J H; Kooij-Winkelaar, Y M; van Luxemburg-Heijs, S A; Goselink, H M; Brand, A

    1994-02-15

    Clinical studies indicated an enhanced antileukemic effect of allogeneic bone marrow transplantation (BMT), as compared with autologous BMT. After allogeneic HLA-identical BMT, donor-derived cytotoxic T lymphocytes (CTLs) directed at minor histocompatibility (mH) antigens on the recipients, tissues can be shown. To evaluate the antileukemic reactivity of mH antigen-specific CTLs, we analyzed the expression of mH antigens on circulating lymphocytic and myeloid leukemic cells. We show that the defined mH specificities HA-1 through HA-5 and H-Y are present on leukemic cells, indicating that mH antigen-specific CTLs are capable of HLA class I-restricted antigen-specific lysis of leukemic cells. Compared with interleukin-2-stimulated normal lymphocytes, leukemic cells of lymphocytic origin are less susceptible to T-cell-mediated cytotoxicity by the HA-2 mH antigen-specific CTL and the anti-HLA-A2 CTL clone. A possible explanation for this phenomenon is impaired expression of the LFA-1 adhesion molecule. Our study suggests that mH antigen-specific HLA class I-restricted CD8+ CTLs may be involved in the graft-versus-leukemia reactivity after allogeneic BMT.

  17. The Tumor Antigen NY-ESO-1 Mediates Direct Recognition of Melanoma Cells by CD4+ T Cells after Intercellular Antigen Transfer

    PubMed Central

    Fonteneau, Jean Francois; Brilot, Fabienne; Münz, Christian

    2016-01-01

    NY-ESO-1–specific CD4+ T cells are of interest for immune therapy against tumors, because it has been shown that their transfer into a patient with melanoma resulted in tumor regression. Therefore, we investigated how NY-ESO-1 is processed onto MHC class II molecules for direct CD4+ T cell recognition of melanoma cells. We could rule out proteasome and autophagy-dependent endogenous Ag processing for MHC class II presentation. In contrast, intercellular Ag transfer, followed by classical MHC class II Ag processing via endocytosis, sensitized neighboring melanoma cells for CD4+ T cell recognition. However, macroautophagy targeting of NY-ESO-1 enhanced MHC class II presentation. Therefore, both elevated NY-ESO-1 release and macroautophagy targeting could improve melanoma cell recognition by CD4+ T cells and should be explored during immunotherapy of melanoma. PMID:26608910

  18. The Tumor Antigen NY-ESO-1 Mediates Direct Recognition of Melanoma Cells by CD4+ T Cells after Intercellular Antigen Transfer.

    PubMed

    Fonteneau, Jean Francois; Brilot, Fabienne; Münz, Christian; Gannagé, Monique

    2016-01-01

    NY-ESO-1-specific CD4(+) T cells are of interest for immune therapy against tumors, because it has been shown that their transfer into a patient with melanoma resulted in tumor regression. Therefore, we investigated how NY-ESO-1 is processed onto MHC class II molecules for direct CD4(+) T cell recognition of melanoma cells. We could rule out proteasome and autophagy-dependent endogenous Ag processing for MHC class II presentation. In contrast, intercellular Ag transfer, followed by classical MHC class II Ag processing via endocytosis, sensitized neighboring melanoma cells for CD4(+) T cell recognition. However, macroautophagy targeting of NY-ESO-1 enhanced MHC class II presentation. Therefore, both elevated NY-ESO-1 release and macroautophagy targeting could improve melanoma cell recognition by CD4(+) T cells and should be explored during immunotherapy of melanoma.

  19. Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47,000 MW acid labile protein in CD4+ T-cell recognition.

    PubMed

    God, Jason M; Zhao, Dan; Cameron, Christine A; Amria, Shereen; Bethard, Jennifer R; Haque, Azizul

    2014-07-01

    While Burkitt lymphoma (BL) has a well-known defect in HLA class I-mediated antigen presentation, the exact role of BL-associated HLA class II in generating a poor CD4(+) T-cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4(+) T cells via the HLA class II pathway. This defect in CD4(+) T-cell recognition was not associated with low levels of co-stimulatory molecules on BL cells, as addition of external co-stimulation failed to elicit CD4(+) T-cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II-peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II-mediated antigen presentation and CD4(+) T-cell recognition. Biochemical analysis showed that these molecules were greater than 30,000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47,000 molecular weight enolase-like molecule that enhances class II-mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.

  20. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  1. Targeting CLEC9A delivers antigen to human CD141+ DC for CD4+ and CD8+T cell recognition

    PubMed Central

    Tullett, Kirsteen M.; Leal Rojas, Ingrid M.; Minoda, Yoshihito; Tan, Peck S.; Zhang, Jian-Guo; Smith, Corey; Shortman, Ken; Caminschi, Irina; Lahoud, Mireille H.; Radford, Kristen J.

    2016-01-01

    DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines.

  2. Targeting CLEC9A delivers antigen to human CD141+ DC for CD4+ and CD8+T cell recognition

    PubMed Central

    Tullett, Kirsteen M.; Leal Rojas, Ingrid M.; Minoda, Yoshihito; Tan, Peck S.; Zhang, Jian-Guo; Smith, Corey; Shortman, Ken; Caminschi, Irina; Lahoud, Mireille H.; Radford, Kristen J.

    2016-01-01

    DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines. PMID:27699265

  3. Use of somatic cell genetics to study chromosomes contributing to antigen plus I recognition by T cell hybridomas

    PubMed Central

    1983-01-01

    Keyhole limpet hemocyanin (KLH)/I region-specific T cell hybridomas have been prepared by fusing KLH/I-specific T cell blasts from mice with single pairs of metacentric chromosomes to the inducible, interleukin 2 (IL-2)-secreting T cell hybridoma FS6-14.13.AG2.1. T cell hybridomas with KLH/I receptors were identified by their ability to secrete IL-2 in response to KLH and the appropriate antigen-presenting cells. After cloning and subcloning, KLH/I reactivity was correlated with the presence or absence of metacentric chromosomes derived from the KLH/I-specific T cell blast parent. Hybridomas were identified that had lost all chromosomes 4 and 6 or 16 and 17 derived from their normal T cell parent, but retained the ability to respond to KLH/I. This suggested that products of genes on these chromosomes did not contribute to the specific portions of T cell Ag/I receptors. These gene products would include, of course, kappa and lambda chains and H- 2. We did not obtain any T cell hybridomas that had lost both metacentric (8.12) chromosomes derived from T cells of the Robertsonian mouse strain Rb(8.12)5, so we could not draw any conclusions about the contributions of products of genes on these chromosomes. T cell hybridomas with KLH/I reactivity were found that contained only one metacentric (8.12) chromosome derived from this strain. Moreover, a T cell hybridoma was found that retained both metacentric (8.12) chromosomes from its normal T cell parent, but had lost KLH/I reactivity. These results suggested that neither two chromosomes 8 nor two chromosomes 12 were required for antigen/I reactivity in normal T cells and that antigen/I reactivity was controlled, at least in part, by genes mapping on chromosomes other than 8 or 12. PMID:6401795

  4. γ-Radiation Promotes Immunological Recognition of Cancer Cells through Increased Expression of Cancer-Testis Antigens In Vitro and In Vivo

    PubMed Central

    Bode, Beata; Wenger, Roland H.; Lehmann, Kuno; Sartori, Alessandro A.; Moch, Holger; Knuth, Alexander

    2011-01-01

    Background γ-radiation is an effective treatment for cancer. There is evidence that radiotherapy supports tumor-specific immunity. It was described that irradiation induces de novo protein synthesis and enhances antigen presentation, we therefore investigated whether γ-radiation results in increased expression of cancer-testis (CT) antigens and MHC-I, thus allowing efficient immunological control. This is relevant because the expression of CT-antigens and MHC-I on tumor cells is often heterogeneous. We found that the changes induced by γ-radiation promote the immunological recognition of the tumor, which is illustrated by the increased infiltration by lymphocytes after radiotherapy. Methods/Findings We compared the expression of CT-antigens and MHC-I in various cancer cell lines and fresh biopsies before and after in vitro irradiation (20 Gy). Furthermore, we compared paired biopsies that were taken before and after radiotherapy from sarcoma patients. To investigate whether the changed expression of CT-antigens and MHC-I is specific for γ-radiation or is part of a generalized stress response, we analyzed the effect of hypoxia, hyperthermia and genotoxic stress on the expression of CT-antigens and MHC-I. In vitro irradiation of cancer cell lines and of fresh tumor biopsies induced a higher or de novo expression of different CT-antigens and a higher expression of MHC-I in a time- and dose-dependent fashion. Importantly, we show that irradiation of cancer cells enhances their recognition by tumor-specific CD8+ T cells. The analysis of paired biopsies taken from a cohort of sarcoma patients before and after radiotherapy confirmed our findings and, in addition showed that irradiation resulted in higher infiltration by lymphocytes. Other forms of stress did not have an impact on the expression of CT-antigens or MHC-I. Conclusions Our findings suggest that γ-radiation promotes the immunological recognition of the tumor. We therefore propose that combining

  5. Tumour-specific CD4 T cells eradicate melanoma via indirect recognition of tumour-derived antigen.

    PubMed

    Shklovskaya, Elena; Terry, Alexandra M; Guy, Thomas V; Buckley, Adrian; Bolton, Holly A; Zhu, Erhua; Holst, Jeff; Fazekas de St. Groth, Barbara

    2016-07-01

    The importance of CD4 T cells in tumour immunity has been increasingly recognised, with recent reports describing robust CD4 T cell-dependent tumour control in mice whose immune-regulatory mechanisms have been disturbed by irradiation, chemotherapy, immunomodulatory therapy and/or constitutive immunodeficiency. Tumour control in such models has been attributed in large part to direct Major Histocompatibility Complex (MHC) class II-dependent CD4 T cell killing of tumour cells. To test whether CD4 T cells can eradicate tumours without directly killing tumour cells, we developed an animal model in which tumour-derived antigen could be presented to T-cell receptor (TCR)-transgenic CD4 T cells by host but not tumour MHC class II molecules. In I-E(+) mice bearing I-E(null) tumours, naive I-E-restricted CD4 T cells proliferated locally in tumour-draining lymph nodes after recognising tumour-derived antigen on migratory dendritic cells. In lymphopaenic but not immunosufficient hosts, CD4 T cells differentiated into polarised T helper type 1 (Th1) cells expressing interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and interleukin (IL)-2 but little IL-17, and cleared established tumours. Tumour clearance was enhanced by higher TCR affinity for tumour antigen-MHC class II and was critically dependent on IFNγ, as demonstrated by early tumour escape in animals treated with an IFNγ blocking antibody. Thus, CD4 T cells and IFNγ can control tumour growth without direct T-cell killing of tumour cells, and without requiring additional adaptive immune cells such as CD8 T cells and B cells. Our results support a role for effective CD4 T cell-dependent tumour immunity against MHC class II-negative tumours. PMID:26837456

  6. Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T cells. I. Population studies.

    PubMed

    Horai, S; van der Poel, J J; Goulmy, E

    1982-01-01

    Human alloimmune cytotoxic T cells, sensitized selectively against the HLA-A2 antigen, were tested on a panel of selected target cells. Five HLA-A2 positive outlier cells could be identified. These outlier cells were only weakly lysed by HLA-A2 specific CTLs, although they were serologically indistinguishable from the other HLA-A2 positive, strongly lysed target cells. Furthermore, it was found that the outlier cells were poor cold target inhibitors in contrast to the other HLA-A2 positive target cells, which showed adequate inhibition of specific lysis of HLA-A2 positive target cells. Population studies indicate that the frequency of such HLA-A2 outlier cells may be approximately 10%. PMID:6183196

  7. Possible involvement of the OKT4 molecule in T cell recognition of class II HLA antigens. Evidence from studies of cytotoxic T lymphocytes specific for SB antigens.

    PubMed

    Biddison, W E; Rao, P E; Talle, M A; Goldstein, G; Shaw, S

    1982-10-01

    A recently described HLA gene, SB, which maps between GLO and HLA-DR, codes for Ia-like molecules that are similar to but distinct from HLA-DR molecules. Cytotoxic T lymphocytes (CTL) specific for SB1, SB2, SB3, and SB4 were compared with HLA-A2-specific CTL with respect to their surface expression of the T cell differentiation antigens OKT3, OKT4, and OKT8. All CTL activity was eliminated by treatment with OKT3 and C'. The SB-specific cytotoxicity was eliminated by OKT4 plus C' but not by OKT8 plus C'. In contrast, HLA-A2-specific killing was completely susceptible to treatment with OKT8 plus C' but not with OKT4 plus C'. Cytotoxicity was analyzed in the presence of OKT8 and a series of monoclonal antibodies (OKT4A, 4B, 4C, and 4D) that react with distinct epitopes on the OKT4 molecule. SB1-, SB3-, and SB4-specific CTL were partially inhibited by OKT4A and 4B (45-75%), whereas HLA-A2-specific CTL were partially inhibited by OKT8 (48-63%) but not by OKT4. SB2-specific CTL were not inhibited (less than 26%) by OKT8 or by any of the OKT4-related antibodies. These results suggest that the OKT4 marker may be expressed on most T cells that recognize allogeneic Ia or self Ia plus foreign antigens; OKT4+ cells do not appear to be functionally homogeneous in that they can act both as helper/inducer and cytotoxic cells. Models are proposed for the functional involvement of the OKT4 molecule in T cell-Ia antigen interactions. PMID:6984061

  8. RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

    PubMed Central

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

    2013-01-01

    T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

  9. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor

    PubMed Central

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A.; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  10. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

  11. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  12. ANTIGEN RECOGNITION AND THE IMMUNE RESPONSE

    PubMed Central

    Bush, Maurice E.; Alkan, Sefik S.; Nitecki, Danute E.; Goodman, Joel W.

    1972-01-01

    L-Tyrosine-p-azobenzenearsonate (RAT) induces cellular immunity without humoral antibody in guinea pigs. Asymmetric bifunctional antigens composed of one RAT moiety and one dinitrophenyl (DNP) group separated by flexible spacers induce anti-RAT cellular immunity and an anti-DNP humoral response. Symmetrical bifunctional antigens of similar design but comprised of two RAT determinants induce cellular immunity without demonstrable anti-RAT antibody. However, when the flexible spacer is replaced by a rigid decaproline chain, humoral anti-RAT responses are provoked. Since RAT contains both electropositive (azo) and electronegative (arsonate) centers, the failure of bifunctional RAT compounds with flexible spacers to induce humoral immunity might be ascribed either to intramolecular stacking, which compromises their bifunctional character, or to interaction of both determinants with receptors on the same cell surface, which would fail to satisfy the requirement for cooperation. In order to distinguish between these alternatives, symmetrical bifunctional antigens composed of two L-tyrosine-p-azophenyltrimethylammonium (TAT) determinants separated by flexible or rigid spacers were synthesized. TAT is immunogenic and does not cross-react with RAT. Furthermore, it contains only electropositive centers and consequently bifunctional molecules do not undergo intramolecular stacking. Immunization with either flexibly or rigidly spaced bifunctional TAT antigens raised anti-TAT antibody. These results conclusively demonstrate that "self-help," cooperation between bone marrow-derived and thymus-derived lymphocytes of identical or similar specificity, can occur, provided the determinants on the antigen are prevented from associating with each other. PMID:4118413

  13. Structures of MART-126/27-35Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

    SciTech Connect

    Borbulevych, Oleg Y; Insaidoo, Francis K; Baxter, Tiffany K; Powell, Jr., Daniel J.; Johnson, Laura A; Restifo, Nicholas P; Baker, Brian M

    2008-09-17

    Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1{sub 26/27-35}-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.

  14. Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

    SciTech Connect

    Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie

    2009-07-10

    Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

  15. Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

    PubMed

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel; Old, Lloyd J; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2014-04-01

    Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

  16. Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

    SciTech Connect

    Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M.

    2012-05-08

    Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

  17. The Structural Basis of Antibody-Antigen Recognition

    PubMed Central

    Sela-Culang, Inbal; Kunik, Vered; Ofran, Yanay

    2013-01-01

    The function of antibodies (Abs) involves specific binding to antigens (Ags) and activation of other components of the immune system to fight pathogens. The six hypervariable loops within the variable domains of Abs, commonly termed complementarity determining regions (CDRs), are widely assumed to be responsible for Ag recognition, while the constant domains are believed to mediate effector activation. Recent studies and analyses of the growing number of available Ab structures, indicate that this clear functional separation between the two regions may be an oversimplification. Some positions within the CDRs have been shown to never participate in Ag binding and some off-CDRs residues often contribute critically to the interaction with the Ag. Moreover, there is now growing evidence for non-local and even allosteric effects in Ab-Ag interaction in which Ag binding affects the constant region and vice versa. This review summarizes and discusses the structural basis of Ag recognition, elaborating on the contribution of different structural determinants of the Ab to Ag binding and recognition. We discuss the CDRs, the different approaches for their identification and their relationship to the Ag interface. We also review what is currently known about the contribution of non-CDRs regions to Ag recognition, namely the framework regions (FRs) and the constant domains. The suggested mechanisms by which these regions contribute to Ag binding are discussed. On the Ag side of the interaction, we discuss attempts to predict B-cell epitopes and the suggested idea to incorporate Ab information into B-cell epitope prediction schemes. Beyond improving the understanding of immunity, characterization of the functional role of different parts of the Ab molecule may help in Ab engineering, design of CDR-derived peptides, and epitope prediction. PMID:24115948

  18. Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.

    PubMed Central

    Barbosa, J A; Mentzer, S J; Minowada, G; Strominger, J L; Burakoff, S J; Biro, P A

    1984-01-01

    The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells. PMID:6390442

  19. Nonclassical T Cells and Their Antigens in Tuberculosis

    PubMed Central

    De Libero, Gennaro; Singhal, Amit; Lepore, Marco; Mori, Lucia

    2014-01-01

    T cells that recognize nonpeptidic antigens, and thereby are identified as nonclassical, represent important yet poorly characterized effectors of the immune response. They are present in large numbers in circulating blood and tissues and are as abundant as T cells recognizing peptide antigens. Nonclassical T cells exert multiple functions including immunoregulation, tumor control, and protection against infections. They recognize complexes of nonpeptidic antigens such as lipid and glycolipid molecules, vitamin B2 precursors, and phosphorylated metabolites of the mevalonate pathway. Each of these antigens is presented by antigen-presenting molecules other than major histocompatibility complex (MHC), including CD1, MHC class I–related molecule 1 (MR1), and butyrophilin 3A1 (BTN3A1) molecules. Here, we discuss how nonclassical T cells participate in the recognition of mycobacterial antigens and in the mycobacterial-specific immune response. PMID:25059739

  20. HIV-infected CD4+ T Cells Use T-bet-dependent Pathway for Production of IL-10 Upon Antigen Recognition.

    PubMed

    Shete, A; Suryawanshi, P; Godbole, S; Pawar, J; Paranjape, R; Thakar, M

    2016-04-01

    Interleukin (IL)-10 has been implicated in persistence of pathogens in a number of chronic infections. Infected CD4+ cells upon reactivation with HIV antigens were also shown to produce IL-10, which might contribute to their persistence. Hence, it is crucial to determine mechanisms regulating IL-10 production after activation with HIV antigens for devising effective blocking strategies. In this study, ERK-, T-bet- and FoxP3-dependent pathways were evaluated for their possible roles in IL-10 production by infected CD4+ cells after reactivation with HIV Env. Intracellular and secreted IL-10 levels were determined by flow cytometry and Bioplex assay after treating PBMCs with PD98059, tipifarnib and cyclosporin A for blocking of ERK-, T-bet-and FoxP3-dependent pathways, respectively. Baseline levels of T-bet, pERK were higher in P24+ CD4+ cells as compared to uninfected CD4+ cells, which increased further after activation with Env. Inhibition of T-bet resulted in 2.3-fold reduction of IL-10 expression whereas ERK and FoxP3 inhibition failed to cause suppression of IL-10 expression. Conversely, IL-10 secreted by PBMCs was inhibited maximally after ERK inhibition suggesting its role in regulation of cytokine secretory pathway. IFN-γ was found to be suppressed after treatment with inhibitors of all these pathways. Thus, the study highlighted need for IL-10 blockade along with the use of antigens for therapeutic vaccinations or latency reversal and identified the T-bet-dependent pathway as an important pathway regulating IL-10 production by infected CD4+ cells. However, simultaneous blockade of IFN-γ precludes use of inhibitor of this pathway as an IL-10 blocking strategy. PMID:27028319

  1. Identification of a crucial energetic footprint on the alpha1 helix of human histocompatibility leukocyte antigen (HLA)-A2 that provides functional interactions for recognition by tax peptide/HLA-A2-specific T cell receptors.

    PubMed

    Baker, B M; Turner, R V; Gagnon, S J; Wiley, D C; Biddison, W E

    2001-03-01

    Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-alpha/betas make multiple contacts with the alpha1 and alpha2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the alpha1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2-specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2-specific TCRs, there is a location on the central portion of the alpha1 helix that provides interactions crucial to their function with the MHC molecule. PMID:11238586

  2. Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

    NASA Astrophysics Data System (ADS)

    Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

    2012-07-01

    Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

  3. Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

    PubMed Central

    2012-01-01

    Background Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-γ production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-α and IFN-γ) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines. PMID:22336002

  4. Heat shock protein derivatives for delivery of antigens to antigen presenting cells.

    PubMed

    Nishikawa, Makiya; Takemoto, Seiji; Takakura, Yoshinobu

    2008-04-16

    Delivery of antigens to antigen presenting cells (APCs) is a key issue for developing effective cancer vaccines. Controlling the tissue distribution of antigens can increase antigen-specific immune responses, including the induction of cytotoxic T lymphocytes (CTL). Heat shock protein 70 (Hsp70) forms complexes with a variety of tumor-related antigens via its polypeptide-binding domain. Because Hsp70 is taken up by APCs through recognition by Hsp receptors, such as CD91 and LOX-1, its application to antigen delivery systems has been examined both in experimental and clinical settings. A tissue distribution study revealed that Hsp70 is mainly taken up by the liver, especially by hepatocytes, after intravenous injection in mice. A significant amount of Hsp70 was also delivered to regional lymph nodes when it was injected subcutaneously, supporting the hypothesis that Hsp70 is a natural targeting system for APCs. Model antigens were complexed with or conjugated to Hsp70, resulting in greater antigen-specific immune responses. Cytoplasmic delivery of Hsp70-antigen further increased the efficacy of the Hsp70-based vaccines. These findings indicate that effective cancer therapy can be achieved by developing Hsp70-based anticancer vaccines when their tissue and intracellular distribution is properly controlled. PMID:17980980

  5. Generalized immunological recognition of the major merozoite surface antigen (gp195) of Plasmodium falciparum

    SciTech Connect

    Chang, S.P.; Hui, G.S.N.; Kato, A.; Siddiqui, W.A. )

    1989-08-01

    The antibody response to the Plasmodium falciparum major merozoite surface antigen (gp195) of congenic mouse strains differing in H-2 haplotype has been examined. All seven strains of mice were capable of producing gp195-specific antibodies. Generalized immune recognition of gp195 by mice of diverse H-2 haplotypes distinguished gp195 from the P. falciparum circumsporozoite protein and the 230-kDa and 48/45-kDa gamete surface antigens. However, the H-2 genetic locus appeared to influence the specificity of gp105-specific antibodies. Immunoblot patterns of mouse sera with parasite antigens revealed a complex pattern of reactivity with terminal and intermediate processing fragments of gp195. The majority of immunoblot bands observed were similar for all of the mouse strains; however, there were several strains that additionally recognized a few unique fragments or displayed more intense reactivities with specific processing fragments. These results suggest that while individuals of diverse major histocompatibility complex makeup are capable of recognizing the gp195 antigen, the recognition of specific gp195 B-cell and T-cell epitopes may be under control of the major histocompatibility complex.

  6. Control of T cell antigen reactivity via programmed TCR downregulation.

    PubMed

    Gallegos, Alena M; Xiong, Huizhong; Leiner, Ingrid M; Sušac, Bože; Glickman, Michael S; Pamer, Eric G; van Heijst, Jeroen W J

    2016-04-01

    The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage. PMID:26901151

  7. Functional Development of the T Cell Receptor for Antigen

    PubMed Central

    Ebert, Peter J.R.; Li, Qi-Jing; Huppa, Johannes B.; Davis, Mark M.

    2016-01-01

    For over three decades now, the T cell receptor (TCR) for antigen has not ceased to challenge the imaginations of cellular and molecular immunologists alike. T cell antigen recognition transcends every aspect of adaptive immunity: it shapes the T cell repertoire in the thymus and directs T cell-mediated effector functions in the periphery, where it is also central to the induction of peripheral tolerance. Yet, despite its central position, there remain many questions unresolved: how can one TCR be specific for one particular peptide-major histocompatibility complex (pMHC) ligand while also binding other pMHC ligands with an immunologically relevant affinity? And how can a T cell’s extreme specificity (alterations of single methyl groups in their ligand can abrogate a response) and sensitivity (single agonist ligands on a cell surface are sufficient to trigger a measurable response) emerge from TCR–ligand interactions that are so low in affinity? Solving these questions is intimately tied to a fundamental understanding of molecular recognition dynamics within the many different contexts of various T cell–antigen presenting cell (APC) contacts: from the thymic APCs that shape the TCR repertoire and guide functional differentiation of developing T cells to the peripheral APCs that support homeostasis and provoke antigen responses in naïve, effector, memory, and regulatory T cells. Here, we discuss our recent findings relating to T cell antigen recognition and how this leads to the thymic development of foreign-antigen-responsive αβT cells. PMID:20800817

  8. Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

    PubMed

    Chawla, Akhil; Alatrash, Gheath; Philips, Anne V; Qiao, Na; Sukhumalchandra, Pariya; Kerros, Celine; Diaconu, Iulia; Gall, Victor; Neal, Samantha; Peters, Haley L; Clise-Dwyer, Karen; Molldrem, Jeffrey J; Mittendorf, Elizabeth A

    2016-06-01

    Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

  9. Acquired loss of red cell Kell antigens.

    PubMed

    Vengelen-Tyler, V; Gonzalez, B; Garratty, G; Kruppe, C; Johnson, C L; Mueller, K A; Marsh, W L

    1987-02-01

    A 19-year-old patient with a long history of idiopathic thrombocytopenic purpura developed a potent antibody against a high-incidence antigen in the Kell blood group system. The direct antiglobulin test on his red cells was negative. His cells exhibited profound depression of Kell blood group antigens, but antigens of other blood groups were normal. Transfusion of incompatible blood was well tolerated and differential agglutination tests, using selected Rh antisera, showed in vivo survival of the transfused red cells for more than 8 weeks. However, the transfused red cells also showed acquired loss of Kell antigens. Five months after the initial findings, Kell-related antibody disappeared and Kell antigens reappeared on his red cells. The patient's serum stored from the initial investigation now reacted with his freshly collected red cells. These data suggest that an environmental agent in the patient's plasma was responsible for the temporary loss of Kell antigens from red cells in his circulation.

  10. Vertebrate Cells Express Protozoan Antigen after Hybridization

    NASA Astrophysics Data System (ADS)

    Crane, Mark St. J.; Dvorak, James A.

    1980-04-01

    Epimastigotes, the invertebrate host stage of Trypanosoma cruzi, the protozoan parasite causing Chagas' disease in man, were fused with vertebrate cells by using polyethylene glycol. Hybrid cells were selected on the basis of T. cruzi DNA complementation of biochemical deficiencies in the vertebrate cells. Some clones of the hybrid cells expressed T. cruzi-specific antigen. It might be possible to use selected antigens obtained from the hybrids as vaccines for immunodiagnosis or for elucidation of the pathogenesis of Chagas' disease.

  11. Antigen presentation by chemically modified splenocytes induces antigen- specific T cell unresponsiveness in vitro and in vivo

    PubMed Central

    1987-01-01

    We investigated the antigen specificity and presentation requirements for inactivation of T lymphocytes in vitro and in vivo. In vitro studies revealed that splenocytes treated with the crosslinker 1-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide (ECDI) and soluble antigen fragments failed to stimulate significant proliferation by normal pigeon cytochrome c-specific T cell clones, suggesting that the chemical treatment inactivated full antigen presentation function. However, T cell clones exposed to ECDI-treated splenocytes and antigen in vitro were rendered unresponsive for at least 8 d to subsequent antigen stimulation with normal presenting cells. As predicted by the in vitro results, specific T cell unresponsiveness was also induced in vivo in B10.A mice injected intravenously with B10.A, but not B10.A(4R), splenocytes coupled with pigeon cytochrome c via ECDI. The antigen and MHC specificity of the induction of this T cell unresponsiveness in vitro and in vivo was identical to that required for T cell activation. These results suggest that nonmitogenic T cell recognition of antigen/MHC on ECDI-modified APCs results in the functional inactivation of T cell clones. PMID:3029267

  12. Recognition of Microbial Glycolipids by Natural Killer T Cells.

    PubMed

    Zajonc, Dirk M; Girardi, Enrico

    2015-01-01

    T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the major histocompatibility complex (MHC) family (MHC I and II), lipids, glycolipids, and lipopeptides can be presented by the non-classical MHC member, CD1. The best studied subset of lipid-reactive T cells are type I natural killer T (iNKT) cells that recognize a variety of different antigens when presented by the non-classical MHCI homolog CD1d. iNKT cells have been shown to be important for the protection against various microbial pathogens, including B. burgdorferi, the causative agents of Lyme disease, and S. pneumoniae, which causes pneumococcal meningitis and community-acquired pneumonia. Both pathogens carry microbial glycolipids that can trigger the T cell antigen receptor (TCR), leading to iNKT cell activation. iNKT cells have an evolutionary conserved TCR alpha chain, yet retain the ability to recognize structurally diverse glycolipids. They do so using a conserved recognition mode, in which the TCR enforces a conserved binding orientation on CD1d. TCR binding is accompanied by structural changes within the TCR binding site of CD1d, as well as the glycolipid antigen itself. In addition to direct recognition of microbial antigens, iNKT cells can also be activated by a combination of cytokines (IL-12/IL-18) and TCR stimulation. Many microbes carry TLR antigens, and microbial infections can lead to TLR activation. The subsequent cytokine response in turn lower the threshold of TCR-mediated iNKT cell activation, especially when weak microbial or even self-antigens are presented during the cause of the infection. In summary, iNKT cells can be directly activated through TCR triggering of strong antigens, while cytokines produced by the innate immune response may be necessary for TCR triggering and iNKT cell activation in the presence of weak antigens. Here, we will review the molecular basis of iNKT cell

  13. Recognition of Microbial Glycolipids by Natural Killer T Cells

    PubMed Central

    Zajonc, Dirk M.; Girardi, Enrico

    2015-01-01

    T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the major histocompatibility complex (MHC) family (MHC I and II), lipids, glycolipids, and lipopeptides can be presented by the non-classical MHC member, CD1. The best studied subset of lipid-reactive T cells are type I natural killer T (iNKT) cells that recognize a variety of different antigens when presented by the non-classical MHCI homolog CD1d. iNKT cells have been shown to be important for the protection against various microbial pathogens, including B. burgdorferi, the causative agents of Lyme disease, and S. pneumoniae, which causes pneumococcal meningitis and community-acquired pneumonia. Both pathogens carry microbial glycolipids that can trigger the T cell antigen receptor (TCR), leading to iNKT cell activation. iNKT cells have an evolutionary conserved TCR alpha chain, yet retain the ability to recognize structurally diverse glycolipids. They do so using a conserved recognition mode, in which the TCR enforces a conserved binding orientation on CD1d. TCR binding is accompanied by structural changes within the TCR binding site of CD1d, as well as the glycolipid antigen itself. In addition to direct recognition of microbial antigens, iNKT cells can also be activated by a combination of cytokines (IL-12/IL-18) and TCR stimulation. Many microbes carry TLR antigens, and microbial infections can lead to TLR activation. The subsequent cytokine response in turn lower the threshold of TCR-mediated iNKT cell activation, especially when weak microbial or even self-antigens are presented during the cause of the infection. In summary, iNKT cells can be directly activated through TCR triggering of strong antigens, while cytokines produced by the innate immune response may be necessary for TCR triggering and iNKT cell activation in the presence of weak antigens. Here, we will review the molecular basis of iNKT cell

  14. Specific recognition of mycobacterial protein and peptide antigens by gamma-delta T cell subsets following infection with virulent Mycobacterium bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Promoting effective immunity to Mycobacterium tuberculosis complex pathogens is a challenge that is of interest to the fields of human and veterinary medicine alike. We report that gamma delta T cells from virulent Mycobacterium bovis-infected cattle respond specifically and directly to complex, pro...

  15. Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

    PubMed Central

    Zeng, Jieming; Wu, Chunxiao; Wang, Shu

    2015-01-01

    Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs. PMID:26471005

  16. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    PubMed

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion.

  17. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    PubMed

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion. PMID:26764596

  18. Recognition of oxidized albumin and thyroid antigens by psoriasis autoantibodies

    PubMed Central

    Al-Shobaili, Hani A.; Ahmed, Ahmed A.; Rasheed, Zafar

    2015-01-01

    Objectives: To investigate the role of reactive-oxygen-species (ROS) induced epitopes on human-serum-albumin (HSA) and thyroid antigens in psoriasis autoimmunity. Methods: This study was performed in the College of Medicine, Qassim University, Buraidah, Saudi Arabia between May 2014 and February 2015. The study was designed to explore the role of ROS-induced epitopes in psoriasis autoimmunity. Singlet-oxygen (or ROS)-induced epitopes on protein (ROS-epitopes-albumin) was characterized by in-vitro and in-vivo. Thyroid antigens were prepared from rabbit thyroid, and thyroglobulin was isolated from thyroid extract. Immunocross-reactions of protein-A purified anti-ROS-epitopes-HSA-immunoglobulin G (IgGs) with thyroid antigen, thyroglobulin, and their oxidized forms were determined. Binding characteristics of autoantibodies in chronic plaque psoriasis patients (n=26) against ROS-epitopes-HSA and also with native and oxidized thyroid antigens were screened, and the results were compared with age-matched controls (n=22). Results: The anti-ROS-epitopes-HSA-IgGs showed cross-reactions with thyroid antigen, thyroglobulin and with their oxidized forms. High degree of specific binding by psoriasis IgGs to ROS-epitopes-HSA, ROS-thyroid antigen and ROS-thyroglobulin was observed. Immunoglobulin G from normal-human-controls showed negligible binding with all tested antigens. Moreover, sera from psoriasis patients had higher levels of carbonyl contents compared with control sera. Conclusion: Structural alterations in albumin, thyroid antigens by ROS, generate unique neo-epitopes that might be one of the factors for the induction of autoantibodies in psoriasis. PMID:26620982

  19. Utilizing Chimeric Antigen Receptors to Direct Natural Killer Cell Activity

    PubMed Central

    Hermanson, David L.; Kaufman, Dan S.

    2015-01-01

    Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains. PMID:25972867

  20. Antigen Presentation by MHC-Dressed Cells

    PubMed Central

    Nakayama, Masafumi

    2015-01-01

    Professional antigen-presenting cells (APCs) such as conventional dendritic cells (DCs) process protein antigens to MHC-bound peptides and then present the peptide–MHC complexes to T cells. In addition to this canonical antigen presentation pathway, recent studies have revealed that DCs and non-APCs can acquire MHC class I (MHCI) and/or MHC class II (MHCII) from neighboring cells through a process of cell–cell contact-dependent membrane transfer called trogocytosis. These MHC-dressed cells subsequently activate or regulate T cells via the preformed antigen peptide–MHC complexes without requiring any further processing. In addition to trogocytosis, intercellular transfer of MHCI and MHCII can be mediated by secretion of membrane vesicles such as exosomes from APCs, generating MHC-dressed cells. This review focuses on the physiological role of antigen presentation by MHCI- or MHCII-dressed cells, and also discusses differences and similarities between trogocytosis and exosome-mediated transfer of MHC. PMID:25601867

  1. CD1 mediated T cell recognition of glycolipids

    PubMed Central

    Zajonc, Dirk M.; Kronenberg, Mitchell

    2007-01-01

    Summary Specialized subsets of T lymphocytes can distinguish the carbohydrate portions of microbial and self-glycolipids when they are presented by proteins in the CD1 family of antigen presenting molecules. Recent immunochemical and structural analyses indicate that the chemical composition of the presented carbohydrate, together with its precise orientation above the CD1 binding groove, determines if a particular T cell is activated. More recently, however, it has been shown that the lipid backbone of the glycolipid, buried inside the CD1 protein, also can have an impact on T cell activation. While glycolipid recognition is a relatively new category of T cell specificity, the powerful combination of microbial antigen discovery and structural biochemistry has provided great insight into the mechanism of carbohydrate recognition. PMID:17951048

  2. Identification and manipulation of antigen specific T-cells with artificial antigen presenting cells.

    PubMed

    Koffeman, Eva; Keogh, Elissa; Klein, Mark; Prakken, Berent; Albani, Salvatore

    2007-01-01

    T-cells specific for a particular antigen represent a small percentage of the overall T-cell population. Detecting the presence of antigen specific T-cells in patients, animal models or populations of cultured cells has presented a challenge to researchers. The T-cell capture method described here utilizes a truly artificial method of antigen presentation and requires only 50,000 cells for the detection of the major histomcompatibility complex (MHC) class II and antigen restricted T-cells. With this method, liposomes, prepared with readily available materials, are loaded with neutravidin "rafts" comprised of MHC/peptide complexes, anti-CD28, a costimulatory molecule, and anti-LFA-1, an adhesion molecule. These artificial APCs are easily manipulated to include any MHC, antibodies to cell surface markers and/or costimulatory signals of interest thereby enabling not only T-cell identification but also the manipulation of mechanisms of T-cell activation. PMID:17983141

  3. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    PubMed

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

  4. Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

    PubMed

    Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

    2015-10-23

    Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.

  5. Antigen Processing and Presentation Mechanisms in Myeloid Cells.

    PubMed

    Roche, Paul A; Cresswell, Peter

    2016-06-01

    Unlike B cells, CD8-positive and CD4-positive T cells of the adaptive immune system do not recognize intact foreign proteins but instead recognize polypeptide fragments of potential antigens. These antigenic peptides are expressed on the surface of antigen presenting cells bound to MHC class I and MHC class II proteins. Here, we review the basics of antigen acquisition by antigen presenting cells, antigen proteolysis into polypeptide fragments, antigenic peptide binding to MHC proteins, and surface display of both MHC class I-peptide and MHC class II-peptide complexes.

  6. Red cell antigens: Structure and function

    PubMed Central

    Pourazar, Abbasali

    2007-01-01

    Landsteiner and his colleagues demonstrated that human beings could be classified into four groups depending on the presence of one (A) or another (B) or both (AB) or none (O) of the antigens on their red cells. The number of the blood group antigens up to 1984 was 410. In the next 20 years, there were 16 systems with 144 antigens and quite a collection of antigens waiting to be assigned to systems, pending the discovery of new information about their relationship to the established systems. The importance of most blood group antigens had been recognized by immunological complications of blood transfusion or pregnancies; their molecular structure and function however remained undefined for many decades. Recent advances in molecular genetics and cellular biochemistry resulted in an abundance of new information in this field of research. In this review, we try to give some examples of advances made in the field of ‘structure and function of the red cell surface molecules.’ PMID:21938229

  7. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  8. A comprehensive platform for ex vivo T-cell expansion based on biodegradable polymeric artificial antigen-presenting cells.

    PubMed

    Steenblock, Erin R; Fahmy, Tarek M

    2008-04-01

    Efficient T-cell stimulation and proliferation in response to specific antigens is a goal of immunotherapy against infectious disease and cancer. Manipulation of this response can be accomplished by adoptive immunotherapy involving the infusion of antigen-specific T-cell populations expanded ex vivo with antigen presenting cells. We mimicked physiological antigen presentation on a biodegradable microparticle constructed from poly(lactide-co-glycolide) (PLGA), a polymer system whose safety has been established for use in humans. These particles present a high density of adaptor elements for attaching both recognition ligands and co-stimulatory ligands to a biodegradable core encapsulating the cytokine interleukin-2 (IL-2). We demonstrate the utility of this system in efficient polyclonal and antigen-specific T-cell stimulation and expansion, showing that sustained release of IL-2 in the vicinity of T-cell contacts dramatically improves the stimulatory capacity of these acellular systems, as compared to the effect of exogenous addition of cytokine. This results in a 45-fold enhancement in T-cell expansion. In addition, this mode of antigen presentation skews the expansion toward the CD8(+) T-cell phenotype. This comprehensive acellular platform, capable of delivering recognition, co-stimulatory, and cytokine signals, represents a promising new technology for artificial antigen presentation. PMID:18334990

  9. Antigen presenting cells - diversity, differentiation, and regulation

    SciTech Connect

    Schook, L.B. ); Tew, J.G. )

    1988-01-01

    This book contains 35 papers. Some of the titles are: DNA-mediated gene transfer as a tool for analyzing Ia structure-function relationships and antigen presentation; Regulation of immune-associated genes during macrophage differentiation; Presentation of arsonate-tyrosine to cloned T-cells by L-Cells transfected with class II genes; and The duration of class II MHC glycoprotein expression by mononuclear phagocytes is regulated by the Bcg gene.

  10. Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation.

    PubMed

    Meyer, Randall A; Sunshine, Joel C; Perica, Karlo; Kosmides, Alyssa K; Aje, Kent; Schneck, Jonathan P; Green, Jordan J

    2015-04-01

    Non-spherical nanodimensional artificial antigen presenting cells (naAPCs) offer the potential to systemically induce an effective antigen-specific immune response. In this report it is shown biodegradable ellipsoidal naAPCs mimic the T-Cell/APC interaction better than equivalent spherical naAPCs. In addition, it is demonstrated ellipsoidal naAPCs offer reduced non-specific cellular uptake and a superior pharmacokinetic profile compared to spherical naAPCs. PMID:25641795

  11. Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells.

    PubMed

    Rossini, Valerio; Radulovic, Katarina; Riedel, Christian U; Niess, Jan Hendrik

    2016-01-01

    Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG(-/-) recipients) are reconstituted with naive CD4(+) T cells from healthy wild type hosts. This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4(+) T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG(-/-) hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria. PMID:27684040

  12. Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells

    PubMed Central

    Verhagen, Johan; Wraith, David C.

    2014-01-01

    Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy. PMID:25108241

  13. [Mucose associated lymphoid tissue. Antigen presenting cells].

    PubMed

    Luzardo-Baptista, Mario J; Luzardo, José Rafael

    2013-12-01

    We studied samples of normal and abnormal human mucosae, including oral tissue and uterine cervix, using electron microscopy. Special attention was given to the functions and mechanisms of defense carried out by the epithelial (EC) and dendritic cells (DC). Activated epithelial cells posses the capacity to uptake and process antigens, in order to present them, subsequently, to the dendritic cells. The structures and elements of the cells intervening on this function are: micropinocytic vesicles, multivesicular bodies, lysosomes, phagosomes, clathrin-covered vesicles, dense granules covered by a unit membrane, granules with onion likes leaves, microbodies, and dense granules with acid phosphatase activity. When they first arrive within the epithelial layers, the DC are clear with long cytoplasmic projections, which later become short, and the density of their cytoplasm increases. They possess mycropinocytic vesicles, some clathrine-covered vesicles, lysososmes and Birbeck granules. At this moment, they are known as Langerhans cells. EC and DC present many surface folds rich in micropynocytic vesicles. Between EC and DC there are many contacts (close junctions or tight junctions), through which antigens, phagocitized and processed by the EC, are given to the DC. These cells join them to major histocompatibility complex molecules or to other molecules with similar functions (CD1). Then the Langerhans cells travel to the lymphatic node to activate T cells and continue the immunologic task. So, in this way, both the EC and the DC are a link between the natural and the acquired immunological mechanisms. PMID:24502183

  14. Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus

    SciTech Connect

    Choi, Jae-Mun; Hutson, Anne M.; Estes, Mary K.; Prasad, B.V. Venkataram

    2008-07-28

    Members of Norovirus, a genus in the family Caliciviridae, are causative agents of epidemic diarrhea in humans. Susceptibility to several noroviruses is linked to human histo-blood type, and its determinant histo-blood group antigens (HBGAs) are regarded as receptors for these viruses. Specificity for these carbohydrates is strain-dependent. Norwalk virus (NV) is the prototype genogroup I norovirus that specifically recognizes A- and H-type HBGA, in contrast to genogroup II noroviruses that exhibit a more diverse HBGA binding pattern. To understand the structural basis for how HBGAs interact with the NV capsid protein, and how the specificity is achieved, we carried out x-ray crystallographic analysis of the capsid protein domain by itself and in complex with A- and H-type HBGA at a resolution of {approx}1.4 {angstrom}. Despite differences in their carbohydrate sequence and linkage, both HBGAs bind to the same surface-exposed site in the capsid protein and project outward from the capsid surface, substantiating their possible role in initiating cell attachment. Precisely juxtaposed polar side chains that engage the sugar hydroxyls in a cooperative hydrogen bonding and a His/Trp pair involved in a cation-p interaction contribute to selective and specific recognition of A- and H-type HBGAs. This unique binding epitope, confirmed by mutational analysis, is highly conserved, but only in the genogroup I noroviruses, suggesting that a mechanism by which noroviruses infect broader human populations is by evolving different sites with altered HBGA specificities.

  15. Viral cell recognition and entry.

    PubMed Central

    Rossmann, M. G.

    1994-01-01

    Rhinovirus infection is initiated by the recognition of a specific cell-surface receptor. The major group of rhinovirus serotypes attach to intercellular adhesion molecule-1 (ICAM-1). The attachment process initiates a series of conformational changes resulting in the loss of genomic RNA from the virion. X-ray crystallography and sequence comparisons suggested that a deep crevice or canyon is the site on the virus recognized by the cellular receptor molecule. This has now been verified by electron microscopy of human rhinovirus 14 (HRV14) and HRV16 complexed with a soluble component of ICAM-1. A hydrophobic pocket underneath the canyon is the site of binding of various hydrophobic drug compounds that can inhibit attachment and uncoating. This pocket is also associated with an unidentified, possibly cellular in origin, "pocket factor." The pocket factor binding site overlaps the binding site of the receptor. It is suggested that competition between the pocket factor and receptor regulates the conformational changes required for the initiation of the entry of the genomic RNA into the cell. PMID:7849588

  16. Multivalent Antigens for Promoting B and T Cell Activation

    PubMed Central

    Bennett, Nitasha R.; Zwick, Daniel B.; Courtney, Adam H.; Kiessling, Laura L.

    2015-01-01

    Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines. PMID:25970017

  17. The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition.

    PubMed

    Hagerman, Lauren M; Law, Brandon F; Bledsoe, Toni A; Hettick, Justin M; Kashon, Michael L; Lemons, Angela R; Wisnewski, Adam V; Siegel, Paul D

    2016-11-01

    Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.

  18. Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen.

    PubMed Central

    Deckhut, A M; Lippolis, J D; Tevethia, S S

    1992-01-01

    Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL. PMID:1370091

  19. CELL SURFACE ANTIGENS OF A MOUSE TESTICULAR TERATOMA

    PubMed Central

    Gooding, Linda R.; Edidin, Michael

    1974-01-01

    Rabbit antisera to a mouse testicular teratoma, absorbed with normal mouse tissues, react by immunofluorescence with plasma membrane antigens of a variety of transplantable mouse tumor cells and transformed fibroblast cell lines including Clone 1D, SV-40-3T3, and 3T12. Trypsin treatment of cells of "normal" lines, 3T3 and FR-SV-3T3, uncovers reactivity on these as well. Early passage mouse embryo fibroblast cell cultures do not react even after trypsinization. By cross-absorbtion studies, the anti-teratoma serum appears to react with an antigen common to most tumor cells investigated thus far. When this antigen on Clone 1D cells is "capped," H-2 antigens collect with the teratoma antigens in the cap indicating a physical association between the molecules. Molecules specified by both the H-2D and H-2K regions are bound to the teratoma antigens in the Clone 1D plasma membrane. This antigen is also found in soluble tumor cell fractions where it is believed to be free of H-2. A second cell surface antigen defined by anti-teratoma serum is expressed only by hepatoma and teratoma itself. This second antigen is apparently a secretory product of teratoma cells. A third surface antigen defined by anti-teratoma serum appears to be specific for the teratoma. PMID:4365513

  20. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  1. Artificial antigen presenting cells for use in adoptive immunotherapy

    PubMed Central

    Turtle, Cameron J.; Riddell, Stanley R.

    2010-01-01

    The observation that T cells can recognize and specifically eliminate cancer cells has spurred interest in the development of efficient methods to generate large numbers of T cells with specificity for tumor antigens that can be harnessed for use in cancer therapy. Recent studies have demonstrated that during encounter with tumor antigen, the signals delivered to T cells by professional antigen presenting cells can affect T cell programming and their subsequent therapeutic efficacy. This has stimulated efforts to develop artificial antigen presenting cells that allow optimal control over the signals provided to T cells. In this review, we will discuss the advantages and disadvantages of cellular and acellular artificial antigen presenting cell systems and their use in T cell adoptive immunotherapy for cancer. PMID:20693850

  2. Artificial antigen-presenting cells for use in adoptive immunotherapy.

    PubMed

    Turtle, Cameron J; Riddell, Stanley R

    2010-01-01

    The observation that T cells can recognize and specifically eliminate cancer cells has spurred interest in the development of efficient methods to generate large numbers of T cells with specificity for tumor antigens that can be harnessed for use in cancer therapy. Recent studies have demonstrated that during encounter with tumor antigen, the signals delivered to T cells by professional antigen-presenting cells can affect T-cell programming and their subsequent therapeutic efficacy. This has stimulated efforts to develop artificial antigen-presenting cells that allow optimal control over the signals provided to T cells. In this review, we will discuss the advantages and disadvantages of cellular and acellular artificial antigen-presenting cell systems and their use in T-cell adoptive immunotherapy for cancer. PMID:20693850

  3. Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

    PubMed Central

    Hickman, C J; Stover, C K; Joseph, S W; Oaks, E V

    1993-01-01

    A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development. Images PMID:8478055

  4. Antibody recognition of an immunogenic influenza hemagglutinin-human leukocyte antigen class II complex

    PubMed Central

    1991-01-01

    The A/Japan/57 influenza hemagglutin (HA) peptide HA 128-145, when bound by human histocompatibility leukocyte antigen-DRw11 cells, is recognized by the human CD4+ T cell clone V1. A rabbit antiserum has been raised against HA 128-145 which recognizes not only the free peptide, but also the HA 128-145/DRw11 complex on a solid matrix, in solution, or on the surface of viable cells. The detection of these complexes on viable cells was shown to be class II specific, DRw11 restricted, and commensurate with the level of DRw11 expression. The identity of DRw11 as the cell surface molecule binding HA 128-145 was confirmed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping. Using this antiserum HA 128-145/DRw11 complexes could be detected on the cell surface as soon as 30 min after the peptide was added, and increased up to 24 h. Dissociation kinetics showed these complexes were long-lived, with a half-life of approximately 14 h. This anti-HA peptide antiserum represents the first direct means of studying antigenic peptide-human leukocyte antigen class II complexes on the surface of living cells without the addition of a non-amino acid moiety to the peptide. The properties of this antiserum thus provide the potential to study naturally processed antigenic peptides as well as the mechanism of processing itself in a physiologically relevant system. PMID:2056278

  5. Toxocara canis glycans influence antigen recognition by mouse IgG1 and IgM antibodies.

    PubMed

    Długosz, Ewa; Wiśniewski, Marcin

    2016-01-01

    The impact of sugar moieties of Toxocara canis glycoprotein antigens on their recognition by infected mouse antibodies was investigated in this study. Native TES and recombinant Toxocara mucins generated in Pichia pastoris yeast as well as their deglycosylated forms were used in ELISA. TES and recombinant mucins were equally recognized by T. canis infected mouse IgG1 antibodies. IgM immunoglobulins predominantly recognized TES antigens. Among mucins recognition of Tc-MUC-4 was the most significant. Deglycosylation of antigens resulted in significant loss of IgM and IgG1 reactivity to TES, mucins, Tc-MUC-3 and Tc-MUC-4. The presence of sugar moieties had no influence on IgE binding to native or recombinant T. canis antigens. Our results suggest that glycans are involved in epitope formation what should be taken into consideration in production of recombinant helminth antigens for diagnostic purposes. PMID:26751891

  6. Two genetically identical antigen-presenting cell clones display heterogeneity in antigen processing.

    PubMed Central

    Michalek, M T; Benacerraf, B; Rock, K L

    1989-01-01

    Evidence from various antigen systems suggests that antigen processing can be one factor that determines the repertoire of immunogenic peptides. Thus, processing events may account for some of the disparity between the available and expressed helper T-cell repertoires. In this report, we demonstrate that the immunodominant T-cell determinant in ovalbumin [p323-339; ovalbumin-(323-339) heptadecapeptide] is processed differently by two genetically identical antigen-presenting cell lines, M12 and A20. The ovalbumin-specific T-cell-T-cell hybridomas, DO-11.10 and 3DO-54.8, were used to detect processed antigen. These T-T hybridomas have different fine specificities for the p323-339 determinant. A20 cells presented native ovalbumin well to both T-T hybridomas, whereas M12 cells presented native ovalbumin well to 3DO-54.8 but very inefficiently to DO-11.10. M12 and A20 cells effectively stimulated both T-T hybridomas with the same concentrations of the immunogenic synthetic peptide p323-339. Therefore, M12 cells and DO-11.10 can interact with each other, and both T-T hybridomas have similar sensitivities for the same immunogenic peptide. We conclude that genetically identical antigen-presenting cells can display heterogeneity in the fine processing of an immunodominant T-cell determinant, and synthetic model peptides that represent the minimal stimulatory sequence of a T-cell determinant are not necessarily identical to the structure of in vivo processed antigen. Heterogeneity in antigen processing by individual antigen-presenting cells would serve to increase the repertoire of immunogenic peptides that are presented to T cells. PMID:2470101

  7. Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

    PubMed Central

    Giannasca, P J; Boden, J A; Monath, T P

    1997-01-01

    The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal

  8. The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

    PubMed Central

    Schmitt, Thomas M.; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N.; Bleakley, Marie; Turtle, Cameron J.; Chang, Wen-Chung; Greisman, Harvey A.; Wood, Brent; Maloney, David G.; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2010-01-01

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells. PMID:20702778

  9. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells

    PubMed Central

    Vela Ramirez, J.E.; Roychoudhury, R.; Habte, H.H.; Cho, M. W.; Pohl, N. L. B.; Narasimhan, B.

    2015-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells, and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by dendritic cells. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and antigen presenting cells and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  10. NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability.

    PubMed

    Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

    2013-03-15

    The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.

  11. Characterization of antigen-presenting properties of tumour cells using virus-specific cytotoxic T lymphocytes.

    PubMed

    Spierings, D C; Agsteribbe, E; Wilschut, J; Huckriede, A

    2000-04-01

    Immunotherapy of tumours by induction of tumour-specific cytotoxic T-lymphocytes (CTLs) will only be effective for tumours with a functional antigen processing and presentation machinery. However, many tumours are known to down-regulate expression of major histocompatibility complex (MHC) class I molecules and/or to impair antigen processing. It is therefore desirable to evaluate the ability of a given tumour to present antigenic epitopes before developing an immunotherapy protocol. In this study we have used influenza virus as a tool to determine the antigen-presenting capacities of the murine neuroblastoma C1300 cell line NB41A3, a frequently used model for human neuroblastoma. Immunofluorescence analyses revealed low and moderate expression of MHC class I molecules Dd and Kk respectively. Nevertheless, infected NB41 A3 cells were lysed efficiently by influenza-specific CTLs. These results demonstrate that all steps of the antigen-processing pathway function properly in the NB tumour cells, and that the limited MHC class I expression suffices for efficient recognition by CTLs. In addition, lysis of the NB tumour cells shows that the cells are susceptible to CTL-induced apoptosis, a pathway that is often impaired in tumour cells. These characteristics make neuroblastoma a suitable target for immunotherapy. The presented assay allows evaluation of various immunological properties of tumour cells and, thus, represents a valuable tool to assess whether a given tumour will be susceptible to immunotherapy or not.

  12. Targeting Antigens to Dendritic Cell Receptors for Vaccine Development

    PubMed Central

    Apostolopoulos, Vasso; Thalhammer, Theresia; Tzakos, Andreas G.

    2013-01-01

    Dendritic cells (DCs) are highly specialized antigen presenting cells of the immune system which play a key role in regulating immune responses. Depending on the method of antigen delivery, DCs stimulate immune responses or induce tolerance. As a consequence of the dual function of DCs, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. In vaccine development, a major aim is to induce strong, specific T-cell responses. This is achieved by targeting antigen to cell surface molecules on DCs that efficiently channel the antigen into endocytic compartments for loading onto MHC molecules and stimulation of T-cell responses. The most attractive cell surface receptors, expressed on DCs used as targets for antigen delivery for cancer and other diseases, are discussed. PMID:24228179

  13. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    PubMed

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  14. B cells do not present antigen covalently linked to microspheres.

    PubMed Central

    Galelli, A; Charlot, B; Dériaud, E; Leclerc, C

    1993-01-01

    B cells have been shown to present antigen to T cells very efficiently through their capacity to capture antigens by their membrane immunoglobulin. This direct cognate interaction of T and B cells results in the proliferation and differentiation of B cells. This concept has been established using soluble proteins. However, most of the antigens to which the immune system is exposed are included in complex particulate structures such as bacteria or parasites. The capacity of B cells to present these large and complex antigens is still unclear. To address this question we have studied the presentation by trinitrophenyl (TNP)-specific B cells of the same antigen TNP-KLH (keyhole limpet haemocyanin), either in a soluble form or covalently linked to poly(acrolein) microspheres, from 0.25 to 1.5 microns in diameter. In the presence of irradiated splenocytes or purified macrophages as a source of antigen-presenting cells (APC), KLH-specific T cells proliferated in response to soluble TNP-KLH or to TNP-KLH coupled to beads. In contrast, TNP-specific memory B cells were totally ineffective in presenting the TNP-KLH beads to KLH-specific T cells whereas they presented very efficiently soluble TNP-KLH. Similar results were obtained with the A20 B lymphoma or with lipopolysaccharide (LPS)-activated TNP-specific B cells. These results therefore indicate that B cells are unable to present large size particulate antigens such as bacteria or parasites. PMID:8509143

  15. Recognition Strategies of Group 3 Innate Lymphoid Cells

    PubMed Central

    Killig, Monica; Glatzer, Timor; Romagnani, Chiara

    2014-01-01

    During the early phase of an inflammatory response, innate cells can use different strategies to sense environmental danger. These include the direct interaction of specific activating receptors with pathogen-encoded/danger molecules or the engagement of cytokine receptors by pro-inflammatory mediators produced by antigen presenting cells in the course of the infection. These general recognition strategies, which have been extensively described for innate myeloid cells, are shared by innate lymphoid cells (ILC), such as Natural Killer (NK) cells. The family of ILC has recently expanded with the discovery of group 2 (ILC2) and group 3 ILC (ILC3), which play an important role in the defense against extracellular pathogens. Although ILC3 and NK cells share some phenotypic characteristics, the recognition strategies employed by the various ILC3 subsets have been only partially characterized. In this review, we will describe and comparatively discuss how ILC3 sense environmental cues and how the triggering of different receptors may regulate their functional behavior during an immune response. PMID:24744763

  16. Recognition strategies of group 3 innate lymphoid cells.

    PubMed

    Killig, Monica; Glatzer, Timor; Romagnani, Chiara

    2014-01-01

    During the early phase of an inflammatory response, innate cells can use different strategies to sense environmental danger. These include the direct interaction of specific activating receptors with pathogen-encoded/danger molecules or the engagement of cytokine receptors by pro-inflammatory mediators produced by antigen presenting cells in the course of the infection. These general recognition strategies, which have been extensively described for innate myeloid cells, are shared by innate lymphoid cells (ILC), such as Natural Killer (NK) cells. The family of ILC has recently expanded with the discovery of group 2 (ILC2) and group 3 ILC (ILC3), which play an important role in the defense against extracellular pathogens. Although ILC3 and NK cells share some phenotypic characteristics, the recognition strategies employed by the various ILC3 subsets have been only partially characterized. In this review, we will describe and comparatively discuss how ILC3 sense environmental cues and how the triggering of different receptors may regulate their functional behavior during an immune response.

  17. Proliferating cell nuclear antigen in neutrophil fate.

    PubMed

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  18. Nanoscale Artificial Antigen Presenting Cells for T Cell Immunotherapy

    PubMed Central

    Perica, Karlo; De León Medero, Andrés; Durai, Malarvizhi; Chiu, Yen Ling; Bieler, Joan Glick; Sibener, Leah; Niemöller, Michaela; Assenmacher, Mario; Richter, Anne; Edidin, Michael; Oelke, Mathias; Schneck, Jonathan

    2014-01-01

    Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50–100 nm in diameter) and avidin-coated quantum dot nanocrystals, (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. PMID:23891987

  19. TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors

    PubMed Central

    Doorduijn, Elien M.; Sluijter, Marjolein; Querido, Bianca J.; Oliveira, Cláudia C.; Achour, Adnane; Ossendorp, Ferry; van der Burg, Sjoerd H.; van Hall, Thorbald

    2016-01-01

    Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies. PMID:26784543

  20. TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors.

    PubMed

    Doorduijn, Elien M; Sluijter, Marjolein; Querido, Bianca J; Oliveira, Cláudia C; Achour, Adnane; Ossendorp, Ferry; van der Burg, Sjoerd H; van Hall, Thorbald

    2016-02-01

    Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the "self" TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies. PMID:26784543

  1. Pattern recognition monitoring of PEM fuel cell

    DOEpatents

    Meltser, Mark Alexander

    1999-01-01

    The CO-concentration in the H.sub.2 feed stream to a PEM fuel cell stack is monitored by measuring current and voltage behavior patterns from an auxiliary cell attached to the end of the stack. The auxiliary cell is connected to the same oxygen and hydrogen feed manifolds that supply the stack, and discharges through a constant load. Pattern recognition software compares the current and voltage patterns from the auxiliary cell to current and voltage signature determined from a reference cell similar to the auxiliary cell and operated under controlled conditions over a wide range of CO-concentrations in the H.sub.2 fuel stream.

  2. Pattern recognition monitoring of PEM fuel cell

    DOEpatents

    Meltser, M.A.

    1999-08-31

    The CO-concentration in the H{sub 2} feed stream to a PEM fuel cell stack is monitored by measuring current and voltage behavior patterns from an auxiliary cell attached to the end of the stack. The auxiliary cell is connected to the same oxygen and hydrogen feed manifolds that supply the stack, and discharges through a constant load. Pattern recognition software compares the current and voltage patterns from the auxiliary cell to current and voltage signature determined from a reference cell similar to the auxiliary cell and operated under controlled conditions over a wide range of CO-concentrations in the H{sub 2} fuel stream. 4 figs.

  3. Mannan-decorated thiolated Eudragit microspheres for targeting antigen presenting cells via nasal vaccination.

    PubMed

    Li, Hui-Shan; Singh, Bijay; Park, Tae-Eun; Hong, Zhong-Shan; Kang, Sang-Kee; Cho, Chong-Su; Choi, Yun-Jaie

    2015-12-01

    Mucosal vaccination of protein as an antigen requires appropriate delivery or adjuvant systems to deliver antigen to mucosal immune cells efficiently and generate valid immune responses. For successful nasal immunization, the obstacles imposed by the normal process of mucociliary clearance which limits residence time of applied antigens and low antigen delivery to antigen presenting cells (APCs) in nasal associated lymphoid tissue (NALT) need to be overcome for the efficient vaccination. Here, we prepared mucoadhesive and mannan-decorated thiolated Eudragit microspheres (Man-TEM) as a nasal vaccine carrier to overcome the limitations. Mucoadhesive thiolated Eudragit (TE) were decorated with mannan for targeting mannose receptors (MR) in antigen presenting cells (APCs) to obtain efficient immune responses. The potential adjuvant ability of Man-TEM for intranasal immunization was confirmed by in vitro and in vivo experiments. In mechanistic study using APCs in vitro, we obtained that Man-TEM enhanced the receptor-mediated endocytosis by stimulating the MR receptors of APCs. The nasal vaccination of OVA-loaded Man-TEM in mice showed higher levels of serum IgG and mucosal sIgA than the soluble OVA group due to the specific recognition of MR of APCs by the mannan in the Man-TEM. These results suggest that mucoadhesive and Man-TEM may be a promising candidate for nasal vaccine delivery system to elicit systemic and mucosal immunity. PMID:26415829

  4. Mannan-decorated thiolated Eudragit microspheres for targeting antigen presenting cells via nasal vaccination.

    PubMed

    Li, Hui-Shan; Singh, Bijay; Park, Tae-Eun; Hong, Zhong-Shan; Kang, Sang-Kee; Cho, Chong-Su; Choi, Yun-Jaie

    2015-12-01

    Mucosal vaccination of protein as an antigen requires appropriate delivery or adjuvant systems to deliver antigen to mucosal immune cells efficiently and generate valid immune responses. For successful nasal immunization, the obstacles imposed by the normal process of mucociliary clearance which limits residence time of applied antigens and low antigen delivery to antigen presenting cells (APCs) in nasal associated lymphoid tissue (NALT) need to be overcome for the efficient vaccination. Here, we prepared mucoadhesive and mannan-decorated thiolated Eudragit microspheres (Man-TEM) as a nasal vaccine carrier to overcome the limitations. Mucoadhesive thiolated Eudragit (TE) were decorated with mannan for targeting mannose receptors (MR) in antigen presenting cells (APCs) to obtain efficient immune responses. The potential adjuvant ability of Man-TEM for intranasal immunization was confirmed by in vitro and in vivo experiments. In mechanistic study using APCs in vitro, we obtained that Man-TEM enhanced the receptor-mediated endocytosis by stimulating the MR receptors of APCs. The nasal vaccination of OVA-loaded Man-TEM in mice showed higher levels of serum IgG and mucosal sIgA than the soluble OVA group due to the specific recognition of MR of APCs by the mannan in the Man-TEM. These results suggest that mucoadhesive and Man-TEM may be a promising candidate for nasal vaccine delivery system to elicit systemic and mucosal immunity.

  5. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

    PubMed Central

    Tabarin, Thibault; Yamamoto, Yui; Ma, Yuanqing; Bridgeman, John S.; Cohnen, André; Benzing, Carola; Gao, Yijun; Crowther, Michael D.; Tungatt, Katie; Dolton, Garry; Sewell, Andrew K.; Price, David A.; Acuto, Oreste; Parton, Robert G.; Gooding, J. Justin; Rossy, Jérémie; Rossjohn, Jamie; Gaus, Katharina

    2016-01-01

    Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination. PMID:27573839

  6. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

    PubMed

    Pageon, Sophie V; Tabarin, Thibault; Yamamoto, Yui; Ma, Yuanqing; Bridgeman, John S; Cohnen, André; Benzing, Carola; Gao, Yijun; Crowther, Michael D; Tungatt, Katie; Dolton, Garry; Sewell, Andrew K; Price, David A; Acuto, Oreste; Parton, Robert G; Gooding, J Justin; Rossy, Jérémie; Rossjohn, Jamie; Gaus, Katharina

    2016-09-13

    Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination. PMID:27573839

  7. The TCR’s sensitivity to self-peptide–MHC dictates the ability of naïve CD8+ T cells to respond to foreign antigens

    PubMed Central

    Fulton, Ross B.; Hamilton, Sara E.; Xing, Yan; Best, J. Adam; Goldrath, Ananda W.; Hogquist, Kristin A.; Jameson, Stephen C.

    2014-01-01

    The strength of self-peptide–major histocompatibility complex (MHC) recognition dictates naïve CD8+ T cell homeostasis, but its effect on foreign antigen reactivity is controversial. As CD5 expression correlates with self-recognition, we studied CD5lo and CD5hi naïve CD8+ T cells. Gene expression characteristics suggested CD5hi cells were better poised for reactivity and differentiation compared to the CD5lo population, and we found that the CD5hi pool exhibited more efficient clonal recruitment and expansion, as well as enhanced reactivity to inflammatory cues, during recognition of foreign antigen. Yet foreign peptide–MHC recognition was similar for both subsets. Thus, CD8+ T cells with higher self-reactivity dominate the immune response against foreign antigens, with implications for T cell repertoire diversity and autoimmunity. PMID:25419629

  8. Calnexin induces expansion of antigen-specific CD4+ T cells that confer immunity to fungal ascomycetes via conserved epitopes

    PubMed Central

    Wüthrich, Marcel; Brandhorst, Tristan T.; Sullivan, Thomas D.; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S.; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A.; Jenkins, Marc K.; Klein, Bruce

    2015-01-01

    Fungal infections remain a threat due to the lack of broad spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown, but conserved antigen. Using transgenic CD4+ T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats induces expansion of calnexin-specific CD4+ T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4+ T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogeneticity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  9. Calnexin induces expansion of antigen-specific CD4(+) T cells that confer immunity to fungal ascomycetes via conserved epitopes.

    PubMed

    Wüthrich, Marcel; Brandhorst, Tristan T; Sullivan, Thomas D; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A; Jenkins, Marc K; Klein, Bruce

    2015-04-01

    Fungal infections remain a threat due to the lack of broad-spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown but conserved antigen. Using transgenic CD4(+) T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae, and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes, including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats, induces expansion of calnexin-specific CD4(+) T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4(+) T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogenicity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  10. Phylogeny of immune recognition: antigen processing/presentation in channel catfish immune responses to hemocyanins.

    PubMed

    Vallejo, A N; Miller, N W; Jørgensen, T; Clem, L W

    1990-10-15

    Studies were conducted to address the role(s) of antigen (Ag) processing/presentation in channel catfish immune responses. Vigorous and specific secondary in vitro proliferative and antibody (Ab) responses were obtained to keyhole limpet and Limulus polyphemus hemocyanins with peripheral blood leukocytes (PBL) from catfish previously primed in vivo with Ag. In addition, such antigen-specific in vitro proliferative and Ab responses were efficiently elicited by antigen-pulsed and subsequently paraformaldehyde-fixed autologous PBL used as putative antigen-presenting cells (APC) but not by APC fixed prior to Ag pulsing. Treatment of these putative APC with lysosomotropic agents, protease inhibitors, or the ionophore monensin prior to or during pulsing with Ag significantly inhibited both in vitro responses. Furthermore, the use of radiolabeled protein indicated that both untreated and inhibitor-treated PBL but not erythrocytes take up Ag; however, only untreated PBL were able to degrade Ag. Immune restriction was indicated by the use of allogeneic PBL as APC in that only strong MLRs were generated with no detectable antibodies produced in vitro. Finally, the employment of isolated leukocyte subpopulations demonstrated that both catfish B (sIg+) lymphocytes and monocytes were efficient Ag presentors. PMID:2208303

  11. Antigen suppression of the in vitro development of plaque-forming cells to autologous erythrocyte antigens.

    PubMed

    Lord, E M; Dutton, R W

    1975-12-01

    Peritoneal cell cultures from NZB and C57BL/6 mice develop large numbers of PFC directed against antigens present on bromelain-treated isologous erythrocytes. The development of these autoimmune PFC can be suppressed by the addition of small numbers of BrMRBC at the start of the culture period. The possibility that the development of the plaque is prevented by the presence of antigen in vivo is discussed.

  12. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells.

    PubMed

    Vela Ramirez, J E; Roychoudhury, R; Habte, H H; Cho, M W; Pohl, N L B; Narasimhan, B

    2014-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  13. Fibroblasts as Efficient Antigen-Presenting Cells in Lymphoid Organs

    NASA Astrophysics Data System (ADS)

    Kundig, Thomas M.; Bachmann, Martin F.; Dipaolo, Claudio; Simard, John J. L.; Battegay, Manuel; Lother, Heinz; Gessner, Andre; Kuhlcke, Klaus; Ohashi, Pamela S.; Hengartner, Hans; Zinkernagel, Rolf M.

    1995-06-01

    Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

  14. Identifying protective Streptococcus pyogenes vaccine antigens recognized by both B and T cells in human adults and children

    PubMed Central

    Mortensen, Rasmus; Nissen, Thomas Nørrelykke; Fredslund, Sine; Rosenkrands, Ida; Christensen, Jan Pravsgaard; Andersen, Peter; Dietrich, Jes

    2016-01-01

    No commercial vaccine exists against Group A streptococci (GAS; Streptococcus pyogenes) and only little is known about anti-GAS protective immunity. In our effort to discover new protective vaccine candidates, we selected 21 antigens based on an in silico evaluation. These were all well-conserved among different GAS strains, upregulated in host-pathogen interaction studies, and predicted to be extracellular or associated with the surface of the bacteria. The antigens were tested for both antibody recognition and T cell responses in human adults and children. The antigenicity of a selected group of antigens was further validated using a high-density peptide array technology that also identified the linear epitopes. Based on immunological recognition, four targets were selected and tested for protective capabilities in an experimental GAS infection model in mice. Shown for the first time, three of these targets (spy0469, spy1228 and spy1801) conferred significant protection whereas one (spy1643) did not. PMID:26911649

  15. T cell recognition of beryllium.

    PubMed

    Dai, Shaodong; Falta, Michael T; Bowerman, Natalie A; McKee, Amy S; Fontenot, Andrew P

    2013-12-01

    Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by a hypersensitivity to beryllium and characterized by the accumulation of beryllium-specific CD4(+) T cells in the lung. Genetic susceptibility to beryllium-induced disease is strongly associated with HLA-DP alleles possessing a glutamic acid at the 69th position of the β-chain (βGlu69). The structure of HLA-DP2, the most prevalent βGlu69-containing molecule, revealed a unique solvent-exposed acidic pocket that includes βGlu69 and represents the putative beryllium-binding site. The delineation of mimotopes and endogenous self-peptides that complete the αβTCR ligand for beryllium-specific CD4(+) T cells suggests a unique role of these peptides in metal ion coordination and the generation of altered self-peptides, blurring the distinction between hypersensitivity and autoimmunity.

  16. The Level of Viral Antigen Presented by Hepatocytes Influences CD8 T-Cell Function▿

    PubMed Central

    Gehring, Adam J.; Sun, Dianxing; Kennedy, Patrick T. F.; Nolte-'t Hoen, Esther; Lim, Seng Gee; Wasser, Shanthi; Selden, Clare; Maini, Mala K.; Davis, Dan M.; Nassal, Michael; Bertoletti, Antonio

    2007-01-01

    CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-γ and TNF-α production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes. PMID:17202217

  17. Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

    PubMed

    Proff, Julia; Walterskirchen, Christian; Brey, Charlotte; Geyeregger, Rene; Full, Florian; Ensser, Armin; Lehner, Manfred; Holter, Wolfgang

    2016-01-01

    In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins.

  18. Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner

    PubMed Central

    Proff, Julia; Walterskirchen, Christian; Brey, Charlotte; Geyeregger, Rene; Full, Florian; Ensser, Armin; Lehner, Manfred; Holter, Wolfgang

    2016-01-01

    In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins. PMID:27375569

  19. Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

    PubMed

    Proff, Julia; Walterskirchen, Christian; Brey, Charlotte; Geyeregger, Rene; Full, Florian; Ensser, Armin; Lehner, Manfred; Holter, Wolfgang

    2016-01-01

    In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins. PMID:27375569

  20. Carbohydrate-Mediated Targeting of Antigen to Dendritic Cells Leads to Enhanced Presentation of Antigen to T Cells

    PubMed Central

    Adams, Eddie W.; Ratner, Daniel M.; Seeberger, Peter H.; Hacohen, Nir

    2009-01-01

    The unique therapeutic value of dendritic cells (DCs) for the treatment of allergy, autoimmunity and transplant rejection is predicated upon our ability to selectively deliver antigens, drugs or nucleic acids to DCs in vivo. Here we describe a method for delivering whole protein antigens to DCs based on carbohydrate-mediated targeting of DC-expressed lectins. A series of synthetic carbohydrates was chemically-coupled to a model antigen, ovalbumin (OVA), and each conjugate was evaluated for its ability to increase the efficiency of antigen presentation by murine DCs to OVA-specific T cells (CD4+ and CD8+). In vitro data are presented that demonstrate that carbohydrate modification of OVA leads to a 50-fold enhancement of presentation of antigenic peptide to CD4+ T cells. A tenfold enhancement is observed for CD8+ T cells; this indicates that the targeted lectin(s) can mediate cross-presentation of antigens on MHC class I. Our data indicate that the observed enhancements in antigen presentation are unique to OVA that is conjugated to complex oligosaccharides, such as a high-mannose nonasaccharide, but not to monosaccharides. Taken together, our data suggest that a DC targeting strategy that is based upon carbohydrate-lectin interactions is a promising approach for enhancing antigen presentation via class I and class II molecules. PMID:18186095

  1. MHC Class II Association with Lipid Rafts on the Antigen Presenting Cell Surface

    PubMed Central

    Anderson, Howard A.; Roche, Paul A.

    2014-01-01

    MHC class II (MHC-II) molecules function by binding peptides derived from either self-or foreign proteins and expressing these peptides on the surface of antigen presenting cells (APCs) for recognition by CD4 T cells. MHC-II is known to exist on clusters on the surface of APCs, and a variety of biochemical and functional studies have suggested that these clusters represent lipid raft microdomain-associated MHC-II. This review will summarize data exploring the biosynthesis of raft-associated MHC-II and the role that lipid raft association plays in regulating T cell activation by APCs. PMID:25261705

  2. Podocytes Are Nonhematopoietic Professional Antigen-Presenting Cells

    PubMed Central

    Burkard, Miriam; Ölke, Martha; Daniel, Christoph; Amann, Kerstin; Hugo, Christian; Kurts, Christian; Steinkasserer, Alexander; Gessner, André

    2013-01-01

    Podocytes are essential to the structure and function of the glomerular filtration barrier; however, they also exhibit increased expression of MHC class II molecules under inflammatory conditions, and they remove Ig and immune complexes from the glomerular basement membrane (GBM). This finding suggests that podocytes may act as antigen-presenting cells, taking up and processing antigens to initiate specific T cell responses, similar to professional hematopoietic cells such as dendritic cells or macrophages. Here, MHC–antigen complexes expressed exclusively on podocytes of transgenic mice were sufficient to activate CD8+ T cells in vivo. In addition, deleting MHC class II exclusively on podocytes prevented the induction of experimental anti-GBM nephritis. Podocytes ingested soluble and particulate antigens, activated CD4+ T cells, and crosspresented exogenous antigen on MHC class I molecules to CD8+ T cells. In conclusion, podocytes participate in the antigen-specific activation of adaptive immune responses, providing a potential target for immunotherapies of inflammatory kidney diseases and transplant rejection. PMID:23539760

  3. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  4. Evaluation of the Antigen-Experienced B-Cell Receptor Repertoire in Healthy Children and Adults

    PubMed Central

    IJspeert, Hanna; van Schouwenburg, Pauline A.; van Zessen, David; Pico-Knijnenburg, Ingrid; Driessen, Gertjan J.; Stubbs, Andrew P.; van der Burg, Mirjam

    2016-01-01

    Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1–CDR3 sequence, but different subclasses. Together, these data suggest that

  5. αβ TCR-mediated recognition: relevance to tumor-antigen discovery and cancer immunotherapy

    PubMed Central

    Reinherz, Ellis L.

    2015-01-01

    αβ T lymphocytes sense perturbations in host cellular body components induced by infectious pathogens, oncogenic transformation or chemical or physical damage. Millions-billions of these lymphocytes are generated through T-lineage development in the thymus, each endowed with a clonally-restricted surface T-cell receptor (TCR). An individual TCR has the capacity to recognize a distinct “foreign” peptide among the myriad of antigens that the mammalian host must be capable of detecting. TCRs explicitly distinguish foreign from self peptides bound to major histocompatibility complex (MHC) molecules. This is a daunting challenge, given that the MHC-linked peptidome consists of thousands of distinct peptides with a relevant non-self target antigen often embedded at low number, among orders of magnitude higher frequency self-peptides. In this Masters of Immunology article, I shall review how TCR structure and attendant mechanobiology involving non-linear responses impact sensitivity as well as specificity to meet this requirement. Assessment of human tumor-cell display using state of the art mass spectrometry physical detection methods that quantify epitope copy number can help inform as to requisite T-cell functional avidity affording protection and/or therapeutic immunity. Future rational CD8 cytotoxic T cell-based vaccines may follow, targeting virally-induced cancers, other non-viral immunogenic tumors, and potentially even non-immunogenic tumors whose peptide display can be purposely altered by MHC-binding drugs to stimulate immune attack. PMID:25847967

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

    PubMed Central

    Newick, Kheng; Moon, Edmund; Albelda, Steven M

    2016-01-01

    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. PMID:27162934

  7. The CD1 family: serving lipid antigens to T cells since the Mesozoic era.

    PubMed

    Zajonc, Dirk M

    2016-08-01

    Class I-like CD1 molecules are in a family of antigen-presenting molecules that bind lipids and lipopeptides, rather than peptides for immune surveillance by T cells. Since CD1 lacks the high degree of polymorphism found in their major histocompatibility complex (MHC) class I molecules, different species express different numbers of CD1 isotypes, likely to be able to present structurally diverse classes of lipid antigens. In this review, we will present a historical overview of the structures of the different human CD1 isotypes and also discuss species-specific adaptations of the lipid-binding groove. We will discuss how single amino acid changes alter the shape and volume of the CD1 binding groove, how these minor changes can give rise to different numbers of binding pockets, and how these pockets affect the lipid repertoire that can be presented by any given CD1 protein. We will compare the structures of various lipid antigens and finally, we will discuss recognition of CD1-presented lipid antigens by antigen receptors on T cells (TCRs).

  8. The CD1 family: serving lipid antigens to T cells since the Mesozoic era.

    PubMed

    Zajonc, Dirk M

    2016-08-01

    Class I-like CD1 molecules are in a family of antigen-presenting molecules that bind lipids and lipopeptides, rather than peptides for immune surveillance by T cells. Since CD1 lacks the high degree of polymorphism found in their major histocompatibility complex (MHC) class I molecules, different species express different numbers of CD1 isotypes, likely to be able to present structurally diverse classes of lipid antigens. In this review, we will present a historical overview of the structures of the different human CD1 isotypes and also discuss species-specific adaptations of the lipid-binding groove. We will discuss how single amino acid changes alter the shape and volume of the CD1 binding groove, how these minor changes can give rise to different numbers of binding pockets, and how these pockets affect the lipid repertoire that can be presented by any given CD1 protein. We will compare the structures of various lipid antigens and finally, we will discuss recognition of CD1-presented lipid antigens by antigen receptors on T cells (TCRs). PMID:27368414

  9. Beyond model antigens: high-dimensional methods for the analysis of antigen-specific T cells

    PubMed Central

    Newell, Evan W.; Davis, Mark M.

    2014-01-01

    Adaptive immune responses often begin with the formation of a molecular complex between a T cell receptor (TCR) and a peptide antigen bound to a major histocompatibility complex (MHC) molecule. These complexes are highly variable, however, due to the polymorphism of MHC genes, the random, inexact recombination of TCR gene segments and the vast array of possible self and pathogen peptide antigens. As a result, it has been very difficult to comprehensively study the TCR repertoire or identify and track more than a few antigen-specific T cells in mice or humans. For mouse studies, this had led to a reliance on model antigens and TCR transgenes. The study of limited human clinical samples, in contrast, requires techniques that can simultaneously survey phenotype, function and reactivity to many T cell epitopes. Thanks to recent advances in single-cell and cytometry methodologies, as well as high-throughput sequencing of the TCR repertoire, we now have or will soon have the tools needed to comprehensively analyze T-cell responses during health and disease. PMID:24441473

  10. Reconstitution of the B cell antigen receptor signaling components in COS cells.

    PubMed

    Saouaf, S J; Kut, S A; Fargnoli, J; Rowley, R B; Bolen, J B; Mahajan, S

    1995-11-10

    To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation. PMID:7592958

  11. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition

    PubMed Central

    Nominé, Yves; Choulier, Laurence; Travé, Gilles; Vernet, Thierry; Altschuh, Danièle

    2015-01-01

    Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv–peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes. PMID:26629896

  12. Five novel cell surface antigens of CNS neoplasms.

    PubMed

    Jennings, M T; Jennings, V D; Asadourian, L L; Rosenblum, M; Albino, A P; Cairncross, J G; Old, L J

    1989-01-01

    Optimal monoclonal antibody-mediated immunotherapy requires the identification of tumor-restricted cell surface antigens. We have identified and partially characterized 5 new monoclonal antibodies generated against malignant astrocytoma, medulloblastoma, neuroblastoma and melanoma which were used to define 5 neuroectodermal tumor antigenic systems. CNT/1 identifies a 57-kDa, heat-stable, trypsin-sensitive neuroblastoma surface antigen, which is expressed intracellularly in many malignant gliomas, medulloblastomas, ependymomas, breast and ovarian carcinomas. CNT/2 reacts with a 130-kDa, heat-labile, trypsin- and neuraminidase-resistant antigen restricted to low-grade astrocytomas and malignant gliomas. CNT/11 reacts with a 70-kDa, heat-labile, trypsin-sensitive antigen coded for by a gene on chromosome 12, and is restricted to astrocytomas, neuroblastomas and sarcomas. CNT/8 identifies a heat-labile, trypsin-sensitive antigen whose gene has been localized to chromosome 15 and is expressed by neuroectodermal and mesodermally derived tumors and few epithelial cancers. The B2.6 antigen is identified only in terms of serologic reactivity with a subset of cultured astrocytomas and melanomas. Neuroectodermal tumor-associated antigens may be categorized as lineage-consistent, lineage-independent and putatively tumor-restricted in their expression. These restricted antibodies may be potentially useful reagents to consider for monoclonal antibody-mediated immunotherapy of CNS neoplasms.

  13. Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells

    PubMed Central

    Ngugi, Daniel; Lizundia, Regina; Hostettler, Isabel; Woods, Kerry; Ballingall, Keith; MacHugh, Niall D.; Morrison, W. Ivan; Weir, Willie; Shiels, Brian; Werling, Dirk

    2016-01-01

    As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles. PMID:27611868

  14. Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells.

    PubMed

    Goh, Shan; Ngugi, Daniel; Lizundia, Regina; Hostettler, Isabel; Woods, Kerry; Ballingall, Keith; MacHugh, Niall D; Morrison, W Ivan; Weir, Willie; Shiels, Brian; Werling, Dirk

    2016-01-01

    As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles. PMID:27611868

  15. NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

    PubMed

    Esser, Ruth; Müller, Tina; Stefes, Dörthe; Kloess, Stephan; Seidel, Diana; Gillies, Stephen D; Aperlo-Iffland, Christel; Huston, James S; Uherek, Christoph; Schönfeld, Kurt; Tonn, Torsten; Huebener, Nicole; Lode, Holger N; Koehl, Ulrike; Wels, Winfried S

    2012-03-01

    Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

  16. NK cells engineered to express a GD2-specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

    PubMed Central

    Esser, Ruth; Müller, Tina; Stefes, Dörthe; Kloess, Stephan; Seidel, Diana; Gillies, Stephen D; Aperlo-Iffland, Christel; Huston, James S; Uherek, Christoph; Schönfeld, Kurt; Tonn, Torsten; Huebener, Nicole; Lode, Holger N; Koehl, Ulrike; Wels, Winfried S

    2012-01-01

    Abstract Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD2, which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD2-specific chimeric antigen receptor (CAR) comprising an anti-GD2 ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD2 expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD2-specific antibody or anti-idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2-specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells. PMID:21595822

  17. Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Stanciu-Gavan, Camelia

    2015-02-01

    New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α-CDs, and 5,10,15,20-tetraphenyl-21H,23H-porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10(7)  s mg(-1)  ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10(8)  s mg(-1)  ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. PMID:25604868

  18. T Cells as Antigen Carriers for Anti-tumor Vaccination.

    PubMed

    Traversari, Catia; Russo, Vincenzo

    2016-01-01

    The exploitation of the physiologic processing and presenting machinery of dendritic cells (DCs) by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. The approach developed by our group was based on the clinical observation that some patients treated with the infusion of donor lymphocytes transduced to express the HSV-TK suicide gene for relapse of hematologic malignancies, after allogeneic hematopoietic stem cell transplantation, developed a T cell-mediated immune response specifically directed against the HSV-TK gene product.We demonstrated that lymphocytes genetically modified to express HSV-TK as well as self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of TRP-2-transduced lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice by cross-presentation of the antigen mediated by the CD11c(+)CD8a(+) DCs subset. A similar approach was applied in a clinical setting. Ten patients affected by MAGE-3(+) metastatic melanoma were treated with autologous lymphocytes retrovirally transduced to express the MAGE-3 tumor antigen. In three patients, the treatment led to the increase of MAGE-3 specific CD8+ and CD4+ effectors and the development of long-term memory, which ultimately correlated with a favorable clinical outcome. Transduced lymphocytes represent an efficient way for in vivo loading of tumor-associated antigens of DCs.

  19. T cell recognition of weak ligands: roles of signaling, receptor number, and affinity

    PubMed Central

    Edwards, Lindsay J.

    2011-01-01

    T cell recognition of antigen is a crucial aspect of the adaptive immune response. One of the most common means of pathogen immune evasion is mutation of T cell epitopes. T cell recognition of such ligands can result in a variety of outcomes including activation, apoptosis and anergy. The ability of a given T cell to respond to a specific peptide–MHC ligand is regulated by a number of factors, including the affinity, on- and off-rates and half-life of the TCR–peptide–MHC interaction. Interaction of T cells with low-potency ligands results in unique signaling patterns and requires engagement with a larger number of T cell receptors than agonist ligands. This review will address these aspects of T cell interaction with weak ligands and the ways in which these ligands have been utilized therapeutically. PMID:21365321

  20. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.

    PubMed Central

    Falo, L D; Haber, S I; Herrmann, S; Benacerraf, B; Rock, K L

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen. PMID:3467371

  1. Activated Brain Endothelial Cells Cross-Present Malaria Antigen

    PubMed Central

    Howland, Shanshan W.; Poh, Chek Meng; Rénia, Laurent

    2015-01-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. PMID:26046849

  2. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy

    PubMed Central

    Dai, Hanren; Wang, Yao; Lu, Xuechun

    2016-01-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  3. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    PubMed

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy.

  4. Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

    PubMed Central

    Tatituri, Raju V.V.; Watts, Gerald F.M.; Bhowruth, Veemal; Leadbetter, Elizabeth A.; Barton, Nathaniel; Cohen, Nadia R.; Hsu, Fong-Fu; Besra, Gurdyal S.

    2011-01-01

    Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections. PMID:21555485

  5. Human cartilage aggrecan CS1 region contains cryptic T-cell recognition sites.

    PubMed Central

    Goodacre, J A; Middleton, S; Lynn, S; Ross, D A; Pearson, J

    1993-01-01

    Cartilage proteoglycan aggregates (PG) are candidate T-cell autoantigens in the pathogenesis of rheumatoid arthritis (RA). We have investigated the possibility that responses to class II-restricted T-cell recognition sites in human cartilage aggrecan (core protein) may depend upon whether these sites are available as free peptide antigens or as part of intact monomers. Analysis of mouse T-cell responses to intact or deglycosylated monomers, purified from human articular cartilage, and to synthetic peptides of the chondroitin sulphate (CS) attachment region homologous repeat sequence showed that recognition of T-cell epitopes in the CS1 region was strongly dependent upon the form of antigen used. The results show that the CS1 region contains cryptic T-cell recognition sites and raise the possibility that fragments of PG, released through the action of extracellular proteases in inflamed joints, may be capable of activating T cells with specificities for epitopes which are not made available following processing of intact PG. T cells with specificities for cryptic epitopes in PG may play a role in the pathogenesis of RA. PMID:8388364

  6. Unusual Water-mediated Antigenic Recognition of the Proinflammatory Cytokine Interleukin-18

    SciTech Connect

    Argiriadi, Maria A.; Xiang, Tao; Wu, Chengbin; Ghayur, Tariq; Borhani, David W.

    2009-10-21

    The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 {angstrom} resolution; the 125-2H Fab (2.3 {angstrom}); and the ABT-325 Fab (1.5 {angstrom}). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (>10 {angstrom}) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966-971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific antibody

  7. Unusual Water-mediated Antigenic Recognition of the Proinflammatory Cytokine Interleukin-18

    PubMed Central

    Argiriadi, Maria A.; Xiang, Tao; Wu, Chengbin; Ghayur, Tariq; Borhani, David W.

    2009-01-01

    The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 Å resolution; the 125-2H Fab (2.3 Å); and the ABT-325 Fab (1.5 Å). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (>10 Å) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966–971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific antibody binding may in some cases be

  8. Antigen conformation determines processing requirements for T-cell activation.

    PubMed Central

    Streicher, H Z; Berkower, I J; Busch, M; Gurd, F R; Berzofsky, J A

    1984-01-01

    We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. PMID:6333686

  9. Enrichment of antigen-specific B lymphocytes by the direct removal of B cells not bearing specificity for the antigen

    PubMed Central

    1977-01-01

    Antigen-specific B cells (ASC) were purified from other B cells by prior incubation with specific antigen followed by rosetting with erythrocytes conjugated with anti-mouse Ig and sedimenting on Ficoll- Isopaque. This procedure allowed the removal of most of the B cells, while those speicifc for the antigen used in incubation were retained. Relative to the B-cell content, ASC were enriched 64- to 132-fold. The method is highly specific in that B cells primed to two different antigens, turkey gamma globulin and sheep erythrocytes, could be separated from each other. The advantages of this indirect purification procedure over purification procedures which obtain ASC directly are the simplicity of obtaining the ASC and the ability of the ASC of respond to antigen without the addition of other cells. PMID:69002

  10. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells

    PubMed Central

    Fischer, Karsten; Scotet, Emmanuel; Niemeyer, Marcus; Koebernick, Heidrun; Zerrahn, Jens; Maillet, Sophie; Hurwitz, Robert; Kursar, Mischo; Bonneville, Marc; Kaufmann, Stefan H. E.; Schaible, Ulrich E.

    2004-01-01

    A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-γ production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d. PMID:15243159

  11. Synthesis of Antihemophilic Factor Antigen by Cultured Human Endothelial Cells

    PubMed Central

    Jaffe, Eric A.; Hoyer, Leon W.; Nachman, Ralph L.

    1973-01-01

    Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system. Images PMID:4583980

  12. An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

    PubMed Central

    Nathan, P D; Walker, L; Hardie, D; Richardson, P; Khan, M; Johnson, G D; Ling, N R

    1986-01-01

    A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens. Images Fig. 4 PMID:3024883

  13. Regulatory T Cells Are Dispensable for Tolerance to RBC Antigens

    PubMed Central

    Richards, Amanda L.; Kapp, Linda M.; Wang, Xiaohong; Howie, Heather L.; Hudson, Krystalyn E.

    2016-01-01

    Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated. While the basic disease pathology of AIHA is well studied, the underlying mechanism(s) behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs) in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved.

  14. Regulatory T Cells Are Dispensable for Tolerance to RBC Antigens

    PubMed Central

    Richards, Amanda L.; Kapp, Linda M.; Wang, Xiaohong; Howie, Heather L.; Hudson, Krystalyn E.

    2016-01-01

    Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated. While the basic disease pathology of AIHA is well studied, the underlying mechanism(s) behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs) in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved. PMID:27698653

  15. Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

    PubMed Central

    Wang, Jing; Tang, Shan; Wan, Zhengpeng; Gao, Yiren; Cao, Yiyun; Yi, Junyang; Si, Yanyan; Zhang, Haowen; Liu, Lei; Liu, Wanli

    2016-01-01

    Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens. PMID:26764382

  16. Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

    PubMed

    Mitra-Kaushik, S; Shaila, M S; Karande, A K; Nayak, R

    2001-05-01

    Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.

  17. Ethanol Metabolism Alters Major Histocompatibility Complex Class I-Restricted Antigen Presentation In Liver Cells

    PubMed Central

    Osna, Natalia A.; White, Ronda L.; Thiele, Geoffrey M.; Donohue, Terrence M.

    2009-01-01

    The proteasome is a major enzyme that cleaves proteins for antigen presentation. Cleaved peptides traffic to the cell surface, where they are presented in the context of MHC class I. Recognition of these complexes by cytotoxic T lymphocytes is crucial for elimination of cells bearing “non-self” proteins. Our previous studies revealed that ethanol suppresses proteasome function in ethanol-metabolizing liver cells. We hypothesized that proteasome suppression reduces the hydrolysis of antigenic peptides, thereby decreasing the presentation of the peptide-MHC class I-complexes on the cell surface. To test this, we used the mouse hepatocyte cell line (CYP2E1/ADH-transfected HepB5 cells) or primary mouse hepatocytes, both derived from livers of C57Bl/6 mice, which present the ovalbumin peptide, SIINFEKL, complexed with H2Kb. To induce H2Kb expression, HepB5 cells were treated with interferon gamma (IFNγ) and then exposed to ethanol. In these cells, ethanol metabolism decreased not only proteasome activity, but also hydrolysis of the C-extended peptide, SIINFEKL-TE and the presentation of SIINFEKL-H2Kb complexes measured after the delivery of SIINFEKL-TE to cytoplasm. The suppressive effects of ethanol were, in part, attributed to ethanol-elicited impairment of IFNγ signaling. However, in primary hepatocytes, even in the absence of IFNγ, we observed a similar decline in proteasome activity and antigen presentation after ethanol exposure. We conclude that proteasome function is directly suppressed by ethanol metabolism and indirectly, by preventing the activating effects of IFNγ. Ethanol-elicited reduction in proteasome activity contributes to the suppression of SIINFEKL-H2Kb presentation on the surface of liver cells. Immune response to viral antigens plays a crucial role in the pathogenesis of hepatitis C or B viral infections (HCV and HBV, respectively). Professional antigen-presenting cells (dendritic cells and macrophages) are responsible for priming the

  18. Molecular and cellular dynamics at the early stages of antigen encounter: the B-cell immunological synapse.

    PubMed

    Carrasco, Yolanda R

    2010-01-01

    The recent development and application of sophisticated technology in the study of the initial stages of the B-cell immune response has lead to a tremendous revolution in the field. The use of real-time confocal microscopy, total interference reflection fluorescence (TIRF) microscopy and in vitro models has revealed the molecular details of the antigen recognition process by B cells. Moreover, experimental models that allow tracking of antigen in vivo in concert with multiphoton microscopy have provided critical information as to the how, where, and when naïve B cells encounter antigen in vivo. This review focuses on the latest data regarding the early phase of the humoral immune response at molecular and cellular levels.

  19. Merocytic dendritic cells break T cell tolerance to beta cell antigens in NOD mouse diabetes1

    PubMed Central

    Katz, Jonathan D; Ondr, Jennifer K; Opoka, Robert J; Garcia, Zacharias; Janssen, Edith M

    2010-01-01

    In type 1 diabetes (T1D), the breach of central and peripheral tolerance results in autoreactive T cells destroying insulin-producing, pancreatic beta cells. Herein, we identify a critical sub-population of dendritic cells responsible for mediating both the cross-presentation of islet antigen to CD8+ T cells and the direct presentation of beta cell antigen to CD4+ T cells. These cells, termed merocytic dendritic cells (mcDC), are more numerous in nonobese diabetic (NOD) mouse, and when antigen-loaded rescue CD8+ T cells from peripheral anergy and deletion, while stimulating islet-reactive CD4+ T cells. When purified from the pancreatic lymph nodes of overtly diabetic NOD mice, mcDC break peripheral T cell tolerance to beta cells in vivo and induce rapid onset T1D in young NOD mouse. Thus, the mcDC subset appears to represent the long-sought APC responsible for breaking peripheral tolerance to beta cell antigen in vivo. PMID:20644171

  20. Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

    PubMed Central

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P.; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  1. Killer artificial antigen presenting cells (KaAPC) for efficient in vitro depletion of human antigen-specific T cells.

    PubMed

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  2. Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity.

    PubMed

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J N

    2014-05-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through coculture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed 1 ligand that could activate CAR T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated that CARL aAPC propagate CAR T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Using CARL enables 1 aAPC to numerically expand all CAR T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR T cells of any specificity. PMID:24714354

  3. Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

    PubMed Central

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

    2014-01-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

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

    SciTech Connect

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

    2011-08-05

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

  5. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody

    PubMed Central

    1983-01-01

    An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1- 26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties. PMID:6601175

  6. Antigen presentation by non-immune B-cell hybridoma clones: presentation of synthetic antigenic sites reveals clones that exhibit no specificity and clones that present only one epitope

    NASA Technical Reports Server (NTRS)

    Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

    1989-01-01

    Recently, we reported the preparation and antigen-presenting properties of hybridoma B-cell clones obtained after fusing non-secreting, non-antigen presenting Balb/c 653-myeloma cells with non-immune SJL spleen cells. It was found that antigen presentation at the clonal level can be specific or non-specific, depending on the particular B-cell clone. In the present work, one specific and one general presenter B-cell clones were tested for their epitope presentation ability to SJL T-cells that were specific to lysozyme or myoglobin. B-cell clone A1G12, a general presenter which presented both lysozyme and myoglobin to their respective T-cell lines, was found to present all five myoglobin epitopes while clone A1L16, a lysozyme specific presenter presented only one of the three epitopes of lysozyme. The latter reveals a hitherto unknown submolecular specificity (to a given epitope within a protein) for antigen presenting cells at the clonal level. Therefore, the specificity of T-cell recognition does not only derive from the T-cell but may also be dependent on the epitope specificity of the antigen-presenting B-cell.

  7. Monoclonal antibodies directed against human Rh antigens in tests with the red cells of nonhuman primates.

    PubMed

    Socha, W W; Ruffie, J

    1990-01-01

    Monoclonal antibodies against Rh related antigens on human red cells often crossreact with the red cells of the highest subhuman primate species. Depending on specificity of antibody, the species tested, and technique used, these reactions can be either species-specific or type specific. In tests with chimpanzee red cells, some of the latter type reactions have specificities related to the R antigen of the R-C-E-F blood group system of chimpanzee; specificities of some others seem to be unrelated to any known chimpanzee blood groups. Monoclonal anti-D reagents that give uniformly positive reactions with human D-positive (common and rare types) red cells, display wide individual differences in tests with chimpanzee blood. This indicates that there are minute structural variations of antibody molecules from one monoclonal anti-D antibodies apparently have no bearing on recognition of the D combining site on the human red cells, but come into play when in contact with chimpanzee rbcs. Some of the monoclonal antibodies directed against Rh and LW molecules are distinguished by unusually strong reactions with the red cells of the Old World monkeys (macaques and baboons), which is in contrast with negative or weak reactions of the same antibodies with the red cells of anthropoid apes and human bloods. One may recall, that polyclonal anti-Rh sera do not react with the blood of rhesus monkeys, the phenomenon that was the source of controversy surrounding the discovery of the rhesus factor of the human blood.

  8. Cross-Presentation of Cell-Associated Antigens by MHC Class I in Dendritic Cell Subsets

    PubMed Central

    Gutiérrez-Martínez, Enric; Planès, Remi; Anselmi, Giorgio; Reynolds, Matthew; Menezes, Shinelle; Adiko, Aimé Cézaire; Saveanu, Loredana; Guermonprez, Pierre

    2015-01-01

    Dendritic cells (DCs) have the unique ability to pick up dead cells carrying antigens in tissue and migrate to the lymph nodes where they can cross-present cell-associated antigens by MHC class I to CD8+ T cells. There is strong in vivo evidence that the mouse XCR1+ DCs subset acts as a key player in this process. The intracellular processes underlying cross-presentation remain controversial and several pathways have been proposed. Indeed, a wide number of studies have addressed the cellular process of cross-presentation in vitro using a variety of sources of antigen and antigen-presenting cells. Here, we review the in vivo and in vitro evidence supporting the current mechanistic models and disscuss their physiological relevance to the cross-presentation of cell-associated antigens by DCs subsets. PMID:26236315

  9. Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing.

    PubMed

    Mpakali, Anastasia; Saridakis, Emmanuel; Harlos, Karl; Zhao, Yuguang; Papakyriakou, Athanasios; Kokkala, Paraskevi; Georgiadis, Dimitris; Stratikos, Efstratios

    2015-09-15

    Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of different long peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes.

  10. αvβ3-dependent cross-presentation of matrix metalloproteinase–2 by melanoma cells gives rise to a new tumor antigen

    PubMed Central

    Godefroy, Emmanuelle; Moreau-Aubry, Agnes; Diez, Elisabeth; Dreno, Brigitte; Jotereau, Francine; Guilloux, Yannick

    2005-01-01

    A large array of antigens that are recognized by tumor-specific T cells has been identified and shown to be generated through various processes. We describe a new mechanism underlying T cell recognition of melanoma cells, which involves the generation of a major histocompatibility complex class I–restricted epitope after tumor-mediated uptake and processing of an extracellular protein—a process referred to as cross-presentation—which is believed to be restricted to immune cells. We show that melanoma cells cross-present, in an αvβ3-dependent manner, an antigen derived from secreted matrix metalloproteinase–2 (MMP-2) to human leukocyte antigen A*0201-restricted T cells. Because MMP-2 activity is critical for melanoma progression, the MMP-2 peptide should be cross-presented by most progressing melanomas and represents a unique antigen for vaccine therapy of these tumors. PMID:15998788

  11. Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4+ T lymphocytes

    NASA Astrophysics Data System (ADS)

    Marañón, Concepción; Desoutter, Jean-François; Hoeffel, Guillaume; Cohen, William; Hanau, Daniel; Hosmalin, Anne

    2004-04-01

    A better understanding of the antigen presentation pathways that lead to CD8+ T cell recognition of HIV epitopes in vivo is needed to achieve better immune control of HIV replication. Here, we show that cross-presentation of very small amounts of HIV proteins from apoptotic infected CD4+ T lymphocytes by dendritic cells to CD8+ T cells is much more efficient than other known HIV presentation pathways, i.e., direct presentation of infectious virus or cross-presentation of defective virus. Unexpectedly, dendritic cells also take up actively antigens into endosomes from live infected CD4+ T lymphocytes and cross-present them as efficiently as antigens derived from apoptotic infected cells. Moreover, live infected CD4+ T cells costimulate cross-presenting dendritic cells in the process. Therefore, dendritic cells can present very small amounts of viral proteins from infected T cells either after apoptosis, which is frequent during HIV infection, or not. Thus, if HIV expression is transiently induced while costimulation is enhanced (for instance after IL-2 and IFN immune therapy), this HIV antigen presentation pathway could be exploited to eradicate latently infected reservoirs, which are poorly recognized by patients' immune systems.

  12. Modulation of cell-surface antigens of a murine neuroblastoma.

    PubMed

    Akeson, R; Herschman, H R

    1974-01-01

    Antisera were produced in rabbits to morphologically differentiated cells from the C1300 murine neuroblastoma (i.e., cells in which process formation was induced by maintenance on serum-free medium for 5 days). These antisera reacted more strongly in the complement fixation reaction with such "differentiated" cells than with "undifferentiated" (nonprocess-bearing) neuroblastoma cells. Adsorption of the antisera with undifferentiated cells removed the reactivity to cells without processes, while the reactivity with serum-free cells which possess processes was retained. Indirect immunofluorescence studies confirmed the results obtained by complement fixation and demonstrated that antibodies to the surface antigens of process-bearing cells could be adsorbed by particulate preparations from brain but not liver, spleen, or kidney. This is the first description of neural-associated cell-surface changes that correlate with the morphological differentiation in culture of neuroblastoma cells.

  13. Immune recognition of AIDS virus antigens by human and murine cytotoxic T lymphocytes.

    PubMed

    Langlade-Demoyen, P; Michel, F; Hoffenbach, A; Vilmer, E; Dadaglio, G; Garicia-Pons, F; Mayaud, C; Autran, B; Wain-Hobson, S; Plata, F

    1988-09-15

    The CTL response to HIV was analyzed in humans and in mice. By using a novel and strictly autologous lymphocyte culture system, human CTL lines were established with PBL from seropositive asymptomatic donors and from patients suffering from AIDS or presenting AIDS-related complex. CTL from HLA-A2 donors recognize and kill murine P815 mastocytoma cells doubly transfected with the human HLA-A2 gene and the HIV env gene; they also kill HLA-compatible human macrophages infected with HIV. CTL specific for the HIV env Ag were also generated in BALB/c mice by immunization with syngeneic murine cells transfected with the HIV env gene. Human and murine HIV-immune CTL populations belong to the CD8 subset of T lymphocytes and are restricted by class I HLA or H-2 transplantation Ag, respectively, in the recognition of HIV env Ag. The two different experimental systems presented here can be used to study CD8 lymphocyte immunity against HIV. The murine model of CTL immunity offers the additional advantage of avoiding the manipulation of infectious virus isolates.

  14. Impaired Tumor Antigen Processing by Immunoproteasome-expressing CD40-Activated B cells and Dendritic Cells

    PubMed Central

    Anderson, Karen S.; Zeng, Wanyong; Sasada, Tetsuro; Su, Mei; Choi, Jaewon; Drakoulakos, Donna; Kang, Yoon-Joong; Brusic, Vladimir; Wu, Catherine; Reinherz, Ellis L.

    2012-01-01

    Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart126–34 (M26) and Cyp1B1239–247 (Cyp239)] and one derived from the Influenza A viral antigen [FluM158–66 (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens. PMID:21400024

  15. The Exonuclease Domain of Lassa Virus Nucleoprotein Is Involved in Antigen-Presenting-Cell-Mediated NK Cell Responses

    PubMed Central

    Russier, Marion; Reynard, Stéphanie; Carnec, Xavier

    2014-01-01

    ABSTRACT Lassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a

  16. Reaching for far-flung antigen: How solid-core podosomes of dendritic cells transform into protrusive structures.

    PubMed

    Baranov, Maksim V; Ter Beest, Martin; van den Bogaart, Geert

    2014-10-01

    We recently identified a novel role for podosomes in antigen sampling. Podosomes are dynamic cellular structures that consist of point-like concentrations of actin surrounded by integrins and adaptor proteins such as vinculin and talin. Podosomes establish cellular contact with the extracellular matrix (ECM) and facilitate cell migration via ECM degradation. In our recent paper, we studied podosomes of human dendritic cells (DCs), major antigen presenting cells (APC) that take-up, process, and present foreign antigen to naive T-cells. We employed gelatin-impregnated porous polycarbonate filters to demonstrate that the mechanosensitive podosomes of DCs selectively localize to regions of low-physical resistance such as the filter pores. After degradation of the gelatin, podosomes increasingly protrude into the lumen of these pores. These protrusive podosome-derived structures contain several endocytic and early endosomal markers such as clathrin, Rab5, and VAMP3, and, surprisingly, also contain C-type lectins, a type of pathogen recognition receptors (PRRs). Finally, we performed functional uptake experiments to demonstrate that these PRRs facilitate uptake of antigen from the opposite side of the filter. Our data provide mechanistic insight in how dendritic cells sample for antigen across epithelial barriers for instance from the lumen of the lung and gut.

  17. Antigen-presenting cells in the female reproductive tract: influence of sex hormones on antigen presentation in the vagina.

    PubMed Central

    Wira, C R; Rossoll, R M

    1995-01-01

    We report here that the stage of the reproductive cycle and the administration of physiological amounts of oestradiol to ovariectomized rats influences antigen presentation by macrophage/dendritic cells in the vagina. Antigen presentation is elevated when oestradiol levels in blood are low, and reduced just prior to ovulation. Of those hormones tested, only oestradiol lowered vaginal antigen presentation. When progesterone was given along with oestradiol, the inhibitory effect of oestradiol on vaginal antigen presentation was reversed. These studies demonstrate that the vagina is an inductive site and that antigen presentation is under hormonal control. Our results suggest that immunization studies designed to enhance mucosal immunity in the female reproductive tract should take into account the stage of the reproductive cycle when antigen is deposited. PMID:7790022

  18. Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

    PubMed Central

    Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

    2013-01-01

    SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

  19. Immunologic ignorance of vascular endothelial cells expressing minor histocompatibility antigen.

    PubMed

    Bolinger, Beatrice; Krebs, Philippe; Tian, Yinghua; Engeler, Daniel; Scandella, Elke; Miller, Simone; Palmer, Douglas C; Restifo, Nicholas P; Clavien, Pierre-Alain; Ludewig, Burkhard

    2008-05-01

    Endothelial cells (ECs) presenting minor histocompatibility antigen (mhAg) are major target cells for alloreactive effector CD8(+) T cells during chronic transplant rejection and graft-versus-host disease (GVHD). The contribution of ECs to T-cell activation, however, is still a controversial issue. In this study, we have assessed the antigen-presenting capacity of ECs in vivo using a transgenic mouse model with beta-galactosidase (beta-gal) expression confined to the vascular endothelium (Tie2-LacZ mice). In a GVHD-like setting with adoptive transfer of beta-gal-specific T-cell receptor-transgenic T cells, beta-gal expression by ECs was not sufficient to either activate or tolerize CD8(+) T cells. Likewise, transplantation of fully vascularized heart or liver grafts from Tie2-LacZ mice into nontransgenic recipients did not suffice to activate beta-gal-specific CD8(+) T cells, indicating that CD8(+) T-cell responses against mhAg cannot be initiated by ECs. Moreover, we could show that spontaneous activation of beta-gal-specific CD8(+) T cells in Tie2-LacZ mice was exclusively dependent on CD11c(+) dendritic cells (DCs), demonstrating that mhAgs presented by ECs remain immunologically ignored unless presentation by DCs is granted.

  20. Modeling T cell responses to antigenic challenge

    PubMed Central

    Wodarz, Dominik

    2014-01-01

    T cell responses are a crucial part of the adaptive immune system in the fight against infections. This article discusses the use of mathematical models for understanding the dynamics of cytotoxic T lymphocyte (CTL) responses against viral infections. Complementing experimental research, mathematical models have been very useful for exploring new hypotheses, interpreting experimental data, and for defining what needs to be measured to improve understanding. This review will start with minimally parameterized models of CTL responses, which have generated some valuable insights into basic dynamics and correlates of control. Subsequently, more biological complexity is incorporated into this modeling framework, examining different mechanisms of CTL expansion, different effector activities, and the influence of T cell help. Models and results are discussed in the context of data from specific infections. PMID:25269610

  1. Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis.

    PubMed

    Sakhno, Ludmila V; Shevela, Ekaterina Ya; Tikhonova, Marina A; Nikonov, Sergey D; Ostanin, Alexandr A; Chernykh, Elena R

    2015-01-01

    The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generated in vitro macrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity to M. tuberculosis antigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14(+)CD16(+) expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which were in vitro generated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γ coupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generated in vitro from peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γ production in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response to M. tuberculosis was discussed. PMID:26339660

  2. Human CD1-restricted T cell recognition of lipids from pollens.

    PubMed

    Agea, Elisabetta; Russano, Anna; Bistoni, Onelia; Mannucci, Roberta; Nicoletti, Ildo; Corazzi, Lanfranco; Postle, Anthony D; De Libero, Gennaro; Porcelli, Steven A; Spinozzi, Fabrizio

    2005-07-18

    Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids. PMID:16009719

  3. Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells.

    PubMed

    Eckle, Sidonia B G; Corbett, Alexandra J; Keller, Andrew N; Chen, Zhenjun; Godfrey, Dale I; Liu, Ligong; Mak, Jeffrey Y W; Fairlie, David P; Rossjohn, Jamie; McCluskey, James

    2015-12-18

    Vitamin B2 (riboflavin) is essential for metabolic functions and is synthesized by many bacteria, yeast, and plants, but not by mammals and other animals, which must acquire it from the diet. In mammals, modified pyrimidine intermediates from the microbial biosynthesis of riboflavin are recognized as signature biomarkers of microbial infection. This recognition occurs by specialized lymphocytes known as mucosal associated invariant T (MAIT) cells. The major histocompatibility class I-like antigen-presenting molecule, MR1, captures these pyrimidine intermediates, but only after their condensation with small molecules derived from glycolysis and other metabolic pathways to form short-lived antigens. The resulting MR1-Ag complexes are recognized by MAIT cell antigen receptors (αβ T cell receptors (TCRs)), and the subsequent MAIT cell immune responses are thought to protect the host from pathogens at mucosal surfaces. Here, we review our understanding of how these novel antigens are generated and discuss their interactions with MR1 and MAIT TCRs. PMID:26468291

  4. T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome.

    PubMed

    Bresciani, Anne; Paul, Sinu; Schommer, Nina; Dillon, Myles B; Bancroft, Tara; Greenbaum, Jason; Sette, Alessandro; Nielsen, Morten; Peters, Bjoern

    2016-05-01

    Several mechanisms exist to avoid or suppress inflammatory T-cell immune responses that could prove harmful to the host due to targeting self-antigens or commensal microbes. We hypothesized that these mechanisms could become evident when comparing the immunogenicity of a peptide from a pathogen or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially the polarization) of T-cell responses to a given epitope is influenced and to some extent predictable based on its similarity to self-antigens and commensal antigens.

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

    SciTech Connect

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

    2010-03-15

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

  6. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    PubMed

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the 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. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. 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 1 July 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 benefits 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 and clinical studies, and the effectiveness and drawbacks of this strategy.

  7. Chimeric Antigen Receptor T Cell Therapy in Hematology

    PubMed Central

    Ataca, Pınar; Arslan, Önder

    2015-01-01

    It is well demonstrated that the 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. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. 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 1 July 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 benefits 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 and clinical studies, and the effectiveness and drawbacks of this strategy. PMID:26377367

  8. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  10. Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration

    PubMed Central

    Lopes Pinheiro, Melissa A; Kamermans, Alwin; Garcia-Vallejo, Juan J; van het Hof, Bert; Wierts, Laura; O'Toole, Tom; Boeve, Daniël; Verstege, Marleen; van der Pol, Susanne MA; van Kooyk, Yvette; de Vries, Helga E; Unger, Wendy WJ

    2016-01-01

    Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment. DOI: http://dx.doi.org/10.7554/eLife.13149.001 PMID:27336724

  11. Specific adhesion of carcinoembryonic antigen-bearing colorectal cancer cells to immobilized carcinoembryonic antigen.

    PubMed

    Levin, L V; Griffin, T W

    1991-11-01

    Recent characterization of the genomic structure of carcinoembryonic antigen (CEA) is consistent with that of a cellular adhesion molecule. To examine this function in colorectal cancer, the adherence of cell lines to microtiter wells coated with CEA and well-described adhesive molecules was determined. The CEA-positive cell line LoVo and the CEA-devoid cell line H-Meso-1 did not differ in adherence to the extracellular matrix proteins laminin, collagen and fibronectin, whereas LoVo cells adhered to CEA (10 micrograms/well) in a specific manner (43% bound cells vs. 1.5% bound cells with BSA or alpha-acidglycoprotein controls, P less than 0.01) while H-MESO-1 showed no adhesion to CEA (less than 0.6% bound cells). This adhesion of LoVo cells to CEA was not affected by co-incubation of cells with EDTA, sodium azide, or at 23 degrees C. However, the CEA to CEA adhesive interaction was inhibited by a monoclonal antibody directed against an epitope in the N-terminal domain of the CEA molecule, and decreased by enzymatic removal of CEA from the LoVo cell membrane. The extent of adhesion to immobilized CEA by four CEA-producing cell lines (LoVo, HT29, LS174T and LS174-S), correlated with membrane CEA expression as determined by FACS analysis. The results of these experiments add support to the concept that CEA may function as a specific homotypic cellular adhesion molecule for colorectal cancer cells.

  12. The role of a human antigen specific T8+ cell subset in antigen presentation, helper function and contrasuppression.

    PubMed Central

    Lehner, T; Avery, J; Jones, T

    1985-01-01

    Regulation of the human immune response was studied by sequential separation of subsets of T cells, followed by assessment of their helper and suppressor functions in a series of reconstitution experiments. T8+ lymphocytes were separated by panning on streptococcal antigen (SA) coated plates into T8+ SA-adherent cells (T8+SA+) and T8+ SA-non-adherent cells (T8+SA-). The helper and suppressor functions of the T8+SA+ and T8+SA- cells, reconstituted with T4+ helper cells were then studied by a direct antibody forming cell assay. T4+ cells will not induce helper activity by 1000 ng SA alone but require the accessory function of monocytes (Mo). However, replacing Mo by T8+SA+ cells will elicit a similar helper activity by T4+ cells and SA as that induced by Mo. In addition to the antigen-specific presentation and induction of helper activity, the T8+SA+ subset displays the properties of antigen-specific contrasuppressor cells. Thus, reconstitution of T4+ cells and T8+SA- (suppressor cells) with T8+SA+ and 1000 ng SA induces helper and no suppressor activity. Substitution of Mo for the T8+SA+ cells converts the helper to a predominantly suppressor-cell function. T8+SA- cells elicit suppression with 1 ng SA in the absence of accessory cells and reconstitution with Mo, T8+SA+ or T4+ cells failed to affect the suppressor activity. Total reconstitution of the four principle subsets of T4+, T8+SA+, T8+SA- cells and Mo elicited similar antigen dose-dependent responses as those of the unseparated mononuclear cells. It seems that all four cell subsets are required for optimal immunoregulation. We suggest that the T8+SA+ can present antigen to T4+ helper cells and induce helper activity, but in addition these cells can prevent the suppressor subset of T8+ cells from inhibiting T4+ helper cells and function as contrasuppressor cells. The mechanism of these functions is not known but HLA class II antigens might play an essential role in antigen binding, presentation and

  13. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor–expressing T cells

    PubMed Central

    Lynn, Rachel C.; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S.; Dimitrov, Dimiter S.

    2015-01-01

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ+ AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ+ THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34+ HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

  14. Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

    SciTech Connect

    Higgins, M.; Whitworth, G; El Warry, N; Randriantsoa, M; Samain, E; Burke, R; Vocadlo, D; Boraston, A

    2009-01-01

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-{beta}-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-{beta}-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  15. The T cell antigen receptor: the Swiss army knife of the immune system.

    PubMed

    Attaf, M; Legut, M; Cole, D K; Sewell, A K

    2015-07-01

    The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: αβ TCR and γδ TCR. The αβ TCR is expressed on the majority of peripheral T cells. Most αβ T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other αβ T cell subsets. These 'unconventional' T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). γδ T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for γδ T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors.

  16. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Rivoltini, L; Topalian, S L; Miki, T; Rosenberg, S A

    1994-01-01

    By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma. PMID:8170938

  17. Age-dependent B cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis

    PubMed Central

    McLaughlin, Katherine A.; Chitnis, Tanuja; Newcombe, Jia; Franz, Bettina; Kennedy, Julia; McArdel, Shannon; Kuhle, Jens; Kappos, Ludwig; Rostasy, Kevin; Pohl, Daniela; Gagne, Donald; Ness, Jayne M.; Tenembaum, Silvia; O'Connor, Kevin C.; Viglietta, Vissia; Wong, Susan J.; Tavakoli, Norma P.; de Seze, Jerome; Khoury, Samia J.; Bar-Or, Amit; Hafler, David A.; Banwell, Brenda; Wucherpfennig, Kai W.

    2009-01-01

    Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface antigens in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. While antibodies to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum antibodies that brightly labeled the MOG transfectant. Antibodies to two other myelin surface antigens were largely absent. Affinity purification of MOG antibodies as well as competition of binding with soluble MOG documented their binding specificity. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG antibodies, compared to 14.7% of patients in the 10–18 year age group. B cell autoimmunity to this myelin surface antigen is therefore most common in patients with a very early onset of MS. PMID:19687098

  18. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis.

    PubMed

    Kobierecka, Patrycja A; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M; Jagusztyn-Krynicka, Elżbieta K; Wyszyńska, Agnieszka K

    2016-01-01

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

  19. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis

    PubMed Central

    Kobierecka, Patrycja A.; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M.; Jagusztyn-Krynicka, Elżbieta K.; Wyszyńska, Agnieszka K.

    2016-01-01

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

  20. Lipase Processing of Complex Lipid Antigens.

    PubMed

    Sander, Peter; Becker, Katja; Molin, Michael Dal

    2016-09-22

    Mycobacterium tuberculosis synthesizes a wide variety of complex lipids that can serve as antigens in immune recognition of the bacterium. In this issue of Cell Chemical Biology, Gilleron et al. (2016) identify key enzymes essential for lipid antigen processing, which is required for CD1b-restricted T cell activation. PMID:27662250

  1. Immune complexes that contain HIV antigens activate peripheral blood T cells.

    PubMed

    Korolevskaya, L B; Shmagel, K V; Saidakova, E V; Shmagel, N G; Chereshnev, V A

    2016-07-01

    Uninfected donor T cells were treated in vitro by model immune complexes that contained either HIV or hepatitis C virus (HCV) antigens. Unlike HCV antigen-containing complexes, the immune complexes that contained HIV antigens have been shown to activate peripheral blood T cells of uninfected donors under in vitro conditions. Both the antiviral antibodies and HIV antigen were involved in the activation process. The unique properties of the immune complexes formed by HIV antigens and antiviral antibodies are believed to result from the virus-specific antibody properties and molecular conformation of the antigen-antibody complex. PMID:27595830

  2. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens

    PubMed Central

    Meermeier, Erin W.; Laugel, Bruno F.; Sewell, Andrew K.; Corbett, Alexandra J.; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J.; Franks, Tamera; Gold, Marielle C.; Lewinsohn, David M.

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2+ MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2+ clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  3. Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens.

    PubMed

    Meermeier, Erin W; Laugel, Bruno F; Sewell, Andrew K; Corbett, Alexandra J; Rossjohn, Jamie; McCluskey, James; Harriff, Melanie J; Franks, Tamera; Gold, Marielle C; Lewinsohn, David M

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites. PMID:27527800

  4. The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens.

    PubMed

    Sykes, P J; Hope, R M

    1978-12-01

    Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).

  5. CD4+ T Cell Tolerance to Tissue-Restricted Self Antigens Is Mediated by Antigen-Specific Regulatory T Cells Rather Than Deletion.

    PubMed

    Legoux, Francois P; Lim, Jong-Baeck; Cauley, Andrew W; Dikiy, Stanislav; Ertelt, James; Mariani, Thomas J; Sparwasser, Tim; Way, Sing Sing; Moon, James J

    2015-11-17

    Deletion of self-antigen-specific T cells during thymic development provides protection from autoimmunity. However, it is unclear how efficiently this occurs for tissue-restricted self antigens, or how immune tolerance is maintained for self-antigen-specific T cells that routinely escape deletion. Here we show that endogenous CD4+ T cells with specificity for a set of tissue-restricted self antigens were not deleted at all. For pancreatic self antigen, this resulted in an absence of steady-state tolerance, while for the lung and intestine, tolerance was maintained by the enhanced presence of thymically-derived antigen-specific Foxp3+ regulatory T (Treg) cells. Unlike deletional tolerance, Treg cell-mediated tolerance was broken by successive antigen challenges. These findings reveal that for some tissue-restricted self antigens, tolerance relies entirely on nondeletional mechanisms that are less durable than T cell deletion. This might explain why autoimmunity is often tissue-specific, and it offers a rationale for cancer vaccine strategies targeting tissue-restricted tumor antigens.

  6. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system. PMID:26481477

  7. In vivo maintenance of T-lymphocyte unresponsiveness induced by thymic medullary epithelium requires antigen presentation by radioresistant cells

    PubMed Central

    Hudrisier, Denis; Feau, Sonia; Bonnet, Véronique; Romagnoli, Paola; Van Meerwijk, Joost P M

    2003-01-01

    The T-cell repertoire developing in the thymus is rid of autospecific cells by the process of thymic negative selection. Recognition of major histocompatibility complex (MHC)/self-peptide complexes expressed by thymic antigen-presenting cells (APC) of bone marrow origin leads to induction of apoptotic death of autospecific thymocytes. Induction of tolerance to self-antigens not presented by thymic APC is mediated by medullary thymic epithelial cells (mTEC) which express a very wide range of proteins, e.g. inducible and tissue-specific proteins. The main type of tolerance induced by mTEC is non-deletional and the issue of how it is maintained outside the thymus is therefore of crucial interest. We have previously shown that the non-T-cell receptor (TCR) -transgenic T-cell repertoire developing in conditions in which tolerance to self-MHC/peptide ligands is exclusively induced by mTEC is tolerant to syngeneic targets in vivo but lyses such targets in vitro. Here we report that this non-deletional in vivo self-tolerance is not due to active tolerance assured by known naturally occurring regulatory or immune-modulating T lymphocytes. Importantly, we show that in vivo maintenance of this therefore probably anergic state requires continued interaction of autospecific T cells with self-MHC/peptide ligands expressed by radioresistant cells while APC are incapable of maintaining the tolerant state. Therefore, maintenance of non-deletional T-lymphocyte tolerance to the wide range of self-antigens expressed by mTEC depends on continued interaction with radioresistant cells that very probably express a much more limited repertoire of antigens. Our data may therefore have important consequences for tolerance to tissue-specific and inducible self-antigens. PMID:12519299

  8. Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

    PubMed Central

    Su, Laura F.; del Alcazar, Daniel; Stelekati, Erietta; Wherry, E. John; Davis, Mark M.

    2016-01-01

    The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide–MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity. PMID:27681619

  9. Mechanism of effector-cell blockade. I. Antigen-induced suppression of Ig synthesis in a hybridoma cell line, and correlation with cell- associated antigen

    PubMed Central

    1980-01-01

    A mouse hybridoma cell line, FluIgM-1, which secretes IgM specific for the hapten fluorescein (FLU) was developed to allow detailed analysis of the effector-cell blockade (ECB) phenomenon, in which contact of antibody-forming cells (AFC) with specific antigen results in marked reduction of antibody secretion. Treatment of hybridoma cells with highly substituted FLU conjugates (e.g., Flu20gelatin) resulted in inhibition of plaque formation. The data indicated close parallels with the ECB of normal spleen AFC, both in speed of onset and the dose of antigen required. The inhibition of antibody secretion was confirmed with a biosynthetic-labeling procedure which demonstrated that this was a result of reduced Ig synthesis. The inhibitory effect appeared to be confined to antibody synthesis, in the total protein synthesis, DNA synthesis, and cell-doubling times were unaffected. The association of FLU conjugates with the cells during and following ECB was studied directly using fluorescence microscopy and the fluorescence-activated cell sorter. These experiments showed that FLU conjugates capable of causing blockade aggregated on the cell surface, that the clearance of cell-associated antigen correlated with recovery from ECB, and that at all times when cell associated antigen was detectable, a portion remained bound to the cell surface and was susceptible to enzymatic removal. The latter observations supported previous findings suggesting that ECB was mediated by extracellular antigen. The direct observation of aggregates of antigen on the surface of blockaded cells is consistent with a mechanism involving cross-linking of Ig receptors. Finally, Fc receptors were not present on hybridoma cells, excluding their involvement in induction of ECB. PMID:7381364

  10. Latent Membrane Protein LMP2A Impairs Recognition of EBV-Infected Cells by CD8+ T Cells.

    PubMed

    Rancan, Chiara; Schirrmann, Leah; Hüls, Corinna; Zeidler, Reinhard; Moosmann, Andreas

    2015-06-01

    The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.

  11. Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

    PubMed Central

    Dhiman, Monisha; Zago, Maria Paola; Nunez, Sonia; Amoroso, Alejandro; Rementeria, Hugo; Dousset, Pierre; Burgos, Federico Nunez; Garg, Nisha Jain

    2012-01-01

    Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD50 sensitivity to 30 µM 4-HNE and 100 µM H2O2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H2O2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We

  12. CD94-NKG2A Recognition of Human Leukocyte Antigen (HLA)-E Bound to an HLA Class I Leader Sequence

    SciTech Connect

    Petrie,E.; Clements, C.; Lin, J.; Sullivan, L.; Johnson, D.; Huyton, T.; Heroux, A.; Hoare, H.; Beddoe, T.; et al

    2008-01-01

    The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A-HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a 'lock and key' interaction is typical of innate receptor-ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

  13. Analysis of cell surface antigens by Surface Plasmon Resonance imaging.

    PubMed

    Stojanović, Ivan; Schasfoort, Richard B M; Terstappen, Leon W M M

    2014-02-15

    Surface Plasmon Resonance (SPR) is most commonly used to measure bio-molecular interactions. SPR is used significantly less frequent for measuring whole cell interactions. Here we introduce a method to measure whole cells label free using the specific binding of cell surface antigens expressed on the surface of cancer cells and specific ligands deposited on sensor chips using an IBIS MX96 SPR imager (SPRi). As a model system, cells from the breast cancer cell line HS578T, SKBR3 and MCF7 were used. SPRi responses to Epithelial Cell Adhesion Molecule (EpCAM) antibody and other ligands coated on the sensor chips were measured. SPR curves show a response attributable to the sedimentation of the cells and a specific binding response on top of the initial response, the magnitude of which is dependent on the ligand density and the cell type used. Comparison of SPRi with flow cytometry showed similar EpCAM expression on MCF7, SKBR3 and HS578T cells.

  14. Antigen specific killing assay using CFSE labeled target cells.

    PubMed

    Durward, Marina; Harms, Jerome; Splitter, Gary

    2010-11-09

    Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

  15. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

    PubMed Central

    Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

    2015-01-01

    The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

  16. Innate recognition of cell wall β-glucans drives invariant Natural Killer T (iNKT) cell responses against fungi

    PubMed Central

    Cohen, Nadia R.; Tatituri, Raju V.V.; Rivera, Amariliz; Watts, Gerald F.M.; Kim, Edy Y.; Chiba, Asako; Fuchs, Beth B.; Mylonakis, Eleftherios; Besra, Gurdyal S.; Levitz, Stuart M.; Brigl, Manfred; Brenner, Michael B.

    2016-01-01

    SUMMARY iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids, and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the anti-fungal iNKT cell response does not require fungal lipids. Instead, Dectin-1 and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma and Alternaria, suggesting that this mechanism may broadly define the basis for anti-fungal iNKT cell responses. PMID:22100160

  17. Autologous tumor cells engineered to express bacterial antigens.

    PubMed

    Ramiya, Vijayakumar K; Jerald, Maya M; Lawman, Patricia D; Lawman, Michael J P

    2014-01-01

    Cancer immunotherapies are emerging as promising treatment modalities in the management of the disease. As a result, cancer vaccines are considered to be immensely crucial in preventing recurrence, a well-known nemesis in cancer patients because they have the potential to activate memory antitumor immunity. Due to poor antigenicity and self-tolerance, most tumor antigens require interventional vaccine therapies to provide an adequate "danger" signal to the immune system in order to activate a robust, clinically meaningful antitumor immunity. It has been postulated that this requirement may be achieved by providing bacterial and/or viral immunogens to prime this type of immune response. Briefly, we provide here a method of transfecting whole tumor cells with plasmid DNA encoding an immunogenic bacterial protein such as Emm55, which was derived from Streptococcus pyogenes (S. pyogenes). Subsequent inactivation of the transfected cells by irradiation (100 Gray) prevents replication. This type of whole-cell vaccine, e.g., ImmuneFx™, has demonstrated activity in a murine neuroblastoma model, in canine lymphoma patients with naturally occurring disease, and in many cancer types in companion animals. The protocols described in this chapter provide the necessary materials and methodologies to manufacture such a vaccine.

  18. Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

    PubMed Central

    Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

    2016-01-01

    Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

  19. Defining the HLA class I-associated viral antigen repertoire from HIV-1-infected human cells.

    PubMed

    Ternette, Nicola; Yang, Hongbing; Partridge, Thomas; Llano, Anuska; Cedeño, Samandhy; Fischer, Roman; Charles, Philip D; Dudek, Nadine L; Mothe, Beatriz; Crespo, Manuel; Fischer, William M; Korber, Bette T M; Nielsen, Morten; Borrow, Persephone; Purcell, Anthony W; Brander, Christian; Dorrell, Lucy; Kessler, Benedikt M; Hanke, Tomáš

    2016-01-01

    Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T-cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4(+) T cells and the C8166 human T-cell line. Importantly, one-third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T-cell responses have previously been reported but for which the precise HLA class I-binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T-cell vaccine development. PMID:26467324

  20. Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity.

    PubMed

    Schirmer, David; Grünewald, Thomas G P; Klar, Richard; Schmidt, Oxana; Wohlleber, Dirk; Rubío, Rebeca Alba; Uckert, Wolfgang; Thiel, Uwe; Bohne, Felix; Busch, Dirk H; Krackhardt, Angela M; Burdach, Stefan; Richter, Günther H S

    2016-06-01

    Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8(+) T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1(130) peptide-driven stimulations with HLA-A*02:01(+) dendritic cells (DC), allo-restricted HLA-A*02:01(-) CD8(+) T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01(-) primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2(-/-)γc(-/-) mouse model. Initially generated transgenic T cells specifically recognized STEAP1(130)-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01(+) ES lines more effectively than HLA-A*02:01(-) ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1(130)-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01(+) ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors. PMID:27471654

  1. Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes. II. Variant DK1: evidence for a discrete CTL recognition region.

    PubMed

    Krangel, M S; Biddison, W E; Strominger, J L

    1983-04-01

    Multiple amino acid sequence differences distinguish individual HLA antigens. Those residues important in immune recognition events have not been defined. Recent studies have identified HLA-A2 structural variants that, although serologically indistinguishable from other HLA-A2 antigens, are recognized poorly, if at all, by HLA-A2-restricted, influenza virus-immune, or HLA-A2-specific alloimmune CTL. In this study we utilize double-label tryptic peptide comparisons performed by both reverse-phase HPLC and cation exchange chromatography, in conjunction with conventional and microsequence analysis, to characterize the HLA-A2 heavy chains derived from variant DK1. We detect a single tryptic peptide that distinguishes DK1 HLA-A2 from the predominant HLA-A2 heavy chain species. This peptide spans residues 147 to 157 in the second heavy chain domain, and carries substitutions at positions 149, 152, and 156. Residues in this segment of the polypeptide are also altered in another HLA-A2 variant, as well as one H-2Kb mutant. Thus, this segment appears to be critical in forming determinants important in CTL recognition of class I antigens in general. On the basis of these and other results, we suggest that in contrast to recognition by alloantibodies, a discrete region of class I antigens may be crucial for CTL recognition. PMID:6601143

  2. Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.

    PubMed Central

    Zisman, E; Dayan, M; Sela, M; Mozes, E

    1993-01-01

    Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen. Images PMID:8381541

  3. Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

    PubMed

    Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A

    1999-10-22

    The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the

  4. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    PubMed

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-01

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera. PMID:27386892

  5. A side-by-side comparison of T cell reactivity to fifty-nine Mycobacterium tuberculosis antigens in diverse populations from five continents.

    PubMed

    Carpenter, Chelsea; Sidney, John; Kolla, Ravi; Nayak, Kaustuv; Tomiyama, Helena; Tomiyama, Claudia; Padilla, Oscar A; Rozot, Virginie; Ahamed, Syed F; Ponte, Carlos; Rolla, Valeria; Antas, Paulo R; Chandele, Anmol; Kenneth, John; Laxmi, Seetha; Makgotlho, Edward; Vanini, Valentina; Ippolito, Giuseppe; Kazanova, Alexandra S; Panteleev, Alexander V; Hanekom, Willem; Mayanja-Kizza, Harriet; Lewinsohn, David; Saito, Mayuko; McElrath, M Juliana; Boom, W Henry; Goletti, Delia; Gilman, Robert; Lyadova, Irina V; Scriba, Thomas J; Kallas, Esper G; Murali-Krishna, Kaja; Sette, Alessandro; Lindestam Arlehamn, Cecilia S

    2015-12-01

    We compared T cell recognition of 59 prevalently recognized Mycobacterium tuberculosis (MTB) antigens in individuals latently infected with MTB (LTBI), and uninfected individuals with previous BCG vaccination, from nine locations and populations with different HLA distribution, MTB exposure rates, and standards of TB care. This comparison revealed similar response magnitudes in diverse LTBI and BCG-vaccinated cohorts and significant correlation between responses in LTBIs from the USA and other locations. Many antigens were uniformly recognized, suggesting suitability for inclusion in vaccines targeting diverse populations. Several antigens were similarly immunodominant in LTBI and BCG cohorts, suggesting applicability for vaccines aimed at boosting BCG responses. The panel of MTB antigens will be valuable for characterizing MTB-specific CD4 T cell responses irrespective of ethnicity, infecting MTB strains and BCG vaccination status. Our results illustrate how a comparative analysis can provide insight into the relative immunogenicity of existing and novel vaccine candidates in LTBIs.

  6. [Relation between Ia antigens and Ir gene products. A theory on the development of the immune response].

    PubMed

    Seignalet, J

    1983-11-01

    To explain the relations between Ia and Ir, we propose the following hypothesis. It would exist an antigen specific factor released by T lymphocytes. This factor would be constituted by two parts: a variable fragment forming the antigenic receptor of T cells and coded by Ir genes, a constant fragment transmitting a helper or suppressor signal and carrying Ia antigens. Ia antigens would allow a mutual recognition between macrophages, T lymphocytes and B lymphocytes and a recognition by these cells of some mediators released during immune response. Ir genes products would allow the antigen recognition, if they correspond to the antigenic receptor of T lymphocytes.

  7. [Relations between Ia antigens and the products of Ir genes. A theory of the evolution of the immune response].

    PubMed

    Seignalet, J

    1983-01-01

    To explain the relations between Ia and Ir, we propose the following hypothesis. It would exist an antigen specific factor released by T lymphocytes. This factor would be constituted by two parts: a variable fragment forming the antigenic receptor of T cells and coded by Ir genes, a constant fragment transmitting an helper or suppressor signal and carrying Ia antigens. Ia antigens would allow a mutual recognition between macrophages, T lymphocytes and B lymphocytes and a recognition by these cells of some mediators released during immune response. Ir genes products would allow the antigen recognition, if they correspond to the antigenic receptor of T lymphocytes.

  8. Cell surface origin of antigens shed by Leishmania donovani during growth in axenic culture.

    PubMed Central

    Kaneshiro, E S; Gottlieb, M; Dwyer, D M

    1982-01-01

    Antisera against isolated cell surface preparations (PCSP-As) of Leishmania donovani promastigotes were used to detect extracellular antigens produced during the growth of these organisms in four different growth media. The PCSP-As precipitated two major antigenically identical but electrophoretically distinct components, in addition to several minor antigens. Immunoelectrophoretic studies employing PCSP-As, PCSP-As absorbed with intact, live promastigotes, and PCSP-As absorbed with a major extracellular antigen demonstrated the antigenic identity between the major extracellular antigens and two major components externally disposed at the surface of promastigotes. Growth curve kinetic investigations suggested that the major extracellular antigens did not appear in the growth media primarily as a result of cell lysis or damage. The carbohydrate nature of the major extracellular antigens was indicated by physicochemical characterization. Images PMID:7118250

  9. VH and VL Domains of Polyspecific IgM and Monospecific IgG Antibodies Contribute Differentially to Antigen Recognition and Virus Neutralization Functions.

    PubMed

    Pasman, Y; Kaushik, A K

    2016-07-01

    We analysed contributions of variable heavy (FdVH ) and variable light (FdVL ) domains in comparison to scFv (FdVH +FdVL ) of naturally occurring polyspecific bovine IgM with an exceptionally long CDR3H and an induced monospecific bovine herpes virus-1 (BoHV-1) neutralizing IgG1 antibody in the context of to antigen-binding site and antibody function. Various recombinant FdVH , FdVL and scFv were constructed and expressed in Pichia pastoris from the bovine IgM and IgG1 antibody encoding cDNA. The scFv1H12 showed polyspecific antigen binding similar to parent IgM antibody, though subtle differences, for example, higher thyroglobulin recognition. Such differences reflect influence of the constant region on the antigen-binding site configuration. Unlike, variable light domain FdVL 1H12, the variable heavy domain FdVH 1H12 alone recognized multiple antigens that differed from the recognition pattern of scFv1H12 (FdVH +FdVL ) and the parent IgM antibody. Nonetheless, role of FdVL 1H12 in providing structural support to FdVH in antigen recognition is noted, apart from its intrinsic antigen recognition ability. Surface plasmon resonance analysis revealed low to moderate affinity of scFv1H12 to IgG antigen. By contrast, the individual FdVH 073 and FdVL 074, originating from induced BoHV-1 neutralizing IgG1 antibody, recognized target epitope on BoHV-1 weakly when compared to FdVH +FdVL (scFv3-18L). Interestingly, both the FdVH and FdVL domains of induced IgG antibody are required to achieve BoHV-1 neutralization. To conclude, there exist subtle functional differences in the contribution of FdVH and FdVL to antigen-binding site generation of polyspecific IgM and monospecific IgG antibodies relevant to antigen recognition and virus neutralization functions.

  10. CD8 T-cell recognition of acquired alloantigen promotes acute allograft rejection

    PubMed Central

    Harper, Simon J. F.; Ali, Jason M.; Wlodek, Elizabeth; Negus, Marg C.; Harper, Ines G.; Chhabra, Manu; Qureshi, M. Saeed; Mallik, Mekhola; Bolton, Eleanor; Bradley, J. Andrew; Pettigrew, Gavin J.

    2015-01-01

    Adaptive CD8 T-cell immunity is the principal arm of the cellular alloimmune response, but its development requires help. This can be provided by CD4 T cells that recognize alloantigen “indirectly,” as self-restricted allopeptide, but this process remains unexplained, because the target epitopes for CD4 and CD8 T-cell recognition are “unlinked” on different cells (recipient and donor antigen presenting cells (APCs), respectively). Here, we test the hypothesis that the presentation of intact and processed MHC class I alloantigen by recipient dendritic cells (DCs) (the “semidirect” pathway) allows linked help to be delivered by indirect-pathway CD4 T cells for generating destructive cytotoxic CD8 T-cell alloresponses. We show that CD8 T-cell–mediated rejection of murine heart allografts that lack hematopoietic APCs requires host secondary lymphoid tissue (SLT). SLT is necessary because within it, recipient dendritic cells can acquire MHC from graft parenchymal cells and simultaneously present it as intact protein to alloreactive CD8 T cells and as processed peptide alloantigen for recognition by indirect-pathway CD4 T cells. This enables delivery of essential help for generating cytotoxic CD8 T-cell responses that cause rapid allograft rejection. In demonstrating the functional relevance of the semidirect pathway to transplant rejection, our findings provide a solution to a long-standing conundrum as to why SLT is required for CD8 T-cell allorecognition of graft parenchymal cells and suggest a mechanism by which indirect-pathway CD4 T cells provide help for generating effector cytotoxic CD8 T-cell alloresponses at late time points after transplantation. PMID:26420874

  11. Fever-range whole-body heat treatment stimulates antigen-specific T-cell responses in humans.

    PubMed

    Kobayashi, Yasunobu; Ito, Yusuke; Ostapenko, Valentina V; Sakai, Mayuko; Matsushita, Norimasa; Imai, Kenichiro; Shimizu, Koichi; Aruga, Atsushi; Tanigawa, Keishi

    2014-11-01

    Increase in body temperature has been thought to play an important role in the regulation of immune responses, although its precise mechanisms are still under investigation. Here, we examined the effects of physiologically relevant thermal stress on the cytokine production from human peripheral T cells. Volunteers were heated using a whole-body hyperthermia device, the rectal temperature was maintained above 38.5 °C for more than 60 min, and peripheral blood mononuclear cells (PBMCs) were obtained before and after the treatment. When T cells were stimulated with anti-CD3/CD28 antibodies, marked increases in the production of interferon-γ (IFN-γ) and interleukin-2 were observed in PBMCs prepared immediately after and 24h after the treatment. Similarly, enhanced production of IFN-γ in response to the tuberculin purified protein derivative or antigenic viral peptides was also observed immediately after and 24h after the treatment. Fluorescence photo-bleaching analyses showed heat-induced increase of membrane fluidity in T cells, which probably enables them to induce rapid and efficient cluster formation of molecules involved in antigen recognition and signal transduction for T-cell stimulation. We concluded that physiologically relevant thermal stress could efficiently modify T-cell responsiveness to various stimuli, including enhanced responses to specific antigens.

  12. Fluorine substitutions in an antigenic peptide selectively modulate T-cell receptor binding in a minimally perturbing manner

    SciTech Connect

    Piepenbrink, Kurt H.; Borbulevych, Oleg Y.; Sommese, Ruth F.; Clemens, John; Armstrong, Kathryn M.; Desmond, Clare; Do, Priscilla; Baker, Brian M.

    2010-08-17

    TCR (T-cell receptor) recognition of antigenic peptides bound and presented by MHC (major histocompatibility complex) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide - MHC complexes weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Furthermore, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. In the present study, we examined how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the Tax peptide [HTLV (human T-cell lymphotropic virus)-1 Tax] enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with the A6 TCR independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, we probed the mechanism of fluorine's effect on TCR binding and we conclude that our results were most consistent with a 'polar hydrophobicity' mechanism, rather than a purely hydrophobic- or electrostatic-based mechanism. This finding should have an impact on other attempts to alter molecular recognition with fluorine.

  13. An Overview of B-1 Cells as Antigen-Presenting Cells

    PubMed Central

    Popi, Ana F.; Longo-Maugéri, Ieda M.; Mariano, Mario

    2016-01-01

    The role of B cells as antigen-presenting cells (APCs) has been extensively studied, mainly in relation to the activation of memory T cells. Considering the B cell subtypes, the role of B-1 cells as APCs is beginning to be explored. Initially, it was described that B-1 cells are activated preferentially by T-independent antigens. However, some reports demonstrated that these cells are also involved in a T-dependent response. The aim of this review is to summarize information about the ability of B-1 cells to play a role as APCs and to briefly discuss the role of the BCR and toll-like receptor signals in this process. Furthermore, some characteristics of B-1 cells, such as natural IgM production and phagocytic ability, could interfere in the participation of these cells in the onset of an adaptive response. PMID:27148259

  14. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

    PubMed Central

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-01-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c+, CD141+ and CD16+ myeloid DCs and CD123+ plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141+ DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  15. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

    PubMed

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-06-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  16. Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

    PubMed

    Jiang, Wenzhi; Rosenberg, Julian N; Wauchope, Akelia D; Tremblay, Jacqueline M; Shoemaker, Charles B; Weeks, Donald P; Oyler, George A

    2013-11-01

    Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

  17. Dietary antigens limit mucosal immunity by inducing regulatory T cells in the small intestine.

    PubMed

    Kim, Kwang Soon; Hong, Sung-Wook; Han, Daehee; Yi, Jaeu; Jung, Jisun; Yang, Bo-Gie; Lee, Jun Young; Lee, Minji; Surh, Charles D

    2016-02-19

    Dietary antigens are normally rendered nonimmunogenic through a poorly understood "oral tolerance" mechanism that involves immunosuppressive regulatory T (Treg) cells, especially Treg cells induced from conventional T cells in the periphery (pTreg cells). Although orally introducing nominal protein antigens is known to induce such pTreg cells, whether a typical diet induces a population of pTreg cells under normal conditions thus far has been unknown. By using germ-free mice raised and bred on an elemental diet devoid of dietary antigens, we demonstrated that under normal conditions, the vast majority of the small intestinal pTreg cells are induced by dietary antigens from solid foods. Moreover, these pTreg cells have a limited life span, are distinguishable from microbiota-induced pTreg cells, and repress underlying strong immunity to ingested protein antigens.

  18. Identification of a potent microbial lipid antigen for diverse Natural Killer T cells1

    PubMed Central

    Wolf, Benjamin J.; Tatituri, Raju V. V.; Almeida, Catarina F.; Le Nours, Jérôme; Bhowruth, Veemal; Johnson, Darryl; Uldrich, Adam P.; Hsu, Fong-Fu; Brigl, Manfred; Besra, Gurdyal S.; Rossjohn, Jamie; Godfrey, Dale I.; Brenner, Michael B.

    2016-01-01

    Invariant Natural Killer T (iNKT) cells are a well-characterized CD1d-restricted T cell subset. The availability of potent antigens and tetramers for iNKT cells has allowed this population to be extensively studied and has revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse Natural Killer T (dNKT) cells are poorly understood because the lipid antigens they recognize are largely unknown. We sought to identify dNKT cell lipid antigen(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol (PG) as a microbial antigen that was significantly more potent than a previously characterized dNKT cell antigen, mammalian PG. Further, while mammalian PG loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived PG loaded tetramers did. The structure of Listeria PG was distinct from mammalian PG since it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d binding lipid displacement studies revealed that the microbial PG antigen binds significantly better to CD1d than counterparts with the same headgroup. These data reveal a highly-potent microbial lipid antigen for a subset of dNKT cells and provide an explanation for its increased antigen potency compared to the mammalian counterpart. PMID:26254340

  19. Modes of Antigen Presentation by Lymph Node Stromal Cells and Their Immunological Implications

    PubMed Central

    Hirosue, Sachiko; Dubrot, Juan

    2015-01-01

    Antigen presentation is no longer the exclusive domain of cells of hematopoietic origin. Recent works have demonstrated that lymph node stromal cell (LNSC) populations, such as fibroblastic reticular cells, lymphatic and blood endothelial cells, not only provide a scaffold for lymphocyte interactions but also exhibit active immunomodulatory roles that are critical to mounting and resolving effective immune responses. Importantly, LNSCs possess the ability to present antigens and establish antigen-specific interactions with T cells. One example is the expression of peripheral tissue antigens, which are presented on major histocompatibility complex (MHC)-I molecules with tolerogenic consequences on T cells. Additionally, exogenous antigens, including self and tumor antigens, can be processed and presented on MHC-I complexes, which result in dysfunctional activation of antigen-specific CD8+ T cells. While MHC-I is widely expressed on cells of both hematopoietic and non-hematopoietic origins, antigen presentation via MHC-II is more precisely regulated. Nevertheless, LNSCs are capable of endogenously expressing, or alternatively, acquiring MHC-II molecules. Transfer of antigen between LNSC and dendritic cells in both directions has been recently suggested to promote tolerogenic roles of LNSCs on the CD4+ T cell compartment. Thus, antigen presentation by LNSCs is thought to be a mechanism that promotes the maintenance of peripheral tolerance as well as generates a pool of diverse antigen-experienced T cells for protective immunity. This review aims to integrate the current and emerging literature to highlight the importance of LNSCs in immune responses, and emphasize their role in antigen trafficking, retention, and presentation. PMID:26441957

  20. Direct tumor recognition by a human CD4+ T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses

    PubMed Central

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel F.; Shiku, Hiroshi; Mineno, Junichi; Okamoto, Sachiko; Old, Lloyd J.; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2015-01-01

    Tumor antigen-specific CD4+ T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4+ T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize anti-tumor effects of a unique human CD4+ helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4+ T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8+ T cells by enhancing cytotoxic activity, and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8+ T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TR-CD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity, and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients. PMID:26447332

  1. Cell-type specific regulation of gene expression by simian virus 40 T antigens

    SciTech Connect

    Cantalupo, Paul G.; Saenz-Robles, Maria Teresa; Rathi, Abhilasha V.; Beerman, Rebecca W.; Patterson, William H.; Whitehead, Robert H.; Pipas, James M.

    2009-03-30

    SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.

  2. Dendritic cell preactivation impairs MHC class II presentation of vaccines and endogenous viral antigens

    PubMed Central

    Young, Louise J.; Wilson, Nicholas S.; Schnorrer, Petra; Mount, Adele; Lundie, Rachel J.; La Gruta, Nicole L.; Crabb, Brendan S.; Belz, Gabrielle T.; Heath, William R.; Villadangos, Jose A.

    2007-01-01

    When dendritic cells (DCs) encounter signals associated with infection or inflammation, they become activated and undergo maturation. Mature DCs are very efficient at presenting antigens captured in association with their activating signal but fail to present subsequently encountered antigens, at least in vitro. Such impairment of MHC class II (MHC II) antigen presentation has generally been thought to be a consequence of down-regulation of endocytosis, so it might be expected that antigens synthesized by the DCs themselves (for instance, viral antigens) would still be presented by mature DCs. Here, we show that DCs matured in vivo could still capture and process soluble antigens, but were unable to present peptides derived from these antigens. Furthermore, presentation of viral antigens synthesized by the DCs themselves was also severely impaired. Indeed, i.v. injection of pathogen mimics, which caused systemic DC activation in vivo, impaired the induction of CD4 T cell responses against subsequently encountered protein antigens. This immunosuppressed state could be reversed by adoptive transfer of DCs loaded exogenously with antigens, demonstrating that impairment of CD4 T cell responses was due to lack of antigen presentation rather than to overt suppression of T cell activation. The biochemical mechanism underlying this phenomenon was the down-regulation of MHC II–peptide complex formation that accompanied DC maturation. These observations have important implications for the design of prophylactic and therapeutic DC vaccines and contribute to the understanding of the mechanisms causing immunosuppression during systemic blood infections. PMID:17978177

  3. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  4. Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy.

    PubMed

    Mirzaei, Hamid Reza; Mirzaei, Hamed; Lee, Sang Yun; Hadjati, Jamshid; Till, Brian G

    2016-10-01

    Excitement is growing for therapies that harness the power of patients' immune systems to combat their diseases. One approach to immunotherapy involves engineering patients' own T cells to express a chimeric antigen receptor (CAR) to treat advanced cancers, particularly those refractory to conventional therapeutic agents. Although these engineered immune cells have made remarkable strides in the treatment of patients with certain hematologic malignancies, success with solid tumors has been limited, probably due to immunosuppressive mechanisms in the tumor niche. In nearly all studies to date, T cells bearing αβ receptors have been used to generate CAR T cells. In this review, we highlight biological characteristics of γδ T cells that are distinct from those of αβ T cells, including homing to epithelial and mucosal tissues and unique functions such as direct antigen recognition, lack of alloreactivity, and ability to present antigens. We offer our perspective that these features make γδ T cells promising for use in cellular therapy against several types of solid tumors, including melanoma and gastrointestinal cancers. Engineered γδ T cells should be considered as a new platform for adoptive T cell cancer therapy for mucosal tumors.

  5. Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy.

    PubMed

    Mirzaei, Hamid Reza; Mirzaei, Hamed; Lee, Sang Yun; Hadjati, Jamshid; Till, Brian G

    2016-10-01

    Excitement is growing for therapies that harness the power of patients' immune systems to combat their diseases. One approach to immunotherapy involves engineering patients' own T cells to express a chimeric antigen receptor (CAR) to treat advanced cancers, particularly those refractory to conventional therapeutic agents. Although these engineered immune cells have made remarkable strides in the treatment of patients with certain hematologic malignancies, success with solid tumors has been limited, probably due to immunosuppressive mechanisms in the tumor niche. In nearly all studies to date, T cells bearing αβ receptors have been used to generate CAR T cells. In this review, we highlight biological characteristics of γδ T cells that are distinct from those of αβ T cells, including homing to epithelial and mucosal tissues and unique functions such as direct antigen recognition, lack of alloreactivity, and ability to present antigens. We offer our perspective that these features make γδ T cells promising for use in cellular therapy against several types of solid tumors, including melanoma and gastrointestinal cancers. Engineered γδ T cells should be considered as a new platform for adoptive T cell cancer therapy for mucosal tumors. PMID:27392648

  6. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    PubMed

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway.

  7. The response of human dendritic cells to co-ligation of pattern-recognition receptors.

    PubMed

    Dzopalic, Tanja; Rajkovic, Ivan; Dragicevic, Ana; Colic, Miodrag

    2012-04-01

    Dendritic cells (DCs) are key antigen-presenting cells that express a wide variety of pattern-recognition receptors (PRRs). Triggering of a single PRR, especially Toll-like receptors (TLRs) and C-type lectins, induces maturation of DCs, but cooperativity between multiple PRRs is needed in order to achieve an effective immune response. In this review, we summarize the published data related to the effect of individual and joint PRR agonists on DCs and Langerhans-like cells derived from monocytes (MoDCs and MoLCs, respectively). Our results demonstrate that MoDCs co-stimulated with TLR3/TLR7 and TLR3/Dectin-1 ligands induced superior T helper (Th)1 and Th17 immune responses, compared to effects of single agonists. The opposite outcome was observed after co-ligation of TLR3 and Langerin on MoLCs. These findings may be relevant to improve strategy for tumor immunotherapy.

  8. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells.

    PubMed

    Epel, Malka; Ellenhorn, Joshua D; Diamond, Don J; Reiter, Yoram

    2002-11-01

    Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy. PMID:12384808

  9. Antigenic peptide molecular recognition by the DRB1-DQB1 haplotype modulates multiple sclerosis susceptibility.

    PubMed

    Kumar, Amit; Melis, Paola; Genna, Vito; Cocco, Eleonora; Marrosu, Maria Giovanna; Pieroni, Enrico

    2014-08-01

    Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that has a notably high incidence in Sardinia. Our study focuses on two HLA class II haplotypes associated with the disease in Sardinia, the rare predisposing DRB1*15:01-DQB1*06:02 and the widespread protective DRB1*16:01-DQB1*05:02. This framework enabled the highlighting of HLA binding pocket specificity and peptide recognition mechanisms by employing molecular dynamics simulations of the whole DRB1-DQB1 haplotype interacting with MBP- and EBV-derived peptides. We analyzed peptide-protein interaction networks and temporal evolution of the original complexes and after key amino acid mutations. The mutation G86V of the protective DRB1 allele exerted its effect mainly in the presence of the EBV viral peptide, with local and long range outcomes. However, the V38A mutation of the protective DQB1 showed a long range effect only in the case of the MBP myelin peptide. Our findings also demonstrate a DRB1/DQB1 complementary molecular recognition of peptides. This mechanism could provide a robust synergistic action and a differential role of DRB1 and DQB1 in tissues and in the time-steps towards autoimmunity. In addition, we demonstrate that negatively charged residues in pockets 4 and 9 play a role in MS susceptibility. Our findings are supported by recent experiments using a closely related MS animal model. Overall, our analysis confirms the role of the DRB1-DQB1 haplotype in conferring disease predisposition and could provide a valuable aid in designing optimal therapeutic peptides for MS therapy.

  10. The cell proliferation antigen Ki-67 organises heterochromatin

    PubMed Central

    Sobecki, Michal; Mrouj, Karim; Camasses, Alain; Parisis, Nikolaos; Nicolas, Emilien; Llères, David; Gerbe, François; Prieto, Susana; Krasinska, Liliana; David, Alexandre; Eguren, Manuel; Birling, Marie-Christine; Urbach, Serge; Hem, Sonia; Déjardin, Jérôme; Malumbres, Marcos; Jay, Philippe; Dulic, Vjekoslav; Lafontaine, Denis LJ; Feil, Robert; Fisher, Daniel

    2016-01-01

    Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001 PMID:26949251

  11. Antigens in tea-beverage prime human Vγ2Vδ2 T cells in vitro and in vivo for memory and nonmemory antibacterial cytokine responses

    PubMed Central

    Kamath, Arati B.; Wang, Lisheng; Das, Hiranmoy; Li, Lin; Reinhold, Vernon N.; Bukowski, Jack F.

    2003-01-01

    Human γδ T cells mediate innate immunity to microbes via T cell receptor-dependent recognition of unprocessed antigens with conserved molecular patterns. These nonpeptide alkylamine antigens are shared by tumor cells, bacteria, parasites, and fungi but also by edible plant products such as tea, apples, mushrooms, and wine. Here we show that priming of γδ T cells with alkylamine antigens in vitro results in a memory response to these antigens. Such priming results also in a nonmemory response to whole bacteria and to lipopolysaccharide, characterized by IL-12-dependent secretion of IFN-γ by γδ T cells and by γδ T cell proliferation. Drinking tea, which contains l-theanine, a precursor of the nonpeptide antigen ethylamine, primed peripheral blood γδ T cells to mediate a memory response on reexposure to ethylamine and to secrete IFN-γ in response to bacteria. This unique combination of innate immune response and immunologic memory shows that γδ T cells can function as a bridge between innate and acquired immunity. In addition, these data provide an explanation for the health benefits of tea. PMID:12719524

  12. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells(1). Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules(2). NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity(3-6), and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d (7). The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines(8). Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines(9,10). In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients(11). However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells(12, 13). Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is

  13. Activation of Type II Cells into Regenerative Stem Cell Antigen-1+ Cells during Alveolar Repair

    PubMed Central

    Kumar, Varsha Suresh; Zhang, Wei; Rehman, Jalees; Malik, Asrar B.

    2015-01-01

    The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa–induced lung injury, we identified a population of stem cell antigen (Sca)-1–expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1–expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C+Sca-1+ cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C+Sca-1+ cells in vitro. Thus, P. aeruginosa–mediated lung injury induces the generation of a Sca-1+ subset of type II cells. The progenitor phenotype of the Sp-C+Sca-1+ cells that mediates alveolar epithelial repair might involve Wnt signaling. PMID:25474582

  14. Recognition of a human T-lymphocyte differentiation antigen by an IgM monoclonal antibody.

    PubMed Central

    Bastin, J M; Granger, S; Tidman, N; Janossy, G; McMichael, A J

    1981-01-01

    A monoclonal antibody directed at a determinant on human T cells was produced and characterized. This IgM antibody, MBG6, bound to human peripheral blood T lymphocytes and to medullary thymocytes. It was unreactive with normal B cells, B-cell lines and granulocytes. Apart from T lymphocytes, bone marrow cells (including cells positive for the terminal transferase marker, myeloid colony-forming cells, myeloblasts, and differentiating myeloid and erythroid cells) were negative. Peripheral blood cells that were treated with MBG6 and rabbit complement were no longer capable of proliferating in response to phytohaemagglutinin or concanavalin A; MBG6 did not have any direct mitogenic action on T lymphocytes. Double immunofluorescence studies using IgM MBG6 and OKT3, and IgG2a monoclonal antibody that recognizes all peripheral T cells, showed that these two antibodies identified exactly the same cell populations. Competitive binding studies, however, indicated that MBG6 and OKT3 recognized different epitopes. The antibody may have clinical applications in bone marrow transplantation. PMID:6802543

  15. Clinical significance of serum carcinoembryonic antigen, carbohydrate antigen 19-9, and squamous cell carcinoma antigen levels in esophageal cancer patients.

    PubMed

    Kosugi, Shin-ichi; Nishimaki, Tadashi; Kanda, Tatsuo; Nakagawa, Satoru; Ohashi, Manabu; Hatakeyama, Katsuyoshi

    2004-07-01

    Serum carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, and squamous cell carcinoma (SCC) antigen levels were assessed to determine if their levels are useful for staging esophageal cancer preoperatively and for predicting patient survival after esophagectomy. Hence their seropositivity was investigated for a correlation with resectability, clinicopathologic parameters of tumor progression, and treatment outcomes in patients with unresectable esophageal cancer ( n = 63) and those undergoing esophagectomy for resectable disease ( n = 267). Abnormal elevation of serum SCC antigen levels showed a significant correlation with resectability ( p< 0.0001), depth of tumor invasion ( p < 0.0001), lymph node status ( p = 0.0015), TNM stage ( p < 0.0001), lymphatic invasion ( p = 0.0019), blood vessel invasion ( p = 0.0079), and poor survival after esophagectomy ( p = 0.0061). A significant relation ( p = 0.0145) was found between elevated serum CEA levels and distant metastasis, whereas the seropositivity of CA 19-9 showed no association with resectability, tumor progression, or patient survival. These results indicate that abnormal elevation of serum SCC antigen is a useful predictor of advanced esophageal cancer associated with poor survival after esophagectomy.

  16. Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells

    PubMed Central

    George-Chandy, Annie; Eriksson, Kristina; Lebens, Michael; Nordström, Inger; Schön, Emma; Holmgren, Jan

    2001-01-01

    Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response. PMID:11500448

  17. Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

    PubMed Central

    Carroll-Portillo, Amanda; Cannon, Judy L.; te Riet, Joost; Holmes, Anna; Kawakami, Yuko; Kawakami, Toshiaki; Cambi, Alessandra

    2015-01-01

    Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell–cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell–cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC–DC synapse suggest a new role for intercellular crosstalk in defining the immune response. PMID:26304724

  18. Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells

    PubMed Central

    Cavanagh, Lois L.; Bonasio, Roberto; Mazo, Irina B.; Halin, Cornelia; Cheng, Guiying; van der Velden, Adrianus W. M.; Cariappa, Annaiah; Chase, Catherine; Russell, Paul; Starnbach, Michael N.; Koni, Pandelakis A.; Pillai, Shiv; Weninger, Wolfgang; von Andrian, Ulrich H.

    2006-01-01

    Dendritic cells (DC) carry antigen from peripheral tissues via lymphatics to lymph nodes (LN). We report that differentiated DC can also travel from the periphery into the blood. Circulating DC migrated to the spleen, liver and lung, but not LN. They also homed to the bone marrow (BM) where they were better retained than in most other tissues. DC homing to the BM depended on constitutively expressed VCAM-1 and endothelial selectins in BM microvessels. Two-photon intravital microscopy in BM cavities revealed that DC formed stable antigen-dependent contacts with BM-resident central memory T cells. Moreover, using this novel migratory pathway, antigen-pulsed DC could trigger central memory T cell-mediated recall responses in the BM. PMID:16155571

  19. Antibody recognition in multiple sclerosis and Rett syndrome using a collection of linear and cyclic N-glucosylated antigenic probes.

    PubMed

    Real Fernández, Feliciana; Di Pisa, Margherita; Rossi, Giada; Auberger, Nicolas; Lequin, Olivier; Larregola, Maud; Benchohra, Amina; Mansuy, Christelle; Chassaing, Gerard; Lolli, Francesco; Hayek, Joussef; Lavielle, Solange; Rovero, Paolo; Mallet, Jean-Maurice; Papini, Anna Maria

    2015-09-01

    Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' β-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD  = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD  = 7.1 nM). PMID:25973844

  20. Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.

    PubMed Central

    Falo, L D; Benacerraf, B; Rock, K L

    1986-01-01

    The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed. PMID:3529095

  1. Ex vivo characterization and isolation of rare memory B cells with antigen tetramers.

    PubMed

    Franz, Bettina; May, Kenneth F; Dranoff, Glenn; Wucherpfennig, Kai

    2011-07-14

    Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer(+) B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

  2. Beta cell antigens in type 1 diabetes: triggers in pathogenesis and therapeutic targets

    PubMed Central

    Mauvais, François-Xavier; Diana, Julien; van Endert, Peter

    2016-01-01

    Research focusing on type 1 diabetes (T1D) autoantigens aims to explore our understanding of these beta cell proteins in order to design assays for monitoring the pathogenic autoimmune response, as well as safe and efficient therapies preventing or stopping it. In this review, we will discuss progress made in the last 5 years with respect to mechanistic understanding, diagnostic monitoring, and therapeutic modulation of the autoantigen-specific cellular immune response in T1D. Some technical progress in monitoring tools has been made; however, the potential of recent technologies for highly multiplexed exploration of human cellular immune responses remains to be exploited in T1D research, as it may be the key to the identification of surrogate markers of disease progression that are still wanting. Detailed analysis of autoantigen recognition by T cells suggests an important role of non-conventional antigen presentation and processing in beta cell-directed autoimmunity, but the impact of this in human T1D has been little explored. Finally, therapeutic administration of autoantigens to T1D patients has produced disappointing results. The application of novel modes of autoantigen administration, careful translation of mechanistic understanding obtained in preclinical studies and in vitro with human cells, and combination therapies including CD3 antibodies may help to make autoantigen-based immunotherapy for T1D a success story in the future. PMID:27158463

  3. Beta cell antigens in type 1 diabetes: triggers in pathogenesis and therapeutic targets.

    PubMed

    Mauvais, François-Xavier; Diana, Julien; van Endert, Peter

    2016-01-01

    Research focusing on type 1 diabetes (T1D) autoantigens aims to explore our understanding of these beta cell proteins in order to design assays for monitoring the pathogenic autoimmune response, as well as safe and efficient therapies preventing or stopping it. In this review, we will discuss progress made in the last 5 years with respect to mechanistic understanding, diagnostic monitoring, and therapeutic modulation of the autoantigen-specific cellular immune response in T1D. Some technical progress in monitoring tools has been made; however, the potential of recent technologies for highly multiplexed exploration of human cellular immune responses remains to be exploited in T1D research, as it may be the key to the identification of surrogate markers of disease progression that are still wanting. Detailed analysis of autoantigen recognition by T cells suggests an important role of non-conventional antigen presentation and processing in beta cell-directed autoimmunity, but the impact of this in human T1D has been little explored. Finally, therapeutic administration of autoantigens to T1D patients has produced disappointing results. The application of novel modes of autoantigen administration, careful translation of mechanistic understanding obtained in preclinical studies and in vitro with human cells, and combination therapies including CD3 antibodies may help to make autoantigen-based immunotherapy for T1D a success story in the future. PMID:27158463

  4. Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets.

    PubMed

    Fischbach, Hanna; Döring, Marius; Nikles, Daphne; Lehnert, Elisa; Baldauf, Christoph; Kalinke, Ulrich; Tampé, Robert

    2015-01-01

    Presentation of peptides on major histocompatibility complex class I (MHC I) is essential for the establishment and maintenance of self-tolerance, priming of antigen-specific CD8(+) T cells and the exertion of several T-cell effector functions. Cytosolic proteasomes continuously degrade proteins into peptides, which are actively transported across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). In the ER lumen antigenic peptides are loaded onto MHC I, which is displayed on the cell surface. Here we describe an innovative flow cytometric approach to monitor time-resolved ER compartmentalization of antigenic peptides. This assay allows the analysis of distinct primary human immune cell subsets at reporter peptide concentrations of 1 nM. Thus, this ultrasensitive method for the first time permits quantification of TAP activity under close to physiological conditions in scarce primary cell subsets such as antigen cross-presenting dendritic cells. PMID:25656091

  5. Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets

    PubMed Central

    Fischbach, Hanna; Döring, Marius; Nikles, Daphne; Lehnert, Elisa; Baldauf, Christoph; Kalinke, Ulrich; Tampé, Robert

    2015-01-01

    Presentation of peptides on major histocompatibility complex class I (MHC I) is essential for the establishment and maintenance of self-tolerance, priming of antigen-specific CD8+ T cells and the exertion of several T-cell effector functions. Cytosolic proteasomes continuously degrade proteins into peptides, which are actively transported across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). In the ER lumen antigenic peptides are loaded onto MHC I, which is displayed on the cell surface. Here we describe an innovative flow cytometric approach to monitor time-resolved ER compartmentalization of antigenic peptides. This assay allows the analysis of distinct primary human immune cell subsets at reporter peptide concentrations of 1 nM. Thus, this ultrasensitive method for the first time permits quantification of TAP activity under close to physiological conditions in scarce primary cell subsets such as antigen cross-presenting dendritic cells. PMID:25656091

  6. Systemic administration of antigen-pulsed dendritic cells induces experimental allergic asthma in mice upon aerosol antigen rechallenge.

    PubMed

    Graffi, Sebastian J; Dekan, Gerhard; Stingl, Georg; Epstein, Michelle M

    2002-05-01

    Antigen-pulsed dendritic cells (DCs) have been used extensively as cellular vaccines to induce a myriad of protective immune responses. Adoptive transfer of antigen-pulsed DCs is especially effective at generating Th1 and CD8 immune responses. However, recently this strategy has been shown to induce Th2 cells when DCs are administered locally into the respiratory tract. We sought to address whether systemic rather than local antigen-pulsed DC administration could induce Th2 experimental allergic asthma. We found that OVA-pulsed splenic DCs injected intraperitoneally induced polarized Th2 allergic lung disease upon secondary OVA aerosol challenge. Disease was characterized by eosinophilic lung inflammation, excess mucus production, airway hyperresponsiveness, and OVA-specific IgG1 and IgE. In addition, unusual pathology characterized by macrophage alveolitis and multinucleated giant cells was observed. These data show that systemic administration of antigen-pulsed DCs and subsequent aeroantigen challenge induces Th2 immunity. These findings have important implications for the development of DC-based vaccines.

  7. Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition

    PubMed Central

    Brzostek, Joanna; Gascoigne, Nicholas R. J.; Rybakin, Vasily

    2016-01-01

    B7 proteins CD80 (B7-1) and CD86 (B7-2) are expressed on most antigen-presenting cells and provide critical co-stimulatory or inhibitory input to T cells via their T-cell-expressed receptors: CD28 and CTLA-4. CD28 is expressed on effector T cells and regulatory T cells (Tregs), and CD28-dependent signals are required for optimum activation of effector T cell functions. CD28 ligation on effector T cells leads to formation of distinct molecular patterns and induction of cytoskeletal rearrangements at the immunological synapse (IS). CD28 plays a critical role in recruitment of protein kinase C (PKC)-θ to the effector T cell IS. CTLA-4 is constitutively expressed on the surface of Tregs, but it is expressed on effector T cells only after activation. As CTLA-4 binds to B7 proteins with significantly higher affinity than CD28, B7 ligand recognition by cells expressing both receptors leads to displacement of CD28 and PKC-θ from the IS. In Tregs, B7 ligand recognition leads to recruitment of CTLA-4 and PKC-η to the IS. CTLA-4 plays a role in regulation of T effector and Treg IS stability and cell motility. Due to their important roles in regulating T-cell-mediated responses, B7 receptors are emerging as important drug targets in oncology. In this review, we present an integrated summary of current knowledge about the role of B7 family receptor–ligand interactions in the regulation of spatial and temporal IS dynamics in effector and Tregs. PMID:26870040

  8. Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues

    SciTech Connect

    Terasawa, Masao; Nagata, Kisaburo; Kobayashi, Yoshiro

    2008-12-12

    Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naive T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1{sup -} and Gr-1{sup +} ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.

  9. Natural and adaptive IgM antibodies in the recognition of tumor-associated antigens of breast cancer (Review)

    PubMed Central

    DÍAZ-ZARAGOZA, MARIANA; HERNÁNDEZ-ÁVILA, RICARDO; VIEDMA-RODRÍGUEZ, RUBÍ; ARENAS-ARANDA, DIEGO; OSTOA-SALOMA, PEDRO

    2015-01-01

    For early detection of cancer, education and screening are important, but the most critical factor is the development of early diagnostic tools. Methods that recognize the warning signs of cancer and take prompt action lead to an early diagnosis; simple tests can identify individuals in a healthy population who have the disease but have not developed symptoms. Early detection of cancer is significant and is one of the most promising approaches by which to reduce the growing cancer burden and guide curative treatment. The early diagnosis of patients with breast cancer is challenging, since it is the most common cancer in women worldwide. Despite the advent of mammography in screening for breast cancer, low-resource, low-cost alternative tools must be implemented to complement mammography findings. IgM is part of the first line of defense of an organism and is responsible for recognizing and eliminating infectious particles and removing transformed cells. Most studies on breast cancer have focused on the development of IgG-like molecules as biomarkers or as a treatment for the advanced stages of cancer, but autoantibodies (IgM) and tumor-associated antigens (proteins or carbohydrates with aberrant structures) have not been examined as early diagnostic tools for breast cancer. The present review summarizes the function of natural and adaptive IgM in eliminating cancer cells in the early stages of pathology and their value as early diagnostic tools. IgM, as a component of the immune system, is being used to identify tumor-associated antigens and tumor-associated carbohydrate antigens. PMID:26133558

  10. Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes.

    PubMed Central

    Falo, L D; Sullivan, K; Benacerraf, B; Mescher, M F; Rock, K L

    1985-01-01

    Several amino acid copolymers are potent immunogens under the control of major histocompatibility complex (MHC)-encoded Ir genes. We have further characterized their accessory-cell-dependent, MHC-restricted presentation to T lymphocytes. We initially characterized their processing requirements by investigating the ability of paraformaldehyde-fixed antigen-presenting cells (APC) to present these copolymers. Fixed APC can present poly(Glu56Lys35Phe9) and poly(Glu60Ala30Tyr10) provided that they have been incubated with antigen prior to fixation. The inability of these same fixed preparations to present soluble antigen indicates a fixation-sensitive antigen-processing step. In contrast, the antigens poly(Glu55Lys35Leu10) and poly(Glu55Lys35Tyr10) can be presented by APC fixed before antigen exposure. This differential requirement for antigen processing was exploited to analyze the events of antigen presentation in two related systems. First, the ability of isolated APC membranes to process and present antigen was assessed. APC membranes can present the antigens poly(GluLysLeu) and poly(GluLysTyr) in a specific and MHC-restricted manner. However, the isolated membranes fail to present either poly(GluLysPhe) or poly(GluAlaTyr), suggesting that such preparations can present but not process antigen. Second, the distinct properties of the various copolymers were used with fixed APC to test the effects of antigen processing on the phenomenon of antigen competition. APC that had processed poly(GluLysPhe) or poly(GluAlaTyr) were subsequently fixed and used to present antigen in the presence or absence of various antagonists. Under these conditions, poly(GluLysLeu) and poly(Glu50Tyr50) could effect specific inhibition, clearly indicating that antigen competition occurs distal to and does not require antigen processing. In contrast, native antigen with an absolute processing requirement is not capable of competing with preprocessed antigen on fixed APC. Taken together, these

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

    PubMed Central

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

    2016-01-01

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

  12. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    PubMed

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

  13. CD1-restricted recognition of exogenous and self-lipid antigens by duodenal gammadelta+ T lymphocytes.

    PubMed

    Russano, Anna M; Bassotti, Gabrio; Agea, Elisabetta; Bistoni, Onelia; Mazzocchi, Alessandro; Morelli, Antonio; Porcelli, Steven A; Spinozzi, Fabrizio

    2007-03-15

    Gammadelta T cells are present in the mucosal intestinal epithelia and secrete factors necessary to maintain tissue integrity. Ags recognized by these cells are poorly defined, although in mice non-classical MHC class I molecules have been implicated. Since MHC class I-like CD1 receptors are widely expressed at the surface of epithelial and dendritic intestinal cells and have the capacity to present lipid Ags to T cells, we hypothesized that these molecules might present autologous and/or exogenous phospholipids to intestinal gammadelta T lymphocytes. Intraepithelial T lymphocytes from normal human duodenal mucosal biopsies were cloned and exposed to natural and synthetic phospholipids using CD1a-, CD1b-, CD1c- or CD1d-transfected C1R lymphoblastoid or HeLa cell lines as APCs. Their cytolytic properties and regulatory cytokine secretion were also examined. Most clones obtained from duodenal mucosa (up to 70%) were TCRalphabeta+, and either CD4+ or CD8+, whereas 20% were CD4-CD8- (6 clones) or TCRgammadelta+ (12 clones). A relevant percentage (up to 66%) of TCRgammadelta+ but few (<5%) TCRalphabeta+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [(3)H]thymidine incorporation and IL-4 release assays. A Th1-like cytolytic and functional activity along with the ability to secrete regulatory cytokines was observed in most phospholipid-specific gammadelta T cell clones. Thus, a substantial percentage of TCRgammadelta+ but few TCRalphabeta+ from human duodenal mucosa recognize exogenous phospholipids in a CD1-restricted fashion. This adaptive response could contribute to mucosal homeostasis, but could also favor the emergence of inflammatory or allergic intestinal diseases. PMID:17339459

  14. Biased T-Cell Antigen Receptor Repertoire in Lyme Arthritis

    PubMed Central

    Roessner, Karen; Trivedi, Harsh; Gaur, Lakshmi; Howard, Diantha; Aversa, John; Cooper, Sheldon M.; Sigal, Leonard H.; Budd, Ralph C.

    1998-01-01

    A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete, Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation with B. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) β-chain variable (Vβ) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vβ2 and Vβ6 in the latter. This is remarkably similar to our previous findings in studies of rheumatoid arthritis and to other reports on psoriatic skin lesions. However, stimulation of synovial fluid T cells with B. burgdorferi provoked active proliferation but not a statistically significant increase in expression of any TCR Vβ, including Vβ2 and Vβ6. Collectively, the findings suggest that the skewing of the TCR repertoire of fresh synovial fluid in Lyme arthritis may represent more a synovium-tropic or nonspecific inflammatory response, similar to that occurring in rheumatoid arthritis or psoriasis, rather than a specific Borrelia reaction. PMID:9488400

  15. Activation requirements of circulating antigen-specific human CD8(+) memory T cells probed with insect cell-based artificial antigen-presenting cells.

    PubMed

    Guelly, Christian; Küpcü, Zaruhi; Zalusky, Doris; Karner, Margarete; Zehetner, Margit; Schweighoffer, Tamás

    2002-01-01

    We sought to define the molecular setup of an antigen-presenting cell that elicits antigen-specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single-chain HLA was complemented step-by-step with costimulatory molecules, including CD54 and CD80, by co-infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha-3 domain of the HLA heavy chain molecule was replaced by its murine K(b) counterpart. Circulating T cells that respond to the EBV-derived HLA-A2-restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient to elicit IFN-gamma secretion from these freshly isolated CD8(+) T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen-specific CTL clones also reacted in a strictly antigen-specific manner, but required CD54 for quantitative responses. The amount of IFN-gamma produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8(+) T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines.

  16. Activation requirements of circulating antigen-specific human CD8(+) memory T cells probed with insect cell-based artificial antigen-presenting cells.

    PubMed

    Guelly, Christian; Küpcü, Zaruhi; Zalusky, Doris; Karner, Margarete; Zehetner, Margit; Schweighoffer, Tamás

    2002-01-01

    We sought to define the molecular setup of an antigen-presenting cell that elicits antigen-specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single-chain HLA was complemented step-by-step with costimulatory molecules, including CD54 and CD80, by co-infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha-3 domain of the HLA heavy chain molecule was replaced by its murine K(b) counterpart. Circulating T cells that respond to the EBV-derived HLA-A2-restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient to elicit IFN-gamma secretion from these freshly isolated CD8(+) T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen-specific CTL clones also reacted in a strictly antigen-specific manner, but required CD54 for quantitative responses. The amount of IFN-gamma produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8(+) T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines. PMID:11754359

  17. Janus particles as artificial antigen-presenting cells for T cell activation.

    PubMed

    Chen, Bo; Jia, Yilong; Gao, Yuan; Sanchez, Lucero; Anthony, Stephen M; Yu, Yan

    2014-01-01

    Here we show that the multifunctionality of Janus particles can be exploited for in vitro T cell activation. We engineer bifunctional Janus particles on which the spatial distribution of two ligands, anti-CD3 and fibronectin, mimics the "bull's eye" protein pattern formed in the membrane junction between a T cell and an antigen-presenting cell. Different levels of T cell activation can be achieved by simply switching the spatial distribution of the two ligands on the surfaces of the "bull's eye" particles. We find that the ligand pattern also affects clustering of intracellular proteins. This study demonstrates that anisotropic particles, such as Janus particles, can be developed as artificial antigen-presenting cells for modulating T cell activation. PMID:25343426

  18. Nonhuman TRIM5 Variants Enhance Recognition of HIV-1-Infected Cells by CD8+ T Cells

    PubMed Central

    Jimenez-Moyano, Esther; Ruiz, Alba; Kløverpris, Henrik N.; Rodriguez-Plata, Maria T.; Peña, Ruth; Blondeau, Caroline; Selwood, David L.; Izquierdo-Useros, Nuria; Moris, Arnaud; Clotet, Bonaventura; Goulder, Philip; Towers, Greg J.

    2016-01-01

    ABSTRACT Tripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8+ T cells. We illustrate how TRIM5 restriction improves CD8+ T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)–CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1β expression in HIV-1-specific CD8+ T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8+ T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8+ T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for nonhuman TRIM5 variants in cellular immunity. We hypothesize that TRIM5 can couple innate viral sensing and CD8+ T-cell activation to increase species barriers against retrovirus infection. IMPORTANCE New therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The nonhuman TRIM5 variants, rhesus TRIM5α (RhT5) and TRIM

  19. Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells.

    PubMed

    Schütz, Christian; Fleck, Martin; Mackensen, Andreas; Zoso, Alessia; Halbritter, Dagmar; Schneck, Jonathan P; Oelke, Mathias

    2008-04-01

    Several cell-based immunotherapy strategies have been developed to specifically modulate T cell-mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell-based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (kappaaAPCs) by coupling an apoptosis-inducing alpha-Fas (CD95) IgM mAb together with HLA-A2 Ig molecules onto beads. These kappaaAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)-dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of kappaaAPCs and independent of activation-induced cell death (AICD). kappaaAPCs represent a novel technology that can control T cell-mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection. PMID:18096763

  20. The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

    PubMed Central

    1982-01-01

    Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues. PMID:6178742

  1. T cell recognition of an HLA-A2-restricted epitope derived from a cleaved signal sequence

    PubMed Central

    1994-01-01

    An alternative pathway for class I-restricted antigen presentation has been suggested on the basis of peptides bound to HLA-A2 molecules in cells lacking the transporter for antigen presentation (TAP). Most of these peptides were derived from signal sequences for translocation into the endoplasmic reticulum (ER). However, it is not known whether these peptides can be presented to T cells. The hydrophobic nature of an HLA-A2-restricted T cell epitope (M1 58-66) was exploited to test whether it could be presented to T cells when derived from a signal sequence. Replacing the signal sequence of the influenza virus hemagglutinin molecule H3 with an artificial sequence containing that HLA-A2-restricted T cell epitope resulted in efficient translocation of H3 molecules into the ER and transport to the cell surface. This signal sequence-derived epitope was presented to HLA-A2-restricted T cells. Involvement of cytosolic processing for this presentation is very unlikely, because (a) presentation occurred in cells lacking TAP; (b) expression of H3 molecules with the artificial signal sequence did not produce a detectable cytosolic form of H3; and (c) presentation of the same epitope expressed in cytosolic forms of antigen required TAP. Thus, a peptide derived from a signal sequence cleaved in the ER can provide an epitope for HLA-A2-restricted T cell recognition. PMID:7525846

  2. A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen*

    PubMed Central

    Bianchi, Valentina; Bulek, Anna; Fuller, Anna; Lloyd, Angharad; Attaf, Meriem; Rizkallah, Pierre J.; Dolton, Garry; Sewell, Andrew K.; Cole, David K.

    2016-01-01

    Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination. PMID:26917722

  3. Lipid and glycolipid antigens of CD1d-restricted natural killer T cells

    PubMed Central

    Venkataswamy, Manjunatha M.; Porcelli, Steven A.

    2009-01-01

    In spite of their relatively limited antigen receptor repertoire, CD1d-restricted NKT cells recognize a surprisingly diverse range of lipid and glycolipid antigens. Recent studies of natural and synthetic CD1d presented antigens provide an increasingly detailed picture of how the specific structural features of these lipids and glycolipids influence their ability to be presented to NKT cells and stimulate their diverse immunologic functions. Particularly for synthetic analogues of α-galactosylceramides which have been the focus of intense recent investigation, it is becoming clear that the design of glycolipid antigens with the ability to precisely control the specific immunologic activities of NKT cells is likely to be feasible. The emerging details of the mechanisms underlying the structure-activity relationship of NKT cell antigens will assist greatly in the design and production of immunomodulatory agents for the precise manipulation of NKT cells and the many other components of the immune system that they influence. PMID:19945296

  4. Adoptive T-cell immunotherapy of cancer using chimeric antigen receptor-grafted T cells.

    PubMed

    Davies, David Marc; Maher, John

    2010-06-01

    Harnessing the power of the immune system to target cancer has long been a goal of tumor immunologists. One avenue under investigation is the modification of T cells to express a chimeric antigen receptor (CAR). Expression of such a receptor enables T-cell specificity to be redirected against a chosen tumor antigen. Substantial research in this field has been carried out, incorporating a wide variety of malignancies and tumor-associated antigens. Ongoing investigations will ensure this area continues to expand at a rapid pace. This review will explain the evolution of CAR technology over the last two decades in addition to detailing the associated benefits and disadvantages. The outcome of recent phase I clinical trials and the impact that these have had upon the direction of future research in this field will also be addressed.

  5. A novel beta 4, alpha 6 integrin-associated epithelial cell antigen involved in natural killer cell and antigen-specific cytotoxic T lymphocyte cytotoxicity

    PubMed Central

    1991-01-01

    Efficient immune responses require interactions between cell adhesion molecules on lymphocytes and counter-receptors on antigen presenting cells or target cells. While target-specific receptors or ligands have not been identified for natural killer (NK) cells, cell adhesion molecules have been implicated in the interaction between NK cell effectors and tumor cell targets. Herein, we describe monoclonal antibodies (mAbs) against a carcinoma cell line that efficiently block the cytolytic activity of interleukin 2-activated NK cell lines and clones. L280 mAb reacts with secretory epithelial cells in normal human tissues, but does not react with hematopoietic cells or other tissue types. Biochemical analysis revealed that L280 mAb immunoprecipitates the beta 4, alpha 6 integrin, as well as a novel 98-kD glycoprotein, and probably reacts with a carbohydrate epitope on these molecules. Involvement of the L280 antigen in cellular immunity is not restricted to NK cell-mediated cytotoxicity. L280 mAb also efficiently inhibits alloantigen-specific cytotoxicity against Colo-205 cells mediated by human histocompatibility leukocyte antigen (HLA)-A2 alloantigen specific alpha beta-TCR+ and gamma delta-TCR+ cytotoxic T lymphocyte (CTL) clones. Additionally, we demonstrate that L280 mAb blocks cytotoxicity mediated by influenza peptide-specific HLA-restricted CTL clones. These data indicate that the antigen recognized by L280 mAb is important in both NK and CTL function, and that an as yet unidentified receptor for this epithelial antigen is present on both NK and T lymphocytes. The restricted expression of L280 antigen indicates that this molecule may be important in immune reactions in epithelial tissues. PMID:1744585

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

    PubMed Central

    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

  7. DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

    PubMed

    Liu, Yang; Blanchfield, Lori; Ma, Victor Pui-Yan; Andargachew, Rakieb; Galior, Kornelia; Liu, Zheng; Evavold, Brian; Salaita, Khalid

    2016-05-17

    T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

  8. DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

    PubMed

    Liu, Yang; Blanchfield, Lori; Ma, Victor Pui-Yan; Andargachew, Rakieb; Galior, Kornelia; Liu, Zheng; Evavold, Brian; Salaita, Khalid

    2016-05-17

    T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response. PMID:27140637

  9. A new approach for evaluating antigen-specific T cell responses to myelin antigens during the course of multiple sclerosis

    PubMed Central

    Arbour, Nathalie; Holz, Andreas; Sipe, Jack C.; Naniche, Denise; Romine, John S.; Zyroff, Jack; Oldstone, Michael B.A.

    2016-01-01

    We used a flow cytometry assay to measure proliferation and cytokine production of self-antigen-specific T cells in individual patients during the clinical course of multiple sclerosis (MS). Myelin-associated oligodendrocytic basic protein (MOBP) was selected for proof of principles in the assay, along with myelin basic protein (MBP) to assess specific activated T cells in 10 MS patients over an 18-month period, in parallel with brain magnetic resonance imaging (MRI) scans and clinical rating scale. A positive correlation occurred between antigen-specific T cell proliferation and interferon-γ production with clinical relapses and MRI lesion activity that was absent when the same patients were in remission. PMID:12667664

  10. Identification of novel Mycobacterium tuberculosis CD4 T-cell antigens via high throughput proteome screening

    PubMed Central

    Nayak, Kaustuv; Jing, Lichen; Russell, Ronnie M.; Davies, D. Huw; Hermanson, Gary; Molina, Douglas M.; Liang, Xiaowu; Sherman, David R.; Kwok, William W.; Yang, Junbao; Kenneth, John; Ahamed, Syed F.; Chandele, Anmol; Kaja, Murali-Krishna; Koelle, David M.

    2015-01-01

    Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens. PMID:25857935

  11. The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

    PubMed

    Howe, Savannah E; Lickteig, Duane J; Plunkett, Kyle N; Ryerse, Jan S; Konjufca, Vjollca

    2014-01-01

    Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies.

  12. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    SciTech Connect

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo; Kim, Myoung Hee

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  13. Engineered Nanostructures of Antigen Provide an Effective Means for Regulating Mast Cell Activation

    PubMed Central

    Deng, Zhao; Weng, I-Chun; Li, Jie-Ren; Chen, Huan-Yuan; Liu, Fu-Tong; Liu, Gang-yu

    2011-01-01

    Nanostructures containing 2,4-Dinitrophenyl (DNP) as antigen were designed and produced to investigate antibody-mediated activation of mast cells. The design consists of nanogrids of DNP termini inlaid in alkanethiol self-assembled monolayers (SAMs). Using scanning probe-based nanografting, nanometer precision was attained for designed geometry, size and periodicity. Rat basophilic leukemia (RBL) cells exhibited high sensitivity to the geometry and local environment of DNP presented on these nanostructures. The impact included cellular adherence, spreading, membrane morphology, cytoskeleton structure, and activation. The highest level of spreading and activation was induced by nanogrids of 17 nm line width and 40 nm periodicity, with DNP haptens 1.4 nm above the surroundings. The high efficacy is attributed to two main factors. First, DNP sites in the nanostructure are highly accessible by anti-DNP-IgE during recognition. Second, the arrangement or geometry of DNP termini in nanostructures promotes clustering of FcεRI receptors that are pre-linked to IgE. The clustering effectively initiates Lyn-mediated signaling cascades, ultimately leading to the degranulation of RBL cells. This work demonstrates an important concept, that nanostructures of ligands provide new and effective cues for directing cellular signaling processes. PMID:21999491

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-01-01

    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. PMID:27251373

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

    PubMed Central

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

    2016-01-01

    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. PMID:27251373

  17. Group-specific human granulocyte antigens on a chronic myelogenous leukemia cell line with a Philadelphia chromosome marker.

    PubMed

    Drew, S I; Terasaki, P I; Billing, R J; Bergh, O J; Minowada, J; Klein, E

    1977-05-01

    Group-specific human granulocyte antigens are serologically detectable with granulocytotoxic-positive human alloantisera on a cell line, K562, of chronic myelogenous leukemia origin which bears a Philadelphia chromosomal marker. The same cell line lacks serologically detectable HLA, B2 microglobulin, and B-lymphocyte antigens. Granulocyte antigens are important cell markers for cell lines of suspected myeloid lineage.

  18. Apoptotic, necrotic, or fused tumor cells: an equivalent source of antigen for dendritic cell loading.

    PubMed

    Larmonier, Nicolas; Mérino, Delphine; Nicolas, Alexandra; Cathelin, Dominique; Besson, Angélique; Bateman, Andrew; Solary, Eric; Martin, François; Katsanis, Emmanuel; Bonnotte, Bernard

    2006-09-01

    The identification of the most efficient strategy for tumor antigen loading of dendritic cells (DCs) remains a challenge in cancer immunotherapy protocols. Autologous dead tumor cells have been demonstrated to constitute an acceptable source of multiple tumor-associated antigens (TAA) to pulse DCs. However the optimal approach for inducing cell death that would lead to effective endocytosis and activation of DCs remains controversial. In this study we have induced and defined 3 distinct mechanisms of tumor cell death (apoptosis, necrosis and fusion-mediated cell death), and investigated their differential effects on DCs. Bone marrow-derived DCs demonstrated comparable uptake of primary apoptotic, necrotic, or fused dead tumor cells. Furthermore, the distinct modes of cancer cell death had analogous potential in activating the transcription factors NF-kappaB and STAT1 and in maturing DCs, resulting in an equally effective stimulation of immune T cells. The current study therefore provides further informations on the use of dead whole tumor cells as antigen sources for effective active anti-cancer immunotherapy.

  19. The Hidden Conformation of Lewis x, a Human Histo-Blood Group Antigen, Is a Determinant for Recognition by Pathogen Lectins.

    PubMed

    Topin, Jérémie; Lelimousin, Mickaël; Arnaud, Julie; Audfray, Aymeric; Pérez, Serge; Varrot, Annabelle; Imberty, Anne

    2016-07-15

    Histo-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavors revealing the unusual distortion of histo-blood group antigens by bacterial and fungal lectins. The Lewis x trisaccharide adopts a rigid closed conformation in solution, while crystallography and molecular dynamics reveal several higher energy open conformations when bound to the Ralstonia solanacearum lectin, which is in agreement with thermodynamic and kinetic measurements. Extensive molecular dynamics simulations confirm rare transient Le(x) openings in solution, frequently assisted by distortion of the central N-acetyl-glucosamine ring. Additional directed molecular dynamic trajectories revealed the role of a conserved tryptophan residue in guiding the fucose into the binding site. Our findings show that conformational adaptation of oligosaccharides is of paramount importance in cell recognition and should be considered when designing anti-infective glyco-compounds. PMID:27198630

  20. Speech recognition systems on the Cell Broadband Engine

    SciTech Connect

    Liu, Y; Jones, H; Vaidya, S; Perrone, M; Tydlitat, B; Nanda, A

    2007-04-20

    In this paper we describe our design, implementation, and first results of a prototype connected-phoneme-based speech recognition system on the Cell Broadband Engine{trademark} (Cell/B.E.). Automatic speech recognition decodes speech samples into plain text (other representations are possible) and must process samples at real-time rates. Fortunately, the computational tasks involved in this pipeline are highly data-parallel and can receive significant hardware acceleration from vector-streaming architectures such as the Cell/B.E. Identifying and exploiting these parallelism opportunities is challenging, but also critical to improving system performance. We observed, from our initial performance timings, that a single Cell/B.E. processor can recognize speech from thousands of simultaneous voice channels in real time--a channel density that is orders-of-magnitude greater than the capacity of existing software speech recognizers based on CPUs (central processing units). This result emphasizes the potential for Cell/B.E.-based speech recognition and will likely lead to the future development of production speech systems using Cell/B.E. clusters.

  1. Norovirus Antagonism of B cell Antigen Presentation Results in Impaired Control of Acute Infection

    PubMed Central

    Zhu, Shu; Jones, Melissa K.; Hickman, Danielle; Han, Shuhong; Reeves, Westley; Karst, Stephanie M.

    2016-01-01

    Human noroviruses are a leading cause of gastroenteritis so vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen presenting cells, we tested whether noroviruses regulate B cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8+ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8+ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for activation of this pathway and ultimately only B cells were required for clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8+ T cell responses. PMID:27007673

  2. 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A

    SciTech Connect

    Samelson, L.E.; Harford, J.; Schwartz, R.H.; Klausner, R.D.

    1985-04-01

    Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell receptor.

  3. Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

    PubMed

    Quinn, Laura L; Zuo, Jianmin; Abbott, Rachel J M; Shannon-Lowe, Claire; Tierney, Rosemary J; Hislop, Andrew D; Rowe, Martin

    2014-08-01

    CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>L), interference by BILF1 increases with progression through lytic cycle (IEantigen presentation of L epitopes. Together, these data firstly indicate which potential immune-evasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into

  4. Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

    SciTech Connect

    Hara, T.; Jung, L.K.L.; FU, S.M.

    1986-03-01

    With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% of the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.

  5. Expression profile of novel cell surface molecules on different subsets of human peripheral blood antigen-presenting cells

    PubMed Central

    Damasceno, Daniela; Andrés, Martín Pérez; van den Bossche, Wouter BL; Flores-Montero, Juan; de Bruin, Sandra; Teodosio, Cristina; van Dongen, Jacques JM; Orfao, Alberto; Almeida, Julia

    2016-01-01

    Although major steps have been recently made in understanding the role of the distinct subsets of dendritic cells (DC)/antigen-presenting cells (APC), further studies are required to unravel their precise role, including in-depth immunophenotypic characterisation of these cells. Here, we used eight-colour flow cytometry to investigate the reactivity of a panel of 72 monoclonal antibodies (including those clustered in seven new Cluster of Differentiation, CD) on different subsets of APC in peripheral blood (PB) samples from five healthy adults. These experiments were performed in the context of the Tenth International Workshop on Human Leukocyte Differentiation Antigens (HLDA10). Plasmacytoid DC was the only cell population that expressed CD85g and CD195, whereas they lacked all of the other molecules investigated. In contrast, myeloid DC mostly expressed inhibitory C-type lectin receptors (CLRs) and other inhibitory-associated molecules, whereas monocytes expressed both inhibitory and activating CLRs, together with other phagocytosis-associated receptors. Within monocytes, progressively lower levels of expression were generally observed from classical monocytes (cMo) to SLAN− and SLAN+ non-classical monocytes (ncMo) for most of the molecules expressed, except for the CD368 endocytic receptor. This molecule was found to be positive only in cMo, and the CD369 and CD371 modulating/signalling receptors. In addition, the CD101 inhibitory molecule was found to be expressed at higher levels in SLAN+ vs SLAN− ncMo. In summary, the pattern of expression of the different signalling molecules and receptors analysed in this work varies among the distinct subsets of PB APCs, with similar profiles for molecules within each functional group. These findings suggest unique pattern-recognition and signalling capabilities for distinct subpopulations of APCs, and therefore, diverse functional roles. PMID:27766148

  6. Stem Cell Antigen-1 in Skeletal Muscle Function

    PubMed Central

    Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

    2013-01-01

    Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age. PMID:24042315

  7. COMMON CELL SURFACE ANTIGEN ASSOCIATED WITH MAMMALIAN C-TYPE RNA VIRUSES

    PubMed Central

    Yoshiki, Takashi; Mellors, Robert C.; Hardy, William D.; Fleissner, Erwin

    1974-01-01

    The indirect membrane immunofluorescence test and the absorption analysis of rabbit anti-FeLV, rabbit anti-FeLVp 30, and rabbit anti-MuLVp 30 antisera yielded the following conclusions. An antigen shared by mammalian (murine and feline) C-type RNA leukemia and sarcoma viruses was detected on the surface of cells infected or transformed by C-type viruses. The antigen was characterized as membrane-bound gs antigen bearing two determinants, membrane-bound gs-1, intraspecies-specific antigenic determinant, and membrane-bound gs-3, interspecies-specific antigenic determinant. Membrane-bound gs antigen was located on the cell surface, frequently near the site of virus budding but not on the envelope of murine C-type RNA virus. PMID:4131513

  8. Neisseria gonorrhoeae suppresses dendritic cell-induced, antigen-dependent CD4 T cell proliferation.

    PubMed

    Zhu, Weiyan; Ventevogel, Melissa S; Knilans, Kayla J; Anderson, James E; Oldach, Laurel M; McKinnon, Karen P; Hobbs, Marcia M; Sempowski, Gregory D; Duncan, Joseph A

    2012-01-01

    Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.

  9. Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

    PubMed

    Choi, Bongseo; Moon, Hyojin; Hong, Sung Joon; Shin, Changsik; Do, Yoonkyung; Ryu, Seongho; Kang, Sebyung

    2016-08-23

    In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

  10. Artificial antigen presenting cells that express prevalent HLA alleles: A step towards the broad application of antigen-specific adoptive cell therapies.

    PubMed

    Hasan, Aisha N; Selvakumar, Annamalai; Doubrovina, Ekaterina; Riviere, Isabelle; Sadelain, Michel W; O'Reilly, Richard J

    2009-12-01

    The artificial antigen-presenting cells (AAPCs) described in this review were generated to facilitate the production of virus-specific T-cells for the treatment of infections in patients after bone marrow transplant. These AAPCs consist of murine 3T3 cells genetically modified to express critical human molecules needed for T-cell stimulation, such as the co-stimulatory molecules B7.1, ICAM-1, and LFA-3 and one of a series of 6 common HLA class I alleles. When T-cells were sensitized against cytomegalovirus (CMV) using AAPCs that express a shared HLA allele or using autologous antigen-presenting cells (APCs) loaded with the CMVpp65 antigen, they were activated and expanded to become HLA-restricted CMVpp65-specific T-cells. These T-cells demonstrated functional activity in vitro against CMV by producing IFN-gamma and inducing CMVpp65-specific cytotoxicity. T-cells sensitized with AAPCs recognized antigenic epitopes presented by each HLA allele known to be immunogenic in Man. Sensitization with AAPCs also permitted expansion of IFN-gamma+ cytotoxic T-cells against subdominant epitopes that were not effectively recognized by T-cells sensitized with autologous APCs. This panel of AAPCs provides a source of immediately accessible, standardizable, and replenishable "off the shelf" cellular reagents with the potential to make adoptive immunotherapy widely available for the treatment of lethal infections, cancer, and autoimmune diseases. PMID:20040272

  11. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  12. Formalinized chicken red cell nuclei as a simple antigen for standardized antinuclear factor determination

    PubMed Central

    Veen, J. H. Ten; Feltkamp, T. E. W.

    1969-01-01

    Chicken red cell nuclei treated with formalin appear to be specific and easy to handle nuclear antigens for antinuclear factor determination. They can be kept without cooling or freezing for at least 6 months, and probably considerably longer. As these nuclei can easily be obtained in great amounts it appears feasible to develop an international nuclear standard antigen for standardization in immunofluorescence. They might also be applied as a nuclear antigen in automated antinuclear factor determination. PMID:4904069

  13. Enhanced antigen-presenting capacity of cultured Langerhans' cells is associated with markedly increased expression of Ia antigen

    SciTech Connect

    Shimada, S.; Caughman, S.W.; Sharrow, S.O.; Stephany, D.; Katz, S.I.

    1987-10-15

    Recent studies indicate that when epidermal Langerhans' cells (LC) are cultured for 2 to 3 days they, in comparison to freshly prepared LC, exhibit markedly enhanced ability to stimulate T cell proliferative responses in oxidative mitogenesis and in the mixed epidermal-leukocyte reaction. In this study, we determined whether cultured LC enhance antigen-specific T cell responses, and whether such enhanced stimulatory capacity correlates with the level of Ia antigen expressed on LC. We used C3H/He (Iak) epidermal cells as stimulators and, as responder cells, both the trinitrophenyl-specific clones D8 and SE4, which were assayed for (/sup 3/H)dThd incorporation, and the pigeon cytochrome c specific hybridoma 2C2, which was assayed for interleukin 2 production. Cultured LC induced 10 to 100 times greater proliferation or interleukin 2 production by responder cells than did freshly prepared LC. The intensity of I-Ak and I-Ek, expressed on cultured LC as assessed by immunofluorescence and flow cytometry, was found to be 10 to 36 times greater on a per cell basis than that on freshly prepared LC. Depletion of LC from fresh epidermal cell suspensions by anti-Iak and complement or treatment with 50 mJ/cm/sup 2/ medium range ultraviolet light or cycloheximide before culture abrogated both the increase in Ia expression and antigen-specific clonal proliferation. The results suggest that when LC are removed from their usual epidermal milieu, they express increased amounts of Ia and become more potent stimulators of T cell responses.

  14. Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level

    NASA Astrophysics Data System (ADS)

    Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P.; Devitt, J.; Candeloro, Patrizio; de Angelis, Francesco; Carbone, Ennio; di Fabrizio, Enzo

    2010-03-01

    Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β2-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β2-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm-1, the intensity variation of cells associated with HLA class I molecules could be measured.

  15. Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level.

    PubMed

    Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P; Devitt, J; Candeloro, Patrizio; De Angelis, Francesco; Carbone, Ennio; Di Fabrizio, Enzo

    2010-01-01

    Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (alpha1, alpha2, and alpha3) once folded by peptide and beta(2)-microglobulin show the presence of two alpha-helix streams and one beta-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+beta(2)-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm(-1), the intensity variation of cells associated with HLA class I molecules could be measured.

  16. Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

    PubMed Central

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

  17. Macrophage recognition of immune complexes: development and application of novel cell surface labeling procedures.

    PubMed

    Petty, H R; Dereski, W

    1985-07-16

    A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex has been prepared for cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas [Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]. The conjugate is a heterodimer of Mr230 000 with linkages to either or both of the heavy and light chains of the antibody, as judged by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. Electron microscopy demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and electron microscopy. Immune complexes were masked with "cold" iodine by use of the endogenous LPO activity. The complexes bound to cells at 4 degrees C as shown by electron microscopy and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling has also been conducted under conditions of substrate deficit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4052386

  18. Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells.

    PubMed

    Kima, P E; Soong, L; Chicharro, C; Ruddle, N H; McMahon-Pratt, D

    1996-12-01

    CD4+ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the I4.07 macrophage cell line (H-2k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4+ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4+ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4+ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.

  19. Presence of species-specific, monomorphic antigens on sheep and goat binucleate cells.

    PubMed

    Gall, J G; MacLaren, L A; Anderson, G B

    1995-07-15

    Immunocytochemical assays for sheep and goat species-specific, monomorphic antigens were developed utilizing polyclonal antisera from sheep and goats immunized by interspecific pregnancy. The assays were applied to cell isolates from sheep and goat fetal cotyledons collected from allogeneic pregnancies at Days 35, 40 and 120 of gestation. The isolates contained 7 to 48% binucleate cells (BNC). Using these assays, the sheep-specific antigen was detected on sheep cotyledonary cell isolates on all days of gestation tested (P < 0.001); the assay also detected the antigen on the BNC subset of the cotyledonary cell isolate population (P < 0.001). The caprine-specific antigen was shown to be present on cotyledonary cell isolates (P < 0.05), although the presence of the antigen could not be demonstrated with statistical confidence on goat BNC due to insufficient numbers of discernible cells. Binucleate cells contribute to the formation of the syncytial layer of the placenta by fusing with maternal epithelial cells and with the syncytium. The species-specific antigen (or antigens) is present on BNC at the appropriate time of gestation at which it (they) could play a role in the humoral immune response to interspecific and hybrid pregnancies observed in ewes and does. PMID:16727726

  20. Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease.

    PubMed

    Chen, Xi; Hnida, Kathrin; Graewert, Melissa Ann; Andersen, Jan Terje; Iversen, Rasmus; Tuukkanen, Anne; Svergun, Dmitri; Sollid, Ludvig M

    2015-08-28

    Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.

  1. Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease.

    PubMed

    Chen, Xi; Hnida, Kathrin; Graewert, Melissa Ann; Andersen, Jan Terje; Iversen, Rasmus; Tuukkanen, Anne; Svergun, Dmitri; Sollid, Ludvig M

    2015-08-28

    Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease. PMID:26160175

  2. Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

    NASA Astrophysics Data System (ADS)

    Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

    2013-03-01

    A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

  3. Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus

    PubMed Central

    Gao, Xiang; Esseili, Malak A.; Lu, Zhongyan; Saif, Linda J.

    2016-01-01

    ABSTRACT Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. IMPORTANCE Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new

  4. Detecting Antigen-Specific T Cell Responses: From Bulk Populations to Single Cells.

    PubMed

    Phetsouphanh, Chansavath; Zaunders, John James; Kelleher, Anthony Dominic

    2015-01-01

    A new generation of sensitive T cell-based assays facilitates the direct quantitation and characterization of antigen-specific T cell responses. Single-cell analyses have focused on measuring the quality and breadth of a response. Accumulating data from these studies demonstrate that there is considerable, previously-unrecognized, heterogeneity. Standard assays, such as the ICS, are often insufficient for characterization of rare subsets of cells. Enhanced flow cytometry with imaging capabilities enables the determination of cell morphology, as well as the spatial localization of the protein molecules within a single cell. Advances in both microfluidics and digital PCR have improved the efficiency of single-cell sorting and allowed multiplexed gene detection at the single-cell level. Delving further into the transcriptome of single-cells using RNA-seq is likely to reveal the fine-specificity of cellular events such as alternative splicing (i.e., splice variants) and allele-specific expression, and will also define the roles of new genes. Finally, detailed analysis of clonally related antigen-specific T cells using single-cell TCR RNA-seq will provide information on pathways of differentiation of memory T cells. With these state of the art technologies the transcriptomics and genomics of Ag-specific T cells can be more definitively elucidated. PMID:26274954

  5. High sensitivity of cancer exome-based CD8 T cell neo-antigen identification

    PubMed Central

    van Buuren, Marit M; Calis, Jorg JA; Schumacher, Ton NM

    2014-01-01

    Recent data suggest that T-cell reactivity against tumor-specific neo-antigens may be central to the clinical efficacy of cancer immunotherapy. The development of personalized vaccines designed to boost T-cell reactivity against patient specific neo-antigens has been proposed largely on the basis of these findings. Work from several groups has demonstrated that novel tumor-specific antigens can be discovered through the use of cancer exome sequencing data, thereby providing a potential pipeline for the development of patient-specific vaccines. Importantly though, it has not been established which fraction of cancer neo-antigens that can be recognized by CD8+ T cells is successfully uncovered with the current exome-based epitope prediction strategies. Here, we use a data set comprising human cancer neo-antigens that was previously identified through the use of unbiased, computational-independent strategies to describe the potential of cancer exome-based neo-antigen discovery. This analysis shows a high sensitivity of exome-guided neo-antigen prediction of approximately 70%. We propose that future research should focus on the analysis and optimization of the specificity of neo-antigen prediction, and should undoubtedly entail the clinical evaluation of patient-specific vaccines with the aim of inducing immunoreactivity against tumor-displayed neo-antigens in a physiologically relevant context. PMID:25083320

  6. How antigen specificity directs regulatory T-cell function: self, foreign and engineered specificity.

    PubMed

    Hoeppli, R E; MacDonald, K G; Levings, M K; Cook, L

    2016-07-01

    Regulatory T cells (Tregs) are a suppressive subset of T cells that have important roles in maintaining self-tolerance and preventing immunopathology. The T-cell receptor (TCR) and its antigen specificity play a dominant role in the differentiation of cells to a Treg fate, either in the thymus or in the periphery. This review focuses on the effects of the TCR and its antigen specificity on Treg biology. The role of Tregs with specificity for self-antigen has primarily been studied in the context of autoimmune disease, although recent studies have focused on their role in steady-state conditions. The role of Tregs that are specific for pathogens, dietary antigens and allergens is much less studied, although recent data suggest a significant and previously underappreciated role for Tregs during memory responses to a wide range of foreign antigens. The development of TCR- or chimeric antigen receptor (CAR)-transduced T cells means we are now able to engineer Tregs with disease-relevant antigen specificities, paving the way for ensuring specificity with Treg-based therapies. Understanding the role that antigens play in driving the generation and function of Tregs is critical for defining the pathophysiology of many immune-mediated diseases, and developing new therapeutic interventions. PMID:27256587

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

    PubMed Central

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 106 to 3 × 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. Images

  8. Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity.

    PubMed

    Fujii, Shin-ichiro; Goto, Akira; Shimizu, Kanako

    2009-04-30

    The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of alpha-galactosylceramide (alpha-GalCer)-loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with alpha-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)-mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with alpha-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases. PMID:19164596

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

    PubMed

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran

    2015-01-01

    We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3(+), CD4(+), CD8(+), TCRαβ(+), TCRγδ(+), CD7(+), CD4(+)CD7(+), CD4(+)CD7(-), and CD71(+)), B cells (HLA-DR(+), CD19(+), and HLA-DR(+)CD19(+)), NKT cells (CD3(+)CD16(+)CD56(+)), and NK cells (CD3(-)CD16(+)CD56(+)). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression. PMID:26788525

  10. Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

    PubMed Central

    Skov Sørensen, U B; Blom, J; Birch-Andersen, A; Henrichsen, J

    1988-01-01

    The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed. Images PMID:3397179

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

    PubMed Central

    González, Cecilia; Esteban, Rosa; Canals, Carme; Muñiz-Díaz, Eduardo; Nogués, Núria

    2016-01-01

    Background The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories. Methods We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment. Results TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies. Conclusions Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs. PMID:27603310

  12. Targeting cryptic epitope with modified antigen coupled to the surface of liposomes induces strong antitumor CD8 T-cell immune responses in vivo

    PubMed Central

    HORIUCHI, YUTAKA; TAKAGI, AKIRA; UCHIDA, TETSUYA; AKATSUKA, TOSHITAKA

    2015-01-01

    Active cancer immunotherapy, such as cancer vaccine, is based on the fundamental knowledge that tumor-associated antigens (TAAs) are presented on MHC molecules for recognition by specific T cells. However, most TAAs are self-antigens and are also expressed on normal tissues, including the thymus. This fact raises the issue of the tolerance of the TAA-specific T-cell repertoire and consequently the inability to trigger a strong and efficient antitumor immune response. In the present study, we used antigens chemically coupled to the surface of liposomes to target telomerase reverse transcriptase (TERT), a widely expressed self/tumor antigen. Taking advantage of the high homology between mouse and human TERT, we investigated immunogenicity and antitumor efficiency of the liposomal TERT peptides in HLA-A*0201 transgenic HHD mice. Using the heteroclitical peptide-modifying approach with antigen-coupled liposomes, we identified a novel cryptic epitope with low affinity for HLA*0201 molecules derived from TERT. The heteroclitical variant derived from this novel low affinity peptide exhibited strong affinity for HLA*0201 molecules. However, it induced only weak CD8 T-cell immune responses in HHD mice when emulsified in IFA. By contrast, when coupled to the surface of the liposomes, it induced powerful CD8 T-cell immune responses which cross-reacted against the original cryptic epitope. The induced CD8 T cells also recognized endogenously TERT-expressing tumor cells and inhibited their growth in HHD mice. These data suggest that heteroclitical antigen derived from low affinity epitope of tumor antigens coupled to the surface of liposome may have a role as an effective cancer vaccine candidate. PMID:26398429

  13. Computational Reprogramming of T Cell Antigen Receptor Binding Properties.

    PubMed

    Riley, Timothy P; Singh, Nishant K; Pierce, Brian G; Baker, Brian M; Weng, Zhiping

    2016-01-01

    T-cell receptor (TCR) binding to peptide/MHC is key to antigen-specific cellular immunity, and there has been considerable interest in modulating TCR affinity and specificity for the development of therapeutics and imaging reagents. While in vitro engineering efforts using molecular evolution have yielded remarkable improvements in TCR affinity, such approaches do not offer structural control and can adversely affect receptor specificity, particularly if the attraction towards the MHC is enhanced independently of the peptide. Here we describe an approach to computational design that begins with structural information and offers the potential for more controlled manipulation of binding properties. Our design process models point mutations in selected regions of the TCR and ranks the resulting change in binding energy. Consideration is given to designing optimized scoring functions tuned to particular TCR-peptide/MHC interfaces. Validation of highly ranked predictions can be used to refine the modeling methodology and scoring functions, improving the design process. Our approach results in a strong correlation between predicted and measured changes in binding energy, as well as good agreement between modeled and experimental structures. PMID:27094299

  14. Cognate antigen directs CD8+ T cell migration to vascularized transplants.

    PubMed

    Walch, Jeffrey M; Zeng, Qiang; Li, Qi; Oberbarnscheidt, Martin H; Hoffman, Rosemary A; Williams, Amanda L; Rothstein, David M; Shlomchik, Warren D; Kim, Jiyun V; Camirand, Geoffrey; Lakkis, Fadi G

    2013-06-01

    The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

  15. Common antigenic determinants on human melanoma, glioma, neuroblastoma, and sarcoma cells defined with monoclonal antibodies.

    PubMed

    Seeger, R C; Rosenblatt, H M; Imai, K; Ferrone, S

    1981-07-01

    Antigenic determinants that are common to melanomas, gliomas, neuroblastomas, and sarcomas but that are minimally or not detectably expressed by adult tissues were defined with monoclonal antibodies. Quantitative absorption of monoclonal antibody (Ab 165) with adult tissues followed by testing on antigen-positive UCLA-SO-M14 melanoma cells did not demonstrate antigenic determinant (Ag 165) in brain, lung, liver, kidney, intestine, adrenal, and muscle, Absorption of Ab 376 demonstrated Ag 376 in adult lung but minimal or no antigen in other tissues. Both antigens were associated with a variety of fetal tissues. Assessment of 28 human tumor cell lines with the 131I-staphylococcal Protein A-binding test demonstrated that Ab 165 reacted strongly with melanomas and gliomas and weakly with sarcomas. Ab 376 reacted strongly with melanomas, gliomas, neuroblastomas, and sarcomas. Neither of these antibodies reacted appreciably with carcinoma or teratoma cell lines. Absorption of Ab 165 and Ab 376 with noncultured tumors demonstrated that melanomas, sarcomas, and neuroblastomas can have greater quantities of these antigens in vivo than do normal adult tissues. Qualitative and quantitative antigenic heterogeneity within positive classes of tumors was demonstrated for both cultured and noncultured tumors. The differences in antigen expression in vivo between normal and neoplastic cells suggest potential value for these antibodies in immunodiagnosis and possibly immunotherapy.

  16. Whole-cell antigenicity data support sequence-based kinetoplastid taxonomy.

    PubMed

    Couvreur, Bernard

    2013-04-01

    Early immunological data, obtained by immunodiffusion and immunoelectrophoresis, on the whole-cell antigenicity of kinetoplastid protozoa were retrieved and used to construct a dendrogram of antigenic distances. Remarkably, they supported the same taxonomic conclusions as analyses based on DNA and protein sequence data.

  17. Dendritic Cells in the Periphery Control Antigen-Specific Natural and Induced Regulatory T Cells

    PubMed Central

    Yamazaki, Sayuri; Morita, Akimichi

    2013-01-01

    Dendritic cells (DCs) are specialized antigen-presenting cells that regulate both immunity and tolerance. DCs in the periphery play a key role in expanding naturally occurring Foxp3+ CD25+ CD4+ regulatory T cells (Natural T-regs) and inducing Foxp3 expression (Induced T-regs) in Foxp3− CD4+ T cells. DCs are phenotypically and functionally heterogeneous, and further classified into several subsets depending on distinct marker expression and their location. Recent findings indicate the presence of specialized DC subsets that act to expand Natural T-regs or induce Foxp3+ T-regs from Foxp3− CD4+ T cells. For example, two major subsets of DCs in lymphoid organs act differentially in inducing Foxp3+ T-regs from Foxp3− cells or expanding Natural T-regs with model-antigen delivery by anti-DC subset monoclonal antibodies in vivo. Furthermore, DCs expressing CD103 in the intestine induce Foxp3+ T-regs from Foxp3− CD4+ T cells with endogenous TGF-β and retinoic acid. In addition, antigen-presenting DCs have a capacity to generate Foxp3+ T-regs in the oral cavity where many antigens and commensals exist, similar to intestine and skin. In skin and skin-draining lymph nodes, at least six DC subsets have been identified, suggesting a complex DC-T-reg network. Here, we will review the specific activity of DCs in expanding Natural T-regs and inducing Foxp3+ T-regs from Foxp3− precursors, and further discuss the critical function of DCs in maintaining tolerance at various locations including skin and oral cavity. PMID:23801989

  18. Up-regulation of HLA class-I antigen expression and antigen-specific CTL response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid

    PubMed Central

    Mora-García, María de Lourdes; Duenas-González, Alfonso; Hernández-Montes, Jorge; De la Cruz-Hernández, Erick; Pérez-Cárdenas, Enrique; Weiss-Steider, Benny; Santiago-Osorio, Edelmiro; Ortíz-Navarrete, Vianney Francisco; Rosales, Víctor Hugo; Cantú, David; Lizano-Soberón, Marcela; Rojo-Aguilar, Martha Patricia; Monroy-García, Alberto

    2006-01-01

    Background DNA hypermethylation and histone deacetylation are epigenetic events that contribute to the absence or downregulated expression of different components of the tumor recognition complex. These events affect the processing and presentation of antigenic peptides to CTLs by HLA class-I molecules. In this work evaluated the effect of the DNA hypomethylating agent hydralazine and the histone deacetylase inhibitor valproic acid, on the expression of HLA class-I molecules and on the antigen-specific immune recognition of cervical cancer cells. Methods Cell lines C33A (HPV-), CaSki (HPV-16+) and MS751 (HPV-18+) were treated with hydralazine and valproic acid to assess the expression of HLA class-I molecules by flow cytometry and RT-PCR. Promoter methylation of HLA class-I -A, -B and C, was also evaluated by Methylation-Specific PCR. Primary cervical tumors of four HLA-A*0201 allele patients were typed for HPV and their CTL's stimulated in vitro with the T2 cell line previously loaded with 50 μM of the HPV peptides. Cytotoxicity of stimulated CTL's was assayed against Caski and MS751 cells pre-treated with hydralazine and valproic acid. Results Valproic acid and hydralazine/valproic acid up-regulated the constitutive HLA class-I expression as evaluated by flow cytometry and RT-PCR despite constitutive promoter demethylation at these loci. Hydralazine and valproic acid in combination but no IFN-gamma hyperacetylated histone H4 as evaluated by ChiP assay. The antigenic immune recognition of CaSki and MS751 cells by CTLs specific to HPV-16/18 E6 and E7-derived epitopes, was increased by VA and H/VA and the combination of H/VA/IFN-gamma. Conclusion These results support the potential use of hydralazine and valproic acid as an adjuvant for immune intervention in cervical cancer patients whenever clinical protocols based on tumor antigen recognition is desirable, like in those cases where the application of E6 and E7 based therapeutic vaccines is used. PMID:17192185

  19. Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy

    PubMed Central

    Liu, Lingfeng; Sommermeyer, Daniel; Cabanov, Alexandra; Kosasih, Paula; Hill, Tyler; Riddell, Stanley R

    2016-01-01

    The tactical introduction of Strep-tag II into synthetic antigen receptors provides engineered T cells with a marker for identification and rapid purification, and a functional element for selective antibody coated microbead-driven large-scale expansion. Such receptor designs can be applied to chimeric antigen receptors of different ligand specificities and costimulatory domains, and to T cell receptors to facilitate cGMP manufacturing of adoptive T cell therapies to treat cancer and other diseases. PMID:26900664

  20. Peptide-β2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells

    PubMed Central

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-01-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-β2-microglobulin–MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide–MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9·2 × 108 antigen-specific cytotoxic CD8+ T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8+ T cells, which could be used for adoptive T-cell therapy. PMID:17472719

  1. Peptide-beta2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells.

    PubMed

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-09-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-beta2-microglobulin-MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide-MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9.2 x 10(8) antigen-specific cytotoxic CD8(+) T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8(+) T cells, which could be used for adoptive T-cell therapy.

  2. Parasite calcineurin regulates host cell recognition and attachment by apicomplexans

    PubMed Central

    Paul, Aditya S.; Saha, Sudeshna; Engelberg, Klemens; Jiang, Rays H.Y.; Coleman, Bradley I.; Kosber, Aziz L.; Chen, Chun-Ti; Ganter, Markus; Espy, Nicole; Gilberger, Tim W.; Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2015-01-01

    SUMMARY Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans. PMID:26118996

  3. Lung adenocarcinoma with clear cell features producing carbohydrate antigen 19-9.

    PubMed

    Goto, Taichiro; Hada, Masao; Oyama, Toshio

    2015-10-01

    A 76-year-old man underwent surgery for lung cancer. Histopathologically, most of the resected tumor was composed of polygonal cells with foamy cytoplasm, and the cells were arranged predominantly in acinar patterns. In this case, although the carbohydrate antigen 19-9 level was high before surgery, it normalized after resection. The tumor was considered a carbohydrate antigen 19-9-producing tumor, which was further supported by the results of immunohistochemical analysis. Adenocarcinoma with clear cell features, producing carbohydrate antigen 19-9, is an exceedingly rare entity.

  4. Targeting to porcine sialoadhesin receptor improves antigen presentation to T cells

    PubMed Central

    Revilla, Concepción; Poderoso, Teresa; Martínez, Paloma; Álvarez, Belén; López-Fuertes, Laura; Alonso, Fernando; Ezquerra, Angel; Domínguez, Javier

    2009-01-01

    Antibody-mediated targeting of antigen to specific antigen presenting cells (APC) receptors is an attractive strategy to enhance T cell immune responses to weak immunogenic antigens. Here, we describe the characterization of two monoclonal antibodies (mAb) against different epitopes of porcine sialoadhesin (Sn) and evaluate in vitro the potential of targeting this receptor for delivery of antigens to APC for T cell stimulation. The specificity of these mAb was determined by amino acid sequence analysis of peptides derived from the affinity purified antigen. Porcine Sn is expressed by macrophages present in the border between white and red pulp of the spleen and in the subcapsular sinus of lymph nodes, an appropriate location for trapping blood and lymph-borne antigens. It is also expressed by alveolar macrophages and monocyte-derived dendritic cells (MoDC). Blood monocytes are negative for this molecule, but its expression can be induced by treatment with IFN-a. MAb bound to Sn is rapidly endocytosed. MAb to sialoadhesin induced in vitro T cell proliferation at concentrations 100-fold lower than the non-targeting control mAb when using T lymphocytes from pigs immunized with mouse immunoglobulins as responder cells and IFN-a treated monocytes or MoDC as APC, suggesting a role of sialoadhesin in antigen uptake and/or delivery into the presentation pathway in APC. PMID:19081005

  5. Targeting to porcine sialoadhesin receptor improves antigen presentation to T cells.

    PubMed

    Revilla, Concepción; Poderoso, Teresa; Martínez, Paloma; Alvarez, Belén; López-Fuertes, Laura; Alonso, Fernando; Ezquerra, Angel; Domínguez, Javier

    2009-01-01

    Antibody-mediated targeting of antigen to specific antigen presenting cells (APC) receptors is an attractive strategy to enhance T cell immune responses to weak immunogenic antigens. Here, we describe the characterization of two monoclonal antibodies (mAb) against different epitopes of porcine sialoadhesin (Sn) and evaluate in vitro the potential of targeting this receptor for delivery of antigens to APC for T cell stimulation. The specificity of these mAb was determined by amino acid sequence analysis of peptides derived from the affinity purified antigen. Porcine Sn is expressed by macrophages present in the border between white and red pulp of the spleen and in the subcapsular sinus of lymph nodes, an appropriate location for trapping blood and lymph-borne antigens. It is also expressed by alveolar macrophages and monocyte-derived dendritic cells (MoDC). Blood monocytes are negative for this molecule, but its expression can be induced by treatment with IFN-alpha. MAb bound to Sn is rapidly endocytosed. MAb to sialoadhesin induced in vitro T cell proliferation at concentrations 100-fold lower than the non-targeting control mAb when using T lymphocytes from pigs immunized with mouse immunoglobulins as responder cells and IFN-alpha treated monocytes or MoDC as APC, suggesting a role of sialoadhesin in antigen uptake and/or delivery into the presentation pathway in APC.

  6. Immunochemical studies of streptococcal cell membrane antigens immunologically related to glomerular basement membrane.

    PubMed

    Zelman, M E; Lange, C F

    1995-12-01

    Pursuing an autoimmune model for the etiology of poststreptococcal glomerulonephritis, protein antigens isolated from the cytoplasmic membrane of nephritogenic group A Type 12 Streptococcus pyogenes were immunochemically characterized using antistreptococcal cell membrane (SCM) monoclonal antibody (MAb) cross-reactive with glomerular basement membrane (GBM). Low molecular weight (9.2, 7.0, 4.7, 2.3 kDa) HPLC-purified SCM polypeptide antigens were characterized by competitive inhibition and equilibrium dialysis. Competitive inhibition of the MAb, by different sized SCM polypeptide antigens showed an inverse relationship between the size of these antigens and the molar amount required to obtain 50% inhibition of the MAb, confirming previous observations that suggested that these SCM antigens exhibit increasing epitope concentration with increasing size, that is constant epitope density. The observed changes in epitope concentration correlated with differences in the valence and affinity of the MAb as determined by equilibrium dialysis. The Kds of the MAb for 9.2-, 7.0-, 4.7-, and 2.3-kDa SCM antigens ranged from 7.42 x 10(-7) to 1.15 x 10(-5). The experimentally determined MAb valence for these antigens was 2 for the 9.2-kDa antigen and approached 10 for the smaller antigens. Finally, the similarity of these SCM antigens was reflected in similar amino acid compositions; of note, these data agreed with the compositions previously reported for sized GBM antigens. Concentrations of Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, and Leu paralleled increasing epitope concentration. Apparent N-terminal blocking prevented sequencing of these peptides, but these immunochemical data suggest that intact SCM antigen recognized by the anti-SCM MAb consists of repeating epitopes, an observation consistent with the cytoplasmic membrane source of the antigen.

  7. Red Blood Cell Antigen Genotyping for Sickle Cell Disease, Thalassemia, and Other Transfusion Complications.

    PubMed

    Fasano, Ross M; Chou, Stella T

    2016-10-01

    Since the discovery of the ABO blood group in the early 20th century, more than 300 blood group antigens have been categorized among 35 blood group systems. The molecular basis for most blood group antigens has been determined and demonstrates tremendous genetic diversity, particularly in the ABO and Rh systems. Several blood group genotyping assays have been developed, and 1 platform has been approved by the Food and Drug Administration as a "test of record," such that no phenotype confirmation with antisera is required. DNA-based red blood cell (RBC) phenotyping can overcome certain limitations of hemagglutination assays and is beneficial in many transfusion settings. Genotyping can be used to determine RBC antigen phenotypes in patients recently transfused or with interfering allo- or autoantibodies, to resolve discrepant serologic typing, and/or when typing antisera are not readily available. Molecular RBC antigen typing can facilitate complex antibody evaluations and guide RBC selection for patients with sickle cell disease (SCD), thalassemia, and autoimmune hemolytic anemia. High-resolution RH genotyping can identify variant RHD and RHCE in patients with SCD, which have been associated with alloimmunization. In the future, broader access to cost-efficient, high-resolution RBC genotyping technology for both patient and donor populations may be transformative for the field of transfusion medicine. PMID:27345938

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

    SciTech Connect

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

    1985-07-01

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

  9. Antigen availability determines CD8+ T cell-dendritic cell interaction kinetics and memory fate decisions

    PubMed Central

    Henrickson, Sarah E.; Stutte, Susanne; Quigley, Michael; Alexe, Gabriela; Iannacone, Matteo; Flynn, Michael P.; Omid, Shaida; Jesneck, Jonathan L.; Imam, Sabrina; Mempel, Thorsten R.; Mazo, Irina B.; Haining, William N.; von Andrian, Ulrich H.

    2014-01-01

    Summary T cells are activated by antigen (Ag) bearing dendritic cells (DCs) in lymph nodes in 3 phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8+ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, while higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation, but yielded different transcriptome signatures at 12h and 24h. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics. PMID:24054328

  10. Skin-Resident Antigen-Presenting Cells: Instruction Manual for Vaccine Development

    PubMed Central

    Fehres, Cynthia M.; Garcia-Vallejo, Juan J.; Unger, Wendy W. J.; van Kooyk, Yvette

    2013-01-01

    The induction of antigen-specific effector T cells is driven by proper antigen presentation and co-stimulation by dendritic cells (DCs). For this reason strategies have been developed to instruct DCs for the induction of CD4+ and CD8+ T cell responses. Since DCs are localized, amongst other locations, in peripheral tissues such as the skin, new vaccines are aiming at targeting antigens to DCs in situ. Optimal skin-DC targeting in combination with adequate adjuvant delivery facilitates DC maturation and migration to draining lymph nodes and enhances antigen cross-presentation and T cell priming. In this review we describe what DC subsets populate the human skin, as well as current vaccination strategies based on targeting strategies and alternative administration for the induction of robust long-lived anti-cancer effector T cells. PMID:23801994

  11. The relative ability of four rubella antigens to elicit blast cell transformation of lymphocytes from immune individuals.

    PubMed

    Rossier, E; Phipps, P H; Webb, T; Polley, J

    1977-05-01

    The ability of crude, semi-purified, and purified rubella antigens to elicit a specific and significant blast cell transformation of lymphocytes from immune individuals was investigated in 25 seronegative and 25 seropositive young adults. The type of preparation and the purity of the antigens were critical. Titration of the antigens by complement fixation or hemagglutination was of little value for selecting the best antigen which was a whole virion-purified antigen.

  12. The skin test antigen stimulated killer (STAK) cell mediating NK like CMC is OKM1 positive and OKT3 negative.

    PubMed Central

    Tartof, D; Curran, J J; Levitt, D; Loken, M R

    1983-01-01

    Recently we demonstrated that candida antigen stimulated natural killer cell like cell-mediated cytolysis (NK like CMC) in peripheral blood mononuclear cells (PBMNC) isolated from normal individuals (Tartof et al., 1980). Utilizing monoclonal antibodies directed against human mononuclear cell subpopulations in conjunction with a fluorescence activated cell sorter (FACS) we determined that, similar to the previously described NK cell, the skin test antigen stimulated killer (STAK) cell is a larger OKM1 positive, OKT3 negative cell. We obtained similar results using two different skin test antigens. Thus, stimulation of NK like CMC in PBMNC by skin test antigens probably represents activation of NK or NK like cells. PMID:6360439

  13. Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

    PubMed

    Aboklaish, Ali F; Ahmed, Shatha; McAllister, Douglas; Cassell, Gail; Zheng, Xiaotian T; Spiller, Owen B

    2016-08-01

    Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections. PMID:27208664

  14. Cell shape recognition by colloidal cell imprints: Energy of the cell-imprint interaction

    NASA Astrophysics Data System (ADS)

    Borovička, Josef; Stoyanov, Simeon D.; Paunov, Vesselin N.

    2015-09-01

    The results presented in this study are aimed at the theoretical estimate of the interactions between a spherical microbial cell and the colloidal cell imprints in terms of the Derjaguin, Landau, Vervey, and Overbeek (DLVO) surface forces. We adapted the Derjaguin approximation to take into account the geometry factor in the colloidal interaction between a spherical target particle and a hemispherical shell at two different orientations with respect to each other. We took into account only classical DLVO surface forces, i.e., the van der Waals and the electric double layer forces, in the interaction of a spherical target cell and a hemispherical shell as a function of their size ratio, mutual orientation, distance between their surfaces, their respective surface potentials, and the ionic strength of the aqueous solution. We found that the calculated interaction energies are several orders higher when match and recognition between the target cell and the target cell imprint is achieved. Our analysis revealed that the recognition effect of the hemispherical shell towards the target microsphere comes from the greatly increased surface contact area when a full match of their size and shape is produced. When the interaction between the surfaces of the hemishell and the target cell is attractive, the recognition greatly amplifies the attraction and this increases the likelihood of them to bind strongly. However, if the surface interaction between the cell and the imprint is repulsive, the shape and size match makes this interaction even more repulsive and thus decreases the likelihood of binding. These results show that the surface chemistry of the target cells and their colloidal imprints is very important in controlling the outcome of the interaction, while the shape recognition only amplifies the interaction. In the case of nonmonotonous surface-to-surface interaction we discovered some interesting interplay between the effects of shape match and surface chemistry

  15. Cell shape recognition by colloidal cell imprints: energy of the cell-imprint interaction.

    PubMed

    Borovička, Josef; Stoyanov, Simeon D; Paunov, Vesselin N

    2015-09-01

    The results presented in this study are aimed at the theoretical estimate of the interactions between a spherical microbial cell and the colloidal cell imprints in terms of the Derjaguin, Landau, Vervey, and Overbeek (DLVO) surface forces. We adapted the Derjaguin approximation to take into account the geometry factor in the colloidal interaction between a spherical target particle and a hemispherical shell at two different orientations with respect to each other. We took into account only classical DLVO surface forces, i.e., the van der Waals and the electric double layer forces, in the interaction of a spherical target cell and a hemispherical shell as a function of their size ratio, mutual orientation, distance between their surfaces, their respective surface potentials, and the ionic strength of the aqueous solution. We found that the calculated interaction energies are several orders higher when match and recognition between the target cell and the target cell imprint is achieved. Our analysis revealed that the recognition effect of the hemispherical shell towards the target microsphere comes from the greatly increased surface contact area when a full match of their size and shape is produced. When the interaction between the surfaces of the hemishell and the target cell is attractive, the recognition greatly amplifies the attraction and this increases the likelihood of them to bind strongly. However, if the surface interaction between the cell and the imprint is repulsive, the shape and size match makes this interaction even more repulsive and thus decreases the likelihood of binding. These results show that the surface chemistry of the target cells and their colloidal imprints is very important in controlling the outcome of the interaction, while the shape recognition only amplifies the interaction. In the case of nonmonotonous surface-to-surface interaction we discovered some interesting interplay between the effects of shape match and surface chemistry

  16. Expression of the human mucosal lymphocyte antigen, HML-1, by T cells activated with mitogen or specific antigen in vitro.

    PubMed

    Brew, R; West, D C; Burthem, J; Christmas, S E

    1995-06-01

    Expression of the human mucosal lymphocyte antigen, HML-1 (CD103), recently identified as a novel alpha E beta 7 integrin, was studied on peripheral blood lymphocytes activated with mitogen or specific antigen. HML-1 was up-regulated on PHA activated T-lymphoblasts cultured in 100IU/ml interleukin-2 (IL-2), reaching a peak of > 50% positive cells at day 7, and expression was maintained at this level throughout the 28-day culture period. Following a transient decrease in the percentage of L-selectin cells, expression of this molecule was maintained on most PHA T-lymphoblasts. Cells activated by purified protein derivative of M. tuberculosis (PPD) or in mixed lymphocyte culture also up-regulated and maintained HML-1 expression for 14 days. In contrast, in all cases the percentage of CD25+ cells rose initially but subsequently declined over the same time periods. When freshly isolated cells from tonsil, spleen, mesenteric lymph node and lung were analysed, only lung contained significant numbers (39 +/- 6%) of HML-1+ cells. In both freshly isolated and activated cell populations the great majority of HML-1+ cells co-expressed CD8 although some HML-1+ CD8- cells were also present. Production of TGF-beta 1 peaked early during T-lymphoblast and MLR cultures and was not related to induction of HML-1 expression. Immunoprecipitation studies showed that the HML-1 molecule expressed on 10-day PHA T-lymphoblasts was indistinguishable from that found on intestinal intraepithelial lymphocytes and that no alpha 4 beta 7 integrin was expressed by these cells. Although HML-1 expression is essentially restricted to mucosal leucocytes in vivo, these experiments show that it is readily induced and maintained along with co-expression of L-selectin following CD8+ T-lymphocyte activation in vitro.

  17. Artificial antigen-presenting cells expressing CD80, CD70, and 4-1BB ligand efficiently expand functional T cells specific to tumor-associated antigens.

    PubMed

    Zeng, Wanyong; Su, Mei; Anderson, Karen S; Sasada, Tetsuro

    2014-08-01

    Professional antigen-presenting cells (APCs), notably dendritic cells (DCs), are the most potent for expanding antigen-specific T cells ex vivo. However, the labor-intensive and expensive procedure for customized preparation of autologous APCs has hampered their broad clinical application. Artificial APC (aAPC) systems, which can be readily prepared from off-the-shelf components, have been proposed as a promising alternative to custom-made professional APCs. Here, in order to develop a novel aAPC system, we established K562 erythroleukemia cells expressing different combinations of co-stimulatory molecule ligands, CD80, CD70, and/or 4-1BB ligand (4-1BBL). When nucleofected with in vitro-generated mRNA encoding a tumor-associated antigen, MART-1, the K562 cells expressing all of CD80, CD70, and 4-1BBL were the most efficient for expansion of functional T cells specific to an HLA-A2-restricted immunodominant epitope, MART-126-35. In addition, only the K562 cells expressing all three of these co-stimulatory molecule ligands could clearly expand T cells specific to other less immunogenic antigen epitopes, gp100154-162 and Cyp1B1239-247, through transfection with in vitro generated gp100 and Cyp1B1 mRNA, respectively. These results indicated that non-redundant and synergistic effects of co-stimulation via CD28/CD80, CD27/CD70, and 4-1BB/4-1BBL might be critical for eliciting efficient expansion of T cells; co-stimulation via the 4-1BB/4-1BBL interaction might expand antigen-specific T cells by preventing apoptotic cell death triggered by specific antigens in the presence of the CD28/CD80 and CD27/CD70 signaling. Taken together, our findings suggested that this K562-based aAPC system expressing CD80, CD70, and 4-1BBL would be useful for efficiently stimulating functional antigen-specific T cells ex vivo, in particular when detailed information on the epitope specificities is unavailable. PMID:24713579

  18. Host cell-induced components of the sulfate assimilation pathway are major protective antigens of Mycobacterium tuberculosis.

    PubMed

    Pinto, Rachel; Leotta, Lisa; Shanahan, Erin R; West, Nicholas P; Leyh, Thomas S; Britton, Warwick; Triccas, James A

    2013-03-01

    New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5'-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans. PMID:23225904

  19. Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal Lactobacillus

    PubMed Central

    Nagy, Lauren H.; Grishina, Irina; Macal, Monica; Hirao, Lauren A.; Hu, William K.; Sankaran-Walters, Sumathi; Gaulke, Christopher A.; Pollard, Richard; Brown, Jennifer; Suni, Maria; Baumler, Andreas J.; Ghanekar, Smita; Marco, Maria L.; Dandekar, Satya

    2013-01-01

    Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation. PMID:24023646

  20. Uncoupling protein 2 regulates metabolic reprogramming and fate of antigen-stimulated CD8+ T cells.

    PubMed

    Chaudhuri, Leena; Srivastava, Rupesh K; Kos, Ferdynand; Shrikant, Protul A

    2016-07-01

    Adoptive cell therapy (ACT) employing ex vivo-generated tumor antigen-specific CD8+ T cells shows tumor efficacy when the transferred cells possess both effector and memory functions. New strategies based on understanding of mechanisms that balance CD8+ T cell differentiation toward effector and memory responses are highly desirable. Emerging information confirms a central role for antigen-induced metabolic reprogramming in CD8+ T cell differentiation and clonal expansion. The mitochondrial protein uncoupling protein 2 (UCP2) is induced by antigen stimulation of CD8+ T cells; however, its role in metabolic reprogramming underlying differentiation and clonal expansion has not been reported. Employing genetic (siRNA) and pharmacologic (Genipin) approaches, we note that antigen-induced UCP2 expression reduces glycolysis, fatty acid synthesis and production of reactive oxygen species to balance differentiation with survival of effector CD8+ T cells. Inhibition of UCP2 promotes CD8+ T cell terminal differentiation into short-lived effector cells (CD62L(lo)KLRG1(Hi)IFNγ(Hi)) that undergo clonal contraction. These findings are the first to reveal a role for antigen-induced UCP2 expression in balancing CD8+ T cell differentiation and survival. Targeting UCP2 to regulate metabolic reprogramming of CD8+ T cells is an attractive new approach to augment efficacy of tumor therapy by ACT. PMID:27271549

  1. Antigen selection in B-cell lymphomas--tracing the evidence.

    PubMed

    Sutton, Lesley-Ann; Agathangelidis, Andreas; Belessi, Chrysoula; Darzentas, Nikos; Davi, Frederic; Ghia, Paolo; Rosenquist, Richard; Stamatopoulos, Kostas

    2013-12-01

    While signaling through the B cell receptor (BcR) facilitates B cell development and maintenance, it also carries intertwined risks for the development of lymphomas since malignant B cells can exploit these pathways in order to trigger and fuel clonal expansion. This corruption of the normal B cell response to antigens, leading to sustained BcR signaling, has given great impulse to investigate in detail the role of antigen in lymphomas. Suffice it to conclude from such studies, largely immunogenetics based, that the evidence implicating antigens (exogenous or self) in lymphoma development is substantial and that lymphomagenesis is functionally driven and dynamic, rather than a simple stochastic process. As the paradigm of antigen-driven lymphoma evolves, further investigation will be paramount to the identification of the inciting agent(s) that may be responsible for immunoproliferative neoplasms and also for the development of therapeutic agents targeting effectors of the BcR signaling pathway.

  2. Antigens for CD4 and CD8 T Cells in Tuberculosis

    PubMed Central

    Lindestam Arlehamn, Cecilia S.; Lewinsohn, David; Sette, Alessandro; Lewinsohn, Deborah

    2014-01-01

    Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (MTB), represents an important cause of morbidity and mortality worldwide for which an improved vaccine and immunodiagnostics are urgently needed. CD4+ and CD8+ T cells play an important role in host defense to TB. Definition of the antigens recognized by these T cells is critical for improved understanding of the immunobiology of TB and for development of vaccines and diagnostics. Herein, the antigens and epitopes recognized by classically HLA class I– and II–restricted CD4+ and CD8+ T cells in humans infected with MTB are reviewed. Immunodominant antigens and epitopes have been defined using approaches targeting particular TB proteins or classes of proteins and by genome-wide discovery approaches. Antigens and epitopes recognized by classically restricted CD4+ and CD8+ T cells show extensive breadth and diversity in MTB-infected humans. PMID:24852051

  3. Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

    PubMed

    Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

    2015-02-01

    Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

  4. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    SciTech Connect

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-05-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with /sup 111/In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

  5. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    PubMed

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

  6. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    PubMed

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape. PMID:27427982

  7. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    PubMed

    Kitchen, Scott G; Bennett, Michael; Galić, Zoran; Kim, Joanne; Xu, Qing; Young, Alan; Lieberman, Alexis; Joseph, Aviva; Goldstein, Harris; Ng, Hwee; Yang, Otto; Zack, Jerome A

    2009-01-01

    There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR). Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  8. Hepatitis B surface antigen-specific cell-mediated immune responses in human chronic hepatitis B surface antigen carriers.

    PubMed Central

    Beutner, K R; Tiku, M L; Ogra, P L

    1978-01-01

    The presence of hepatitis B surface antigen (HBsAg) and antibody (anti-HBs), hepatitis B e antigen (HBeAg) and antibody (anti-HBe), the nature of T-cell function, and specific cell-mediated immunity to HBsAg were determined and evaluated serially in groups of subjects with chronic HBsAg carrier states and in seronegative controls. The techniques of in vitro lymphocyte transformation, spontaneous rosette formation, radioimmunoassay, reverse passive hemagglutination, passive hemagglutination, rheophoresis, and liver function tests were employed for these studies. For the lymphocyte transformation assay, multiple concentrations of phytohemagglutinin and purified HBsAg were used as stimulants. Cell-mediated immunity to HBsAg was detectable in 50% of the chronic HBsAg carriers (responders) at one or more concentrations of HBsAg. The remaining carriers (nonresponders) and controls failed to manifest HBsAg-specific lymphocyte transformation activity. The profile of the responders was characterized by elevated serum glutamic pyruvic transaminase levels, the presence of anti-HBe, high HBsAg titers, and the conspicuous absence of HBeAg in the serum. The nonresponders were characterized by normal serum glutamic pyruvic transaminase levels, the presence of HBeAg and anti-HBe, and lower HBsAg titers. These observations demonstrate the presence of specific cell-mediated immunity to HBsAg in chronic HBsAg carriers who manifest biochemical evidence of liver disease. PMID:80380

  9. Antigen-specific CD8(+) T cells fail to respond to Shigella flexneri.

    PubMed

    Jehl, Stephanie P; Doling, Amy M; Giddings, Kara S; Phalipon, Armelle; Sansonetti, Philippe J; Goldberg, Marcia B; Starnbach, Michael N

    2011-05-01

    CD8(+) T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8(+) T cells are not required for protective immunity to the enteric pathogen Shigella flexneri, despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8(+) T cells. To determine why CD8(+) T cells fail to play a role in adaptive immunity to S. flexneri, we investigated whether antigen-specific CD8(+) T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8(+) T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8(+) T-cell epitope via the Shigella type III secretion system and characterized the CD8(+) T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella. Collectively, these results suggest that antigen-specific CD8(+) T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection.

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

    SciTech Connect

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

    1990-09-01

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

  11. Transient induction of a nuclear antigen unrelated to Epstein-Barr nuclear antigen in cells of two human B-lymphoma lines converted by Epstein-Barr virus.

    PubMed

    Fresen, K O; zur Hausen, H

    1977-01-01

    Infection of cells of the Epstein-Barr virus (EBV)-negative human B-lymphoma lines BJAB and Ramos with EBV preparations from P3HR-1 or B 95-8 cells converted these cells to EBV genome carriers expressing Epstein-Barr nuclear antigen (EBNA) in almost 100% of these cells. Induction of these cells as well as of clones from P3HR-1 EBV-converted BJAB cells with iododeoxyuridine, aminopterin, and hypoxanthine resulted in the appearance of a nuclear antigen in about 1-6% of the cells 1-4 days after induction. The antigen is different from known EBV-induced antigens like EBNA, viral capsid antigen (VCA) or the D- and R-subspecificities of the early antigen (EA) complex. It is demonstrated by indirect immunofluorescence and inactivated after acetone fixation. The antigen was not detectable after induction of uninfected BJAB and Ramos cells nor has it been found in noninduced or induced P3HR-1 and Raji cells. Thus, it appears that EBV-infection mediates the expression of this antigen, for which the name TINA (transiently induced nuclear antigen) is suggested. Sera reacting against TINA generally contained high antibody titers against EBV-induced EA. Only a limited number of highly EA-reactive sera, however, were also positive for TINA. Among 200 sera tested thus far, TINA reactivity was most frequently observed in sera of patients with nasopharyngeal carcinoma (7 out of 28), in sera of the only two patients with immunoblastoma tested and occasionally in sera from patients with Hodgkin's disease and chronic lymphatic leukemia. Among 70 sera from nontumor patients, TINA reactivity was observed three times: two patients suffered from "chronic" infectious mononucleosis, the other revealed persistent splenomegaly. PMID:189313

  12. Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site.

    PubMed

    Julien, Jean-Philippe; Bryson, Steve; Nieva, Jose L; Pai, Emil F

    2008-12-12

    2F5 is a monoclonal antibody with potent and broadly neutralizing activity against HIV-1. It targets the membrane-proximal external region (MPER) of the gp41 subunit of the envelope glycoprotein and interferes with the process of fusion between viral and host cell membranes. This study presents eight 2F5 F(ab)' crystal structures in complex with various gp41 peptide epitopes. These structures reveal several key features of this antibody-antigen interaction. (1) Whenever free of contacts caused by crystal artifacts, the extended complementarity-determining region H3 loop is mobile; this is true for ligand-free and epitope-bound forms. (2) The interaction between the antibody and the gp41 ELDKWA epitope core is absolutely critical, and there are also close and specific contacts with residues located N-terminal to the epitope core. (3) Residues located at the C-terminus of the gp41 ELDKWA core do not interact as tightly with the antibody. However, in the presence of a larger peptide containing the gp41 fusion peptide segment, these residues adopt a conformation consistent with the start of an alpha-helix. (4) At high sulfate concentrations, the electron density maps of 2F5 F(ab)'-peptide complexes contain a peak that may mark a binding site for phosphate groups of negatively charged lipid headgroups. The refined atomic-level details of 2F5 paratope-epitope interactions revealed here should contribute to a better understanding of the mechanism of 2F5-based virus neutralization, in general, and prove important for the design of potential vaccine candidates intended to elicit 2F5-like antibody production.

  13. Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

    SciTech Connect

    Wang, B.S.; Lumanglas, A.L.; Silva, J.; Ruszala-Mallon, V.; Durr, F.E.

    1987-04-15

    Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of /sup 111/In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

  14. Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.

    PubMed

    Hegde, Meenakshi; Corder, Amanda; Chow, Kevin K H; Mukherjee, Malini; Ashoori, Aidin; Kew, Yvonne; Zhang, Yi Jonathan; Baskin, David S; Merchant, Fatima A; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Wu, Meng Fen; Liu, Hao; Heslop, Helen E; Gottschalk, Stephen; Gottachalk, Stephen; Yvon, Eric; Ahmed, Nabil

    2013-11-01

    Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

  15. Codelivery of antigen and an immune cell adhesion inhibitor is necessary for efficacy of soluble antigen arrays in experimental autoimmune encephalomyelitis

    PubMed Central

    Sestak, Joshua O; Sullivan, Bradley P; Thati, Sharadvi; Northrup, Laura; Hartwell, Brittany; Antunez, Lorena; Forrest, M Laird; Vines, Charlotte M; Siahaan, Teruna J; Berkland, Cory

    2014-01-01

    Autoimmune diseases such as multiple sclerosis (MS) are typified by the misrecognition of self-antigen and the clonal expansion of autoreactive T cells. Antigen-specific immunotherapies (antigen-SITs) have long been explored as a means to desensitize patients to offending self-antigen(s) with the potential to retolerize the immune response. Soluble antigen arrays (SAgAs) are composed of hyaluronic acid (HA) cografted with disease-specific autoantigen (proteolipid protein peptide) and an ICAM-1 inhibitor peptide (LABL). SAgAs were designed as an antigen-SIT that codeliver peptides to suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Codelivery of antigen and cell adhesion inhibitor (LABL) conjugated to HA was essential for SAgA treatment of EAE. Individual SAgA components or mixtures thereof reduced proinflammatory cytokines in cultured splenocytes from EAE mice; however, these treatments showed minimal to no in vivo therapeutic effect in EAE mice. Thus, carriers that codeliver antigen and a secondary “context” signal (e.g., LABL) in vivo may be an important design criteria to consider when designing antigen-SIT for autoimmune therapy. PMID:26015953

  16. THE MECHANISM OF ANTIGENIC STIMULATION OF PRIMARY AND SECONDARY CLONAL PRECURSOR CELLS

    PubMed Central

    Klinman, Norman R.

    1972-01-01

    Cell transfers to carrier-immunized irradiated mice have permitted an analysis of the in vitro stimulation of clonal precursors of anti-2,4-dinitrophenyl (DNP) antibody-producing cells derived from both immune and nonimmune mice. The results indicate that: (a) carrier-specific enhancement is obligatory for stimulation of primary precursor cells and increases both the size and number of detectable foci derived from secondary precursors. (b) This carrier-specific enhancement is most apparent in the stimulation of precursors of high-affinity antibody producer cells. (c) The antibody produced by primary foci, like that of secondary foci, appears homogeneous. (d) The frequency of clonal precursors in normal spleens is 38% that in spleens from mice 4–8 months after immunization, and the number of such precursors in normal spleens can be reduced fivefold by specific suppression of donor mice with soluble antigen. (e) The average of association constants of primary monofocal antibodies, like that of primary serum antibody produced in carrier-primed mice, is less than 10-fold lower than that of secondary clonal or serum antibody. (f) The affinity of primary monofocal antibodies shows a slight dependence on stimulating antigen concentration; however, a minimum threshold affinity consonant with stimulation is apparent. (g) Free hapten inhibits antigenic stimulation of primary precursor cells at a much lower concentration than is required for the inhibition of secondary precursors. These results are interpreted as indicating that (a) primary stimulation, like secondary stimulation, results from the selective stimulation by antigen of a population of cells differing from one another in their potential antibody product but each having only a single such product; (b) the antigen receptors of primary cells interact with antigen as if they are monovalent while receptors of secondary cells evidence multivalence; (c) antigenic stimulation appears to require both a relatively high

  17. Innate immune pattern recognition: a cell biological perspective.

    PubMed

    Brubaker, Sky W; Bonham, Kevin S; Zanoni, Ivan; Kagan, Jonathan C

    2015-01-01

    Receptors of the innate immune system detect conserved determinants of microbial and viral origin. Activation of these receptors initiates signaling events that culminate in an effective immune response. Recently, the view that innate immune signaling events rely on and operate within a complex cellular infrastructure has become an important framework for understanding the regulation of innate immunity. Compartmentalization within this infrastructure provides the cell with the ability to assign spatial information to microbial detection and regulate immune responses. Several cell biological processes play a role in the regulation of innate signaling responses; at the same time, innate signaling can engage cellular processes as a form of defense or to promote immunological memory. In this review, we highlight these aspects of cell biology in pattern-recognition receptor signaling by focusing on signals that originate from the cell surface, from endosomal compartments, and from within the cytosol.

  18. Targeting cryptic epitope with modified antigen coupled to the surface of liposomes induces strong antitumor CD8 T-cell immune responses in vivo.

    PubMed

    Horiuchi, Yutaka; Takagi, Akira; Uchida, Tetsuya; Akatsuka, Toshitaka

    2015-12-01

    Active cancer immunotherapy, such as cancer vaccine, is based on the fundamental knowledge that tumor‑associated antigens (TAAs) are presented on MHC molecules for recognition by specific T cells. However, most TAAs are self-antigens and are also expressed on normal tissues, including the thymus. This fact raises the issue of the tolerance of the TAA‑specific T‑cell repertoire and consequently the inability to trigger a strong and efficient antitumor immune response. In the present study, we used antigens chemically coupled to the surface of liposomes to target telomerase reverse transcriptase (TERT), a widely expressed self/tumor antigen. Taking advantage of the high homology between mouse and human TERT, we investigated immunogenicity and antitumor efficiency of the liposomal TERT peptides in HLA-A*0201 transgenic HHD mice. Using the heteroclitical peptide-modifying approach with antigen‑coupled liposomes, we identified a novel cryptic epitope with low affinity for HLA*0201 molecules derived from TERT. The heteroclitical variant derived from this novel low affinity peptide exhibited strong affinity for HLA*0201 molecules. However, it induced only weak CD8 T‑cell immune responses in HHD mice when emulsified in IFA. By contrast, when coupled to the surface of the liposomes, it induced powerful CD8 T‑cell immune responses which cross-reacted against the original cryptic epitope. The induced CD8 T cells also recognized endogenously TERT‑expressing tumor cells and inhibited their growth in HHD mice. These data suggest that heteroclitical antigen derived from low affinity epitope of tumor antigens coupled to the surface of liposome may have a role as an effective cancer vaccine candidate.

  19. Targeting Antigens to Dec-205 on Dendritic Cells Induces Immune Protection in Experimental Colitis in Mice

    PubMed Central

    Wadwa, Munisch; Klopfleisch, Robert; Buer, Jan; Westendorf, Astrid M.

    2016-01-01

    The endocytotic c-type lectin receptor DEC-205 is highly expressed on immature dendritic cells. In previous studies, it was shown that antigen-targeting to DEC-205 is a useful tool for the induction of antigen-specific Foxp3+ regulatory T cells and thereby can prevent inflammatory processes. However, whether this approach is sufficient to mediate tolerance in mucosal tissues like the gut is unknown. In this study, we established a new mouse model in which the adoptive transfer of naive hemagglutinin (HA)-specific CD4+Foxp3– T cells into VILLIN-HA transgenic mice leads to severe colitis. To analyze if antigen-targeting to DEC-205 could protect against inflammation of the gut, VILLIN-HA transgenic mice were injected with an antibody–antigen complex consisting of the immunogenic HA110–120 peptide coupled to an α-DEC-205 antibody (DEC-HA) before adoptive T cell transfer. DEC-HA-treated mice showed significantly less signs of intestinal inflammation as was demonstrated by reduced loss of body weight and histopathology in the gut. Strikingly, abrogated intestinal inflammation was mediated via the conversion of naive HA-specific CD4+Foxp3– T cells into HA-specific CD4+Foxp3+ regulatory T cells. In this study, we provide evidence that antigen-targeting to DEC-205 can be utilized for the induction of tolerance in mucosal organs that are confronted with large numbers of exogenous antigens. PMID:27141310

  20. Atomic resolution insight into host cell recognition by Toxoplasma gondii.

    PubMed

    Blumenschein, Tharin M A; Friedrich, Nikolas; Childs, Robert A; Saouros, Savvas; Carpenter, Elisabeth P; Campanero-Rhodes, Maria A; Simpson, Peter; Chai, Wengang; Koutroukides, Theodoros; Blackman, Michael J; Feizi, Ten; Soldati-Favre, Dominique; Matthews, Stephen

    2007-06-01

    The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell-binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high-resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein. We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite-receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies. PMID:17491595

  1. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    PubMed Central

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  2. Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans.

    PubMed

    Pavia, J; Aguado, C; Mormeneo, S; Sentandreu, R

    2001-07-01

    The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls. PMID:11429475

  3. Killer artificial antigen-presenting cells: the synthetic embodiment of a 'guided missile'.

    PubMed

    Schütz, Christian; Oelke, Mathias; Schneck, Jonathan P; Mackensen, Andreas; Fleck, Martin

    2010-07-01

    At present, the treatment of T-cell-dependent autoimmune diseases relies exclusively on strategies leading to nonspecific suppression of the immune systems causing a substantial reduced ability to control concomitant infections or malignancies. Furthermore, long-term treatment with most drugs is accompanied by several serious adverse effects and does not consequently result in cure of the primary immunological malfunction. By contrast, antigen-specific immunotherapy offers the potential to achieve the highest therapeutic efficiency in accordance with minimal adverse effects. Therefore, several studies have been performed utilizing antigen-presenting cells specifically engineered to deplete allo- or antigen-specific T cells ('guided missiles'). Many of these strategies take advantage of the Fas/Fas ligand signaling pathway to efficiently induce antigen-presenting cell-mediated apoptosis in targeted T cells. In this article, we discuss the advantages and shortcomings of a novel non-cell-based 'killer artificial antigen-presenting cell' strategy, developed to overcome obstacles related to current cell-based approaches for the treatment of T-cell-mediated autoimmunity. PMID:20636007

  4. Increased competition for antigen during priming negatively impacts the generation of memory CD4 T cells

    PubMed Central

    Blair, David A.; Lefrançois, Leo

    2007-01-01

    The factors involved in the differentiation of memory CD4 T cells from naïve precursors are poorly understood. We developed a system to examine the effect of increased competition for antigen by CD4 T cells on the generation of memory in response to infection with a recombinant vesicular stomatitis virus. Competition was initially regulated by increasing the precursor frequency of adoptively transferred naïve T cell antigen receptor transgenic CD4 T cells. Despite robust proliferation at high precursor frequencies, memory CD4 T cells did not develop, whereas decreasing the input number of naïve CD4 T cells promoted memory development after infection. The lack of memory development was linked to reduced blastogenesis and poor effector cell induction, but not to initial recruitment or proliferation of antigen-specific CD4 T cells. To prove that availability of antigen alone could regulate memory CD4 T cell development, we used treatment with an mAb specific for the epitope recognized by the transferred CD4 T cells. At high doses, this mAb effectively inhibited the antigen-specific CD4 T cell response. However, at a very low dose of mAb, primary CD4 T cell expansion was unaffected, although memory development was dramatically reduced. Moreover, the induction of effector function was concomitantly inhibited. Thus, competition for antigen during CD4 T cell priming is a major contributing factor to the development of the memory CD4 T cell pool. PMID:17827281

  5. Induction of primary, antiviral cytotoxic, and proliferative responses with antigens administered via dendritic cells.

    PubMed Central

    Nair, S; Babu, J S; Dunham, R G; Kanda, P; Burke, R L; Rouse, B T

    1993-01-01

    Cytotoxic T lymphocytes (CTL) play an essential role in recovery from viral infections, but induction of CTL responses with nonreplicating antigens is difficult to achieve. Exogenous antigens, such as viral proteins and peptides, normally induce CD4+ T-cell responses unless appropriately delivered to the major histocompatibility complex class I antigen presentation pathway. In vitro studies performed to address this issue revealed a similar scenario, and primary CTL induction with nonreplicating antigens has rarely been reported. This study demonstrated primary antiviral CTL induction in vitro with exogenous antigens delivered in vivo to dendritic cells. This study also evaluated the efficacy of glycoprotein B peptide (free or encapsulated in liposomes), peptide-tripalmitoyl-S-glyceryl cysteinyl conjugate (acylpeptide), and glycoprotein B protein encapsulated in pH-sensitive liposomes as antigen delivery vehicles. Our results show that higher levels of cytotoxicity against herpes simplex virus type 1 resulted from exposure of dendritic cells to peptide-tripalmitoyl-S-glyceryl cysteinyl in liposomes. Macrophages treated in a similar manner were not effective stimulators for primary CTL induction. Our data have relevance to the understanding of mechanisms of antigen processing and presentation and the design of antiviral vaccines. PMID:8510217

  6. SEROLOGICAL ANALYSES OF CELLULAR SLIME-MOLD DEVELOPMENT. I. CHANGES IN ANTIGENIC ACTIVITY DURING CELL AGGREATION.

    PubMed

    SONNEBORN, D R; SUSSMAN, M; LEVINE, L

    1964-06-01

    Sonneborn, D. R. (Brandeis University, Waltham, Mass.), M. Sussman, and L. Levine. Serological analysis of cellular slime-mold development. I. Changes in antigenic activity during cell aggregation. J. Bacteriol. 87:1321-1329. 1964.-During aggregation in Dictyostelium discoideum, the concentration of a single antigenic determinant increased markedly, starting from very low or undetectable levels. Subsequently, the determinant appeared to segregate preferentially into the stalks of terminal fruiting bodies. Sera containing the antibody specific for this determinant inhibited the aggregation of D. discoideum without disturbing cell viability. The properties of the antigen during fractionation are consistent with the supposition that it may be a protein associated with the cell membrane. The ability or inability of three species to coaggregate with D. discoideum was correlated with the presence or absence of the antigenic determinant in aggregates of these species.

  7. Successive Administration of Streptococcus Type 5 Group A Antigens and S. typhimurium Antigenic Complex Corrects Elevation of Serum Cytokine Concentration and Number of Bone Marrow Stromal Pluripotent Cells in CBA Mice Induced by Each Antigen Separately.

    PubMed

    Gorskaya, Yu F; Danilova, T A; Grabko, V I; Nesterenko, V G

    2015-12-01

    Administration of bacterial antigens to CBA mice induced an increase in serum concentration of virtually all cytokines with a peak in 4 h after administration of S. typhimurium antigens and in 7 h after administration of streptococcus antigens. In 20 h, cytokine concentrations returned to the control level or were slightly below it. In 4 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, we observed a significant decrease in serum concentrations of IFN-γ, IL-10, GM-CSF, IL-12, and TNF-α, in comparison with injection S. typhimurium antigens alone and IL-5, IL-10, GM-CSF, and TNF-α in comparison with injection of streptococcus antigens alone; the concentrations of IL-2 and IFN-γ, in contrast, increased by 1.5 times in this case. In 20 h after administration of S. typhimurium antigens, the number of multipotential stromal cells (MSC) in the bone marrow and their cloning efficiency (ECF-MSC) increased by 4.8 and 4.4 times, respectively, in comparison with the control, while after administration of streptococcus antigens by 2.6 and 2.4 times, respectively. In 20 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, these parameters increased by 3.2 and 2.9 times, respectively, in comparison with the control, i.e. the observed increase in the level of MSC count and ECF-MSC is more consistent with the response of the stromal tissue to streptococcus antigens. Thus, successive administration of two bacterial antigens corrected both serum cytokine profiles and MSC response to administration of each antigen separately, which indicates changeability of the stromal tissue in response to changes in the immune response.

  8. Molecular Determinants of T Cell Epitope Recognition to the Common Timothy Grass Allergen

    PubMed Central

    Oseroff, Carla; Sidney, John; Kotturi, Maya F.; Kolla, Ravi; Alam, Rafeul; Broide, David H.; Wasserman, Stephen I.; Weiskopf, Daniela; McKinney, Denise M.; Chung, Jo L.; Petersen, Arnd; Grey, Howard; Peters, Bjoern; Sette, Alessandro

    2012-01-01

    We investigated the molecular determinants of allergen-derived T cell epitopes in humans utilizing the Phleum pratense (Timothy grass) allergens (Phl p). PBMCs from allergic individuals were tested in ELISPOT assays with overlapping peptides spanning known Phl p allergens. A total of 43 distinct antigenic regions were recognized, illustrating the large breadth of grass-specific T cell epitopes. Th2 cytokines (as represented by IL-5) were predominant, whereas IFN-γ, IL-10, and IL-17 were detected less frequently. Responses from specific immunotherapy treatment individuals were weaker and less consistent, yet similar in epitope specificity and cytokine pattern to allergic donors, whereas nonallergic individuals were essentially nonreactive. Despite the large breadth of recognition, nine dominant antigenic regions were defined, each recognized by multiple donors, accounting for 51% of the total response. Multiple HLA molecules and loci restricted the dominant regions, and the immunodominant epitopes could be predicted using bioinformatic algorithms specific for 23 common HLA-DR, DP, and DQ molecules. Immunodominance was also apparent at the Phl p Ag level. It was found that 52, 19, and 14% of the total response was directed to Phl p 5, 1, and 3, respectively. Interestingly, little or no correlation between Phl p-specific IgE levels and T cell responses was found. Thus, certain intrinsic features of the allergen protein might influence immunogenicity at the level of T cell reactivity. Consistent with this notion, different Phl p Ags were associated with distinct patterns of IL-5, IFN-γ, IL-10, and IL-17 production. PMID:20554959

  9. Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses.

    PubMed

    Diwan, Manish; Elamanchili, Praveen; Lane, Helena; Gainer, Anita; Samuel, John

    2003-01-01

    Dendritic cells (DCs) in the peripheral tissues act as sentinels of the immune system. They detect and capture pathogens entering the body and present their antigens to T cells to trigger responses directed towards elimination of the pathogen. The induction of peripheral tolerance against self and certain foreign antigens is also believed to be mediated through DCs. The outcome of any immune response is largely controlled by the microenvironment of antigen capture, processing and presentation by DCs. The "context" of antigen delivery to DCs will directly influence the microenvironment of antigen presentation and hence the regulation of immune responses. We report here preliminary investigations describing the formulation of a pharmaceutically acceptable, biodegradable, and strategic nanoparticulate delivery system, and its application for efficient antigen loading of DCs to achieve antigen specific T cell activation. "Pathogen-mimicking" nanoparticles capable of interacting with DCs were fabricated by incorporating monophosphoryl lipid A (MPLA; toll-like receptor (TLR) 4 ligand) or CpG ODN (seq #2006; TLR9 ligand) in biodegradable copolymer, poly(D,L,-lactic-co-glycolic acid) (PLGA). The uptake of PLGA nanoparticles by human umbilical cord blood derived DCs (DCs propagated from CD34 progenitors) was conclusively demonstrated by scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM). Cell phenotype at day 12 of cultures was determined as immature DC using specific cell surface markers, i.e. CD11c (approximately 90%), MHC-II (approximately 70%), CD86 (approximately 20%), CD83 (approximately 5%), CD80 (approximately 40%), CD40 (approximately 40%), and CCR7 (approximately 5%). Tetanus toxoid (TT), a model antigen, was encapsulated in nanoparticles along with an immunomodulator, i.e. either MPLA or CpG ODN. DCs pulsed with various antigen formulations were co-cultured with autologous naïve T cells at

  10. Human cell-based artificial antigen-presenting cells for cancer immunotherapy

    PubMed Central

    Butler, Marcus O.; Hirano, Naoto

    2013-01-01

    Summary Adoptive T-cell therapy, where antitumor T cells are first prepared in vitro, is attractive since it facilitates the delivery of essential signals to selected subsets of antitumor T cells without unfavorable immunoregulatory issues that exist in tumor-bearing hosts. Recent clinical trials have demonstrated that antitumor adoptive T-cell therapy, i.e. infusion of tumor-specific T cells, can induce clinically relevant and sustained responses in patients with advanced cancer. The goal of adoptive cell therapy is to establish antitumor immunological memory, which can result in life-long rejection of tumor cells in patients. To achieve this goal, during the process of in vitro expansion, T-cell grafts used in adoptive T-cell therapy must to be appropriately educated and equipped with the capacity to accomplish multiple, essential tasks. Adoptively transferred T cells must be endowed, prior to infusion, with the ability to efficiently engraft, expand, persist, and traffic to tumor in vivo. As a strategy to consistently generate T-cell grafts with these capabilities, artificial antigen-presenting cells have been developed to deliver the proper signals necessary to T cells to enable optimal adoptive cell therapy. PMID:24329798

  11. Human cell-based artificial antigen-presenting cells for cancer immunotherapy.

    PubMed

    Butler, Marcus O; Hirano, Naoto

    2014-01-01

    Adoptive T-cell therapy, where anti-tumor T cells are first prepared in vitro, is attractive since it facilitates the delivery of essential signals to selected subsets of anti-tumor T cells without unfavorable immunoregulatory issues that exist in tumor-bearing hosts. Recent clinical trials have demonstrated that anti-tumor adoptive T-cell therapy, i.e. infusion of tumor-specific T cells, can induce clinically relevant and sustained responses in patients with advanced cancer. The goal of adoptive cell therapy is to establish anti-tumor immunologic memory, which can result in life-long rejection of tumor cells in patients. To achieve this goal, during the process of in vitro expansion, T-cell grafts used in adoptive T-cell therapy must be appropriately educated and equipped with the capacity to accomplish multiple, essential tasks. Adoptively transferred T cells must be endowed, prior to infusion, with the ability to efficiently engraft, expand, persist, and traffic to tumor in vivo. As a strategy to consistently generate T-cell grafts with these capabilities, artificial antigen-presenting cells have been developed to deliver the proper signals necessary to T cells to enable optimal adoptive cell therapy. PMID:24329798

  12. Recognition and Regulation of T Cells by NK Cells

    PubMed Central

    Pallmer, Katharina; Oxenius, Annette

    2016-01-01

    Regulation of T cell responses by innate lymphoid cells (ILCs) is increasingly documented and studied. Direct or indirect crosstalk between ILCs and T cells early during and after T cell activation can affect their differentiation, polarization, and survival. Natural killer (NK) cells that belong to the ILC1 group were initially described for their function in recognizing and eliminating “altered self” and as source of early inflammatory cytokines, most notably type II interferon. Using signals conveyed by various germ-line encoded activating and inhibitory receptors, NK cells are geared to sense sudden cellular changes that can be caused by infection events, malignant transformation, or cellular stress responses. T cells, when activated by TCR engagement (signal 1), costimulation (signal 2), and cytokines (signal 3), commit to a number of cellular alterations, including entry into rapid cell cycling, metabolic changes, and acquisition of effector functions. These abrupt changes may alert NK cells, and T cells might thereby expose themselves as NK cell targets. Here, we review how activated T cells can be recognized and regulated by NK cells and what consequences such regulation bears for T cell immunity in the context of vaccination, infection, or autoimmunity. Conversely, we will discuss mechanisms by which activated T cells protect themselves against NK cell attack and outline the significance of this safeguard mechanism. PMID:27446081

  13. Comprehensive red blood cell and platelet antigen prediction from whole genome sequencing: proof of principle

    PubMed Central

    Westhoff, Connie M.; Uy, Jon Michael; Aguad, Maria; Smeland‐Wagman, Robin; Kaufman, Richard M.; Rehm, Heidi L.; Green, Robert C.; Silberstein, Leslie E.

    2015-01-01

    BACKGROUND There are 346 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet (PLT) antigens, most of which have known genetic changes in 45 RBC or six PLT genes that correlate with antigen expression. Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA. This makes antigen prediction from next‐generation sequencing data challenging, since it uses genomic coordinates. STUDY DESIGN AND METHODS The conventional cDNA reference sequences for all known RBC and PLT genes that correlate with antigen expression were aligned to the human reference genome. The alignments allowed conversion of conventional cDNA nucleotide positions to the corresponding genomic coordinates. RBC and PLT antigen prediction was then performed using the human reference genome and whole genome sequencing (WGS) data with serologic confirmation. RESULTS Some major differences and alignment issues were found when attempting to convert the conventional cDNA to human reference genome sequences for the following genes: ABO, A4GALT, RHD, RHCE, FUT3, ACKR1 (previously DARC), ACHE, FUT2, CR1, GCNT2, and RHAG. However, it was possible to create usable alignments, which facilitated the prediction of all RBC and PLT antigens with a known molecular basis from WGS data. Traditional serologic typing for 18 RBC antigens were in agreement with the WGS‐based antigen predictions, providing proof of principle for this approach. CONCLUSION Detailed mapping of conventional cDNA annotated RBC and PLT alleles can enable accurate prediction of RBC and PLT antigens from whole genomic sequencing data. PMID:26634332

  14. Microenvironmental stresses induce HLA-E/Qa-1 surface expression and thereby reduce CD8(+) T-cell recognition of stressed cells.

    PubMed

    Sasaki, Takanori; Kanaseki, Takayuki; Shionoya, Yosuke; Tokita, Serina; Miyamoto, Sho; Saka, Eri; Kochin, Vitaly; Takasawa, Akira; Hirohashi, Yoshihiko; Tamura, Yasuaki; Miyazaki, Akihiro; Torigoe, Toshihiko; Hiratsuka, Hiroyoshi; Sato, Noriyuki

    2016-04-01

    Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8(+) T-cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA-E in human and Qa-1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa-1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8(+) T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8(+) T-cell recognition. PMID:26711740

  15. Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

    PubMed Central

    Shotton, David M.; Attaran, Amir

    1998-01-01

    Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity. PMID:9861010

  16. CTLA4 mediates antigen-specific apoptosis of human T cells.

    PubMed Central

    Gribben, J G; Freeman, G J; Boussiotis, V A; Rennert, P; Jellis, C L; Greenfield, E; Barber, M; Restivo, V A; Ke, X; Gray, G S

    1995-01-01

    The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells. Images Fig. 3 PMID:7846057

  17. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies.

    PubMed

    Tang, Jonathan Cy; Drokhlyansky, Eugene; Etemad, Behzad; Rudolph, Stephanie; Guo, Binggege; Wang, Sui; Ellis, Emily G; Li, Jonathan Z; Cepko, Constance L

    2016-01-01

    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. PMID:27205882

  18. Differential presentation of tumor antigen-derived epitopes by MHC-class I and antigen-positive tumor cells.

    PubMed

    Held, Gerhard; Neumann, Frank; Sturm, Christine; Kaestner, Lars; Dauth, Nina; de Bruijn, Diederik R; Renner, Christoph; Lipp, Peter; Pfreundschuh, Michael

    2008-10-15

    SSX2 is a member of the family of cancer/testis antigens. The SSX2 derived peptide SSX2(103-111) has been shown to be presented to cytotoxic T-lymphocytes (CTL) by Major-Histocompatibility (MHC) Class-I complexes after endogenous processing, more precisely by the allele HLA-A*0201. The HLA-A*0201- and SSX2-positive melanoma cell line SK-Mel-37 but not Me275 had been shown to elicit reactivity in SSX2(103-111) specific cytotoxic T-lymphocytes. To analyze the correlation between SSX2(103-111) presentation and T-cell stimulation, we intended to visualize presentation of SSX2(103-111) in these melanoma cell lines. Fab-antibodies were established from a human phage library with specificity for SSX2(103-111)/HLA-A*0201 complexes (but non-reactive with HLA-A*0201 or SSX2(103-111) alone) and used to visualize the presentation of SSX2(103-111) in the context of HLA-A*0201 by fluorescence microscopy. Presentation of SSX2(103-111) the context of HLA-A*0201 was demonstrated for the majority of SK-Mel-37, but for only a small fraction (<1%) of Me275 as indicated by a clear membrane-staining pattern in fluorescence microscopy. The presentation of SSX2(103-111) on SK-Mel37 and Me275, but not the expression of the SSX2 protein correlated with the capability of these cells to stimulate cells of an SSX2(103-111)-specific T-cell clone. MHC-peptide specific antibodies are a valuable tool for the analysis of antigenic peptides in the context of MHC-I molecules and for the structural definition of immunodominant epitopes. PMID:18688854

  19. Crypt cells are involved in kin recognition in larval zebrafish.

    PubMed

    Biechl, Daniela; Tietje, Kristin; Gerlach, Gabriele; Wullimann, Mario F

    2016-01-01

    Zebrafish larvae imprint on visual and olfactory kin cues at day 5 and 6 postfertilization, respectively, resulting in kin recognition later in life. Exposure to non-kin cues prevents imprinting and kin recognition. Imprinting depends on MHC class II related signals and only larvae sharing MHC class II alleles can imprint on each other. Here, we analyzed which type of olfactory sensory neuron (OSN) detects kin odor. The single teleost olfactory epithelium harbors ciliated OSNs carrying OR and TAAR gene family receptors (mammals: main olfactory epithelium) and microvillous OSNs with V1R and V2R gene family receptors (mammals: vomeronasal organ). Additionally, teleosts exhibit crypt cells which possess microvilli and cilia. We used the activity marker pERK (phosphorylated extracellular signal regulated kinase) after stimulating 9 day old zebrafish larvae with either non-kin conspecific or food odor. While food odor activated both ciliated and microvillous OSNs, only the latter were activated by conspecific odor, crypt cells showed no activation to both stimuli. Then, we tested imprinted and non-imprinted larvae (full siblings) for kin odor detection. We provide the first direct evidence that crypt cells, and likely a subpopulation of microvillous OSNs, but not ciliated OSNs, play a role in detecting a kin odor related signal. PMID:27087508

  20. Crypt cells are involved in kin recognition in larval zebrafish

    PubMed Central

    Biechl, Daniela; Tietje, Kristin; Gerlach, Gabriele; Wullimann, Mario F.

    2016-01-01

    Zebrafish larvae imprint on visual and olfactory kin cues at day 5 and 6 postfertilization, respectively, resulting in kin recognition later in life. Exposure to non-kin cues prevents imprinting and kin recognition. Imprinting depends on MHC class II related signals and only larvae sharing MHC class II alleles can imprint on each other. Here, we analyzed which type of olfactory sensory neuron (OSN) detects kin odor. The single teleost olfactory epithelium harbors ciliated OSNs carrying OR and TAAR gene family receptors (mammals: main olfactory epithelium) and microvillous OSNs with V1R and V2R gene family receptors (mammals: vomeronasal organ). Additionally, teleosts exhibit crypt cells which possess microvilli and cilia. We used the activity marker pERK (phosphorylated extracellular signal regulated kinase) after stimulating 9 day old zebrafish larvae with either non-kin conspecific or food odor. While food odor activated both ciliated and microvillous OSNs, only the latter were activated by conspecific odor, crypt cells showed no activation to both stimuli. Then, we tested imprinted and non-imprinted larvae (full siblings) for kin odor detection. We provide the first direct evidence that crypt cells, and likely a subpopulation of microvillous OSNs, but not ciliated OSNs, play a role in detecting a kin odor related signal. PMID:27087508

  1. Dscam-Mediated Cell Recognition Regulates Neural Circuit Formation

    PubMed Central

    Hattori, Daisuke; Millard, S. Sean; Wojtowicz, Woj M.; Zipursky, S. Lawrence

    2009-01-01

    The Dscam family of immunoglobulin cell surface proteins mediates recognition events between neurons that play an essential role in the establishment of neural circuits. The Drosophila Dscam1 locus encodes tens of thousands of cell surface proteins via alternative splicing. These isoforms exhibit exquisite isoform-specific binding in vitro that mediates homophilic repulsion in vivo. These properties provide the molecular basis for self-avoidance, an essential developmental mechanism that allows axonal and dendritic processes to uniformly cover their synaptic fields. In a mechanistically similar fashion, homophilic repulsion mediated by Drosophila Dscam2 prevents processes from the same class of cells from occupying overlapping synaptic fields through a process called tiling. Genetic studies in the mouse visual system support the view that vertebrate DSCAM also promotes both self-avoidance and tiling. By contrast, DSCAM and DSCAM-L promote layer-specific targeting in the chick visual system, presumably through promoting homophilic adhesion. The fly and mouse studies underscore the importance of homophilic repulsion in regulating neural circuit assembly, whereas the chick studies suggest that DSCA Mproteins may mediate a variety of different recognition events during wiring in a context-dependent fashion. PMID:18837673

  2. Polysialylation controls dendritic cell trafficking by regulating chemokine recognition.

    PubMed

    Kiermaier, Eva; Moussion, Christine; Veldkamp, Christopher T; Gerardy-Schahn, Rita; de Vries, Ingrid; Williams, Larry G; Chaffee, Gary R; Phillips, Andrew J; Freiberger, Friedrich; Imre, Richard; Taleski, Deni; Payne, Richard J; Braun, Asolina; Förster, Reinhold; Mechtler, Karl; Mühlenhoff, Martina; Volkman, Brian F; Sixt, Michael

    2016-01-01

    The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification that is mainly known to control the developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for the recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase-deficient mice, manifesting as disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis. PMID:26657283

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

    PubMed Central

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

    2016-01-01

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

  4. Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis.

    PubMed

    Brooks, Suzanne E; Bonney, Stephanie A; Lee, Cindy; Publicover, Amy; Khan, Ghazala; Smits, Evelien L; Sigurdardottir, Dagmar; Arno, Matthew; Li, Demin; Mills, Ken I; Pulford, Karen; Banham, Alison H; van Tendeloo, Viggo; Mufti, Ghulam J; Rammensee, Hans-Georg; Elliott, Tim J; Orchard, Kim H; Guinn, Barbara-ann

    2015-01-01

    Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.

  5. Antigen-dependent and -independent contributions to primary memory CD8 T cell activation and protection following infection.

    PubMed

    Martin, Matthew D; Badovinac, Vladimir P

    2015-12-10

    Memory CD8 T-cell activation, including expression of IFN-γ and granzymeB, can be induced by antigen (Ag)-dependent signals through the T-cell-receptor, or by pathogen-derived inflammatory cytokines in an Ag-independent manner. Recent studies have come to conflicting results regarding the contributions of Ag and/or inflammation to memory CD8 T-cell activation. Additionally, research has indicated that inflammation-driven CD8 T-cell responses during un-related infections (bystander activation) have the potential to provide protection, but whether protection occurs in immuno-competent hosts is unclear. To investigate these questions, we examined activation of virus-specific memory CD8 T-cells following infection with L. monocytogenes either expressing or not cognate Ag. We show that Ag and inflammation act synergistically in vitro to induce memory activation. In vivo, we found that when memory CD8 T-cells significantly contribute to clearance of infection, early activation and continued responses by these cells are enhanced by cognate Ag recognition. Mechanistically, we show that bystander responses by memory are dependent upon the dose of infection and the amount of inflammation elicited following infection and are able to provide protection in IFN-γ deficient mice, but not in immuno-competent hosts. The data elucidate the requirements for memory CD8 T-cell activation and the protective role of bystander responses.

  6. Antigen-dependent and –independent contributions to primary memory CD8 T cell activation and protection following infection

    PubMed Central

    Martin, Matthew D.; Badovinac, Vladimir P.

    2015-01-01

    Memory CD8 T-cell activation, including expression of IFN-γ and granzymeB, can be induced by antigen (Ag)-dependent signals through the T-cell-receptor, or by pathogen-derived inflammatory cytokines in an Ag-independent manner. Recent studies have come to conflicting results regarding the contributions of Ag and/or inflammation to memory CD8 T-cell activation. Additionally, research has indicated that inflammation-driven CD8 T-cell responses during un-related infections (bystander activation) have the potential to provide protection, but whether protection occurs in immuno-competent hosts is unclear. To investigate these questions, we examined activation of virus-specific memory CD8 T-cells following infection with L. monocytogenes either expressing or not cognate Ag. We show that Ag and inflammation act synergistically in vitro to induce memory activation. In vivo, we found that when memory CD8 T-cells significantly contribute to clearance of infection, early activation and continued responses by these cells are enhanced by cognate Ag recognition. Mechanistically, we show that bystander responses by memory are dependent upon the dose of infection and the amount of inflammation elicited following infection and are able to provide protection in IFN-γ deficient mice, but not in immuno-competent hosts. The data elucidate the requirements for memory CD8 T-cell activation and the protective role of bystander responses. PMID:26658291

  7. In situ recognition of cell-surface glycans and targeted imaging of cancer cells

    PubMed Central

    Xu, Xiao-Ding; Cheng, Han; Chen, Wei-Hai; Cheng, Si-Xue; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLex) tetrasaccharide, have great potential for cancer diagnosis and therapy. Studies on water-soluble and biocompatible sensors for in situ recognition of cancer-associated glycans in live cells and targeted imaging of cancer cells are very limited at present. Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells. By screening BPFSs with different structures, it was demonstrated that BPFS1 with a FRGDF peptide could recognize cell-surface glycan of sLex with high specificity and thereafter fluorescently label and discriminate cancer cells through the cooperation with the specific recognition between RGD and integrins. The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis. PMID:24042097

  8. Tubulin and actin interplay at the T cell and antigen-presenting cell interface.

    PubMed

    Martín-Cófreces, Noa Beatriz; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2011-01-01

    T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off.

  9. Tubulin and Actin Interplay at the T Cell and Antigen-Presenting Cell Interface

    PubMed Central

    Martín-Cófreces, Noa Beatriz; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2011-01-01

    T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off. PMID:22566814

  10. Studies on thyroid cell surface antigens using cultured human thyroid cells.

    PubMed Central

    Fenzi, G F; Bartalena, L; Chiovato, L; Marcocci, C; Rotella, C M; Zonefrati, R; Toccafondi, R; Pinchera, A

    1982-01-01

    Human thyroid cells in primary culture were used for studies of thyroid cell surface antibodies in patients with thyroid autoimmune disorders. Radioiodinated IgG preparations containing thyroid microsomal antibody (TMAb), thyroid stimulating antibody (TSAb) and/or thyroglobulin antibody (TgAb) were tested for binding to thyroid cells. Binding was observed with radioiodinated IgG from patients with Graves' disease, Hashimoto's thyroiditis and idiopathic myxoedema containing TMAb, irrespective of the presence of TSAb and TgAb, while negative results were obtained with normal IgG. A dose-dependent inhibition of binding to thyroid cells was produced by the addition of the corresponding unlabelled IgG preparations. Evidence for tissue specificity was provided by the absence of binding to human skin fibroblasts used as controls. Preabsorption with human thyroid microsomes completely abolished the binding to thyroid cells of a radioiodinated TMAb positive IgG preparation, while only incomplete removal of the reactivity to thyroid microsomes was produced by preabsorption with thyroid cells. These data suggest that some but not all microsomal antigenic determinants are expressed on the thyroid cell surface. Binding to thyroid cells was also observed with purified TgAb, indicating that thyroglobulin antigenic determinants are present on the surface of thyroid cells. No evidence of binding was obtained with a TSAb positive Graves' IgG preparation with undetectable TMAb and TgAb. Unlabelled IgG preparations containing TMAb from patients with either Hashimoto's thyroiditis or idiopathic myxoedema were shown to inhibit the binding to thyroid cells of radioiodinated TMAb positive Graves' IgG and vice versa. These data indicate that antibodies present in these thyroid autoimmune disorders share common thyroid cell surface antigens. However, the binding of radioiodinated IgG from a patient with idiopathic myxoedema was only partially inhibited by Graves' or Hashimoto's Ig

  11. Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration

    PubMed Central

    Knight, Laura M.; Stakaityte, Gabriele; Wood, Jennifer, J.; Abdul-Sada, Hussein; Griffiths, David A.; Howell, Gareth J.; Wheat, Rachel; Blair, G. Eric; Steven, Neil M.; Macdonald, Andrew; Blackbourn, David J.

    2014-01-01

    ABSTRACT Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule

  12. Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

    PubMed

    Nashida, Tomoko; Sato, Ritsuko; Haga-Tsujimura, Maiko; Yoshie, Sumio; Yoshimura, Ken; Imai, Akane; Shimomura, Hiromi

    2013-02-01

    Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

  13. Development of immunoglobulin and antibody-synthesizing cells after immunization with different doses of antigen.

    PubMed Central

    Antoine, J C; Petit, C; Avrameas, S

    1976-01-01

    The kinetics of development of antibody-synthesizing cells and of cells synthesizing immunoglobulins without detectable antibody function were studied in rats immunized with different doses (0-1, 1, 10, 100 mg) of horse radish peroxidase, bovine serum albumin, human serum albumin, hen ovalbumin, or human IgG, which had been deaggregated or heat-aggregated. Each antigen was injected once or twice as a solution in saline. Antibody and immunoglobulin-producing cells were detected in draining lymph nodes by immunohistochemical staining. In the primary response a few antibody-synthesizing cells were found whatever the dose injected. No increase or some increase was found with the amount of antigen injected, according to the protein used, but with all doses of antigen injected, the population of cells remained small, except with human IgG where a relatively high number of positive cells was detected even after injection of 1 mg of antigen. In the secondary response a few antibody-forming cells were also detected with the lower doses of antigen, but this population increased after boosting with 100 mg of antigen. With human IgG a greater number of positive cells was induced withall the doses tested. A correlation between the number of cells synthesizing immunoglobulins without antibody function and the amount of antigen injected was observed in the primary and secondary responses. The relative size of these two populations varied with the stage of immunity of the animals. In the primary response, the population of cells synthesizing immunoglobulins without antibody function was larger than the population of antibody-forming cells. The same was true in the secondary response, but if after a booster injection the level of antibody-synthesizing cells exceeded that reached in the primary response, the increase of cells synthesizing Ig without antibody function was smaller than the increase in antibody-forming cells. In general the more immunogenic an antigen was, the smaller

  14. Distribution and functional characteristics of antigen-specific helper T cells arising after Peyer's patch immunization.

    PubMed Central

    Dunkley, M L; Husband, A J

    1987-01-01

    Antigen-specific T-helper cells for IgA responses arise in Peyer's patches (PP) following their immunization by subserosal injection of keyhole limpet haemocyanin (KLH). These are of the W3/25 phenotype and the W3/25 receptor is shown here to be involved in their helper function. These cells originate in PP and migrate via mesenteric lymph nodes (MLN) to thoracic duct lymph, although the MLN appear to be unnecessary for the induction or maturation of antigen-specific helper cells collected in thoracic duct lymph after intra-Peyer's patch (i.p.p.) immunization. KLH-specific helper cells can be detected subsequently in the intraepithelial lymphocyte population and also among lamina propria lymphocytes. The helper cells also relocate to PP distant to their site of origin where they are retained only when antigen is present. While i.p.p. immunization is an efficient route for the induction of IgA helper cells in gut-associated lymphoid tissue, it differs from oral immunization in that concomitant induction of antigen-specific splenic suppressor cells does not occur, indicating a role for epithelial antigen processing in this phenomenon. PMID:2450831

  15. Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells.

    PubMed Central

    Karimi, S. T.; Schloemer, R. H.; Wilde, C. E.

    1989-01-01

    The presence of a chlamydia-specified antigen associated with the plasma membrane of infected cell lines was demonstrated by indirect immunofluorescence staining with a monoclonal antibody, designated 47A2, specific for the chlamydial genus-specific lipopolysaccharide (LPS) antigen. Staining of HeLa, L-929, and McCoy cells infected with the L2 or F serovar of Chlamydia trachomatis was observed either without fixation or following aldehyde fixation and brief drying. The 47A2-reactive antigen appeared to be present on the plasma membrane, on bleb-like structures on the host cell surface, and on proximal processes of neighboring uninfected cells. Antibodies to chlamydial protein antigens such as the major outer membrane protein produced no surface staining under similar conditions. Membrane vesicles elaborated from infected cells were enriched for the 47A2-reactive antigen. Superinfection of chlamydia-infected cells with vesicular stomatitis virus, an enveloped virus which buds from the plasma membrane, allowed purification of progeny virions that were enriched with chlamydial LPS. These results are consistent with the presence of chlamydial LPS in the plasma membranes of infected host cells. Images PMID:2470679

  16. Human macrophages can express the Hodgkin's cell-associated antigen Ki-1 (CD30).

    PubMed Central

    Andreesen, R.; Brugger, W.; Löhr, G. W.; Bross, K. J.

    1989-01-01

    The normal precursor of the neoplastic cell in Hodgkin's lymphoma is still unknown. Previous reports on the expression of the Hodgkin's cell-associated antigen Ki-1, CD30, on normal cells have been limited to activated lymphocytes. This study demonstrates, however, that cells of the macrophage lineage also are able to express the Ki-1 antigen. The Ki-1 antigen is absent from normal blood monocytes but expressed on up to 85% of macrophage-type cells developed during subsequent in vitro differentiation on Teflon membranes. Unlike other maturation-associated antigens, Ki-1 is found only at late stages of the macrophage primary cultures. Its expression can be enhanced by human interferon-gamma in a fashion similar to that of HLA-DR molecules. In addition, freshly explanted tumor cells from three patients with histopathologic and clinical features consistent with the diagnosis of true histiocytic lymphoma or malignant histiocytosis as well as the permanent cell line SU-DHL-1 could be demonstrated to express the Ki-1 antigen. The phenotype of histiocytic malignancy was further evaluated to be HLA-DR+MAX.26+CD25+-EMA+OKT9+Ki-1+. The results could indicate either that Hodgkin's lymphoma may arise not only from the lymphocyte but also from the macrophage lineage or may emphasize a macrophage involvement in the pathogenesis of this disease. Images Figure 3 PMID:2536522

  17. Bronchoalveolar CD4+ T cell responses to respiratory antigens are impaired in HIV-infected adults

    PubMed Central

    Sepako, Enoch; Fullerton, Duncan G; Mzinza, David; Glennie, Sarah; Wright, Adam K; Heyderman, Robert S; Gordon, Stephen B

    2011-01-01

    Rationale HIV-infected adults are at an increased risk of lower respiratory tract infections. HIV infection impairs systemic acquired immunity, but there is limited information in humans on HIV-related cell-mediated immune defects in the lung. Objective To investigate antigen-specific CD4+ T cell responses to influenza virus, Streptococcus pneumoniae and Mycobacterium tuberculosis antigens in bronchoalveolar lavage (BAL) and peripheral blood between HIV-infected individuals and HIV-uninfected Malawian adults. Methods We obtained BAL fluid and blood from HIV-infected individuals (n=21) and HIV-uninfected adults (n=24). We determined the proportion of T cell subsets including naive, memory and regulatory T cells using flow cytometry, and used intracellular cytokine staining to identify CD4+ T cells recognising influenza virus-, S pneumoniae- and M tuberculosis-antigens. Main results CD4+ T cells in BAL were predominantly of effector memory phenotype compared to blood, irrespective of HIV status (p<0.001). There was immune compartmentalisation with a higher frequency of antigen-specific CD4+ T cells against influenza virus, S pneumoniae and M tuberculosis retained in BAL compared to blood in HIV-uninfected adults (p<0.001 in each case). Influenza virus- and M tuberculosis-specific CD4+ T cell responses in BAL were impaired in HIV-infected individuals: proportions of total antigen-specific CD4+ T cells and of polyfunctional IFN-γ and TNF-α-secreting cells were lower in HIV-infected individuals than in HIV-uninfected adults (p<0.05 in each case). Conclusions BAL antigen-specific CD4+ T cell responses against important viral and bacterial respiratory pathogens are impaired in HIV-infected adults. This might contribute to the susceptibility of HIV-infected adults to lower respiratory tract infections such as pneumonia and tuberculosis. PMID:21357587

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

    PubMed Central

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran

    2015-01-01

    We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+), B cells (HLA-DR+, CD19+, and HLA-DR+CD19+), NKT cells (CD3+CD16+CD56+), and NK cells (CD3−CD16+CD56+). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression. PMID:26788525

  19. Identification of T-cell stimulatory antigens of Chlamydia trachomatis using synovial fluid-derived T-cell clones.

    PubMed Central

    Hassell, A B; Reynolds, D J; Deacon, M; Gaston, J S; Pearce, J H

    1993-01-01

    Chlamydia trachomatis is a major cause of sexually transmitted disease, infertility and reactive arthritis in the Western world, and of trachoma in the developing world. There is evidence that the chronic inflammatory reaction seen in diseases associated with chlamydiae represents a delayed-type hypersensitivity response to chlamydial antigens. Little is known about which chlamydial antigens elicit T-cell responses yet such information could have important implications in terms of both immunopathological understanding of these diseases and immunoprophylaxis design. In this study, 61 chlamydia-specific T-cell clones have been produced from the synovial fluid of an individual with sexually acquired reactive arthritis (SARA). Ten clones have been characterized in detail and used to identify T-cell stimulatory antigens of chlamydiae by means of T-cell immunoblotting. Two distinct antigenic fractions have been identified, one recognized by three of the clones (molecular weight 18,000), the other recognized by six of the clones (molecular weight 30,000). The fractions are distinct from the major outer membrane protein, the 57,000 MW stress protein and the 60,000 MW cysteine-rich membrane protein of chlamydiae. The major histocompatibility complex (MHC) restriction of the response to these antigens differed: clones recognizing the 18,000 MW antigen required antigen-presenting cells expressing DR1 subtype DRB1*0101 or DRB1*0102 which only differ at amino acids 85 and 86 on the DR beta-chain; by contrast clones recognizing the 30,000 MW antigen were presented to only by antigen-presenting cells from DRB1*0101 individuals, reflecting extreme sensitivity of these clones to the polymorphism at positions 85 and 86 on the DR beta-chain. Images Figure 4 PMID:7691730

  20. Effect of activated antigen-specific B cells on ES-62-mediated modulation of effector function of heterologous antigen-specific T cells in vivo

    PubMed Central

    Marshall, Fraser A; Watson, Katherine A; Garside, Paul; Harnett, Margaret M; Harnett, William

    2008-01-01

    There is currently great interest in the idea of using helminth-derived molecules for therapeutic purposes and indeed we have shown that ES-62, a filarial nematode-derived phosphorylcholine-containing glycoprotein, significantly reduces the severity of arthritis in a murine model. Clearly, knowledge of mechanism of action is important when considering molecules for use in treating disease and although much is known regarding how ES-62 interacts with the immune system, gaps in our understanding remain. A feature of filarial nematode infection is a defective, T helper 2 (Th2)-polarized antigen-specific T-cell response and in relation to this we have recently shown that ES-62 inhibits clonal expansion and modulates effector function towards a Th2 phenotype, of antigen-specific T cells in vivo. ES-62 is also known to directly modulate B-cell behaviour and hence to determine whether it was mediating these effects on T cells by disrupting B–T-cell co-operation, we have investigated antigen-specific responses using an adoptive transfer system in which traceable numbers of tg ovalbumin (OVA)-specific T cells and hen egg lysozyme (HEL)-specific B cells respond to a chemically coupled form of OVA–HEL that contains linked epitopes that promote cognate T- and B-cell interactions. Surprisingly, these studies indicate that activated B cells restore T-cell expansion and prevent Th2-like polarization. However, ES-62-treated double cell transfer mice demonstrate a more generalized immunosuppression with reduced levels of Th1 and -2 type cytokines and antibody subclasses. Collectively, these results suggest that whilst ES-62 can target B–T-cell co-operation, this does not promote polarizing of T-cell responses towards a Th2-type phenotype. PMID:17961164

  1. Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen.

    PubMed

    Kang, Su-Min; Rhee, Jin-Kyu; Kim, Eui-Joong; Han, Kwang-Hyub; Oh, Jong-Won

    2003-09-26

    Cell surface expression of protein has been widely used to display enzymes and antigens. Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC. Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein. Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA. These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen. PMID:14553932

  2. Antigen/IgG immune complex-primed mucosal mast cells mediate antigen-specific activation of co-cultured T cells

    PubMed Central

    Ding, Jie; Fang, Yu; Xiang, Zou

    2015-01-01

    Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed bone marrow-derived MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, bone marrow-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described as favouring phagocytosis of mast cells by professional antigen-presenting cells. PMID:25196548

  3. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    PubMed

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

  4. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    PubMed

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed. PMID:2973431

  5. Defining the HLA class I‐associated viral antigen repertoire from HIV‐1‐infected human cells

    PubMed Central

    Yang, Hongbing; Partridge, Thomas; Llano, Anuska; Cedeño, Samandhy; Fischer, Roman; Charles, Philip D.; Dudek, Nadine L.; Mothe, Beatriz; Crespo, Manuel; Fischer, William M.; Korber, Bette T. M.; Nielsen, Morten; Borrow, Persephone; Purcell, Anthony W.; Brander, Christian; Dorrell, Lucy; Kessler, Benedikt M.; Hanke, Tomáš

    2015-01-01

    Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High‐throughput definition of HLA class I‐associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T‐cell responses against pathogens such as HIV‐1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo‐assisted database searching to define the HLA class I‐associated immunopeptidome of HIV‐1‐infected human cells. We here report for the first time the identification of 75 HIV‐1‐derived peptides bound to HLA class I complexes that were purified directly from HIV‐1‐infected human primary CD4+ T cells and the C8166 human T‐cell line. Importantly, one‐third of eluted HIV‐1 peptides had not been previously known to be presented by HLA class I. Over 82% of the identified sequences originated from viral protein regions for which T‐cell responses have previously been reported but for which the precise HLA class I‐binding sequences have not yet been defined. These results validate and expand the current knowledge of virus‐specific antigenic peptide presentation during HIV‐1 infection and provide novel targets for T‐cell vaccine development. PMID:26467324

  6. Mutations in pattern recognition receptor genes modulate seroreactivity to microbial antigens in patients with inflammatory bowel disease

    PubMed Central

    Henckaerts, Liesbet; Pierik, Marie; Joossens, Marie; Ferrante, Marc; Rutgeerts, Paul; Vermeire, Séverine

    2007-01-01

    Background and aims A number of antibodies against microbial epitopes or self‐antigens have been associated with Crohn's disease. The development of antibodies reflects a loss of tolerance to intestinal bacteria that underlies Crohn's disease, resulting in an exaggerated adaptive immune response to these bacteria. It was hypothesised that the development of antimicrobial antibodies is influenced by the presence of genetic variants in pattern recognition receptor genes. The aim of this study was therefore to investigate the influence of mutations in these innate immune receptor genes (nucleotide oligomerisation domain (NOD) 2/caspase recruitment domain (CARD) 15, NOD1/CARD4, TUCAN/CARDINAL/CARD8, Toll‐like receptor (TLR) 4, TLR2, TLR1 and TLR6) on the development of antimicrobial and antiglycan antibodies in inflammatory bowel disease (IBD). Materials and methods A cohort of 1163 unrelated patients with IBD (874 Crohn's disease, 259 ulcerative colitis, 30 indeterminate colitis) and 312 controls were analysed for anti‐Saccharomyces cerevisiae antibodies (gASCA) IgG, anti‐laminaribioside antibodies (ALCA) IgG, anti‐chitobioside antibodies (ACCA) IgA, anti‐mannobioside antibodies (AMCA) IgG and outer membrane porin (Omp) IgA and were genotyped for variants in NOD2/CARD15, TUCAN/CARDINAL/CARD8, NOD1/CARD4, TLR4, TLR1, TLR2 and TLR6. Results When compared with Crohn's disease patients without CARD15 mutations, the presence of at least one CARD15 variant in Crohn's disease patients more frequently led to gASCA positivity (66.1% versus 51.5%, p < 0.0001) and ALCA positivity (43.3% versus 34.9%, p  =  0.018) and higher gASCA titers (85.7 versus 51.8 ELISA units, p < 0.0001), independent of ileal involvement. A gene dosage effect, with increasing gASCA and ALCA positivity for patients carrying none, one and two CARD15 variants, respectively, was seen for both markers. Similarly, Crohn's disease patients carrying NOD1/CARD4 indel had a higher

  7. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor

    PubMed Central

    MacDonald, Katherine G.; Hoeppli, Romy E.; Huang, Qing; Gillies, Jana; Luciani, Dan S.; Orban, Paul C.; Broady, Raewyn; Levings, Megan K.

    2016-01-01

    Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2–specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR–expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR–mediated stimulation. In mouse models, human A2-CAR–expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases. PMID:26999600

  8. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

    PubMed

    MacDonald, Katherine G; Hoeppli, Romy E; Huang, Qing; Gillies, Jana; Luciani, Dan S; Orban, Paul C; Broady, Raewyn; Levings, Megan K

    2016-04-01

    Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases. PMID:26999600

  9. Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

    PubMed

    Xu, Yingping; Liu, Yi; Yang, Chunqing; Kang, Li; Wang, Meixiang; Hu, Jingxia; He, Hao; Song, Wengang; Tang, Hua

    2016-10-01

    Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis. PMID:27278624

  10. Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

    PubMed

    Xu, Yingping; Liu, Yi; Yang, Chunqing; Kang, Li; Wang, Meixiang; Hu, Jingxia; He, Hao; Song, Wengang; Tang, Hua

    2016-10-01

    Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis.

  11. Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion.

    PubMed Central

    Rathjen, F G; Schachner, M

    1984-01-01

    Monoclonal and polyclonal L1 antibodies react by indirect immunofluorescence with the cell surface of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, but not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. During cerebellar development L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after 3 days in culture. In sections of the early post-natal cerebellum, L1 antigen is found on pre-migratory neurons in the internal, but not in the external part of the external granular layer. In the adult cerebellum, L1 antigen is predominantly localized in the molecular layer and around Purkinje cells. Fibers in white matter and the granular layer are also L1 antigen-positive. Granule cell bodies and synaptic glomeruli are weakly antigen-positive. Several cell lines derived from neuroblastoma C1300 also express L1 antigen. The antigen is not detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are found in brains of rat, guinea pig, hamster, chicken, rabbit and man, but not in frog, while monoclonal antibody reacts detectably only with mouse brain. The molecular species recognized by both monoclonal and polyclonal antibodies display two prominent bands by SDS-PAGE under reducing and non-reducing conditions with apparent mol. wts. of 140 and 200 kd. L1 antigen isolated from cultured cerebellar cells consists mainly of a band in the 200-kd range and a faint one at 140 kd. L1 antigen from neuroblastoma N2A shows two bands with slightly higher apparent mol. wts. All molecular forms of L1 antigen can be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continuous cell line N2A derived from the murine

  12. Antigen dynamics govern the induction of CD4(+) T cell tolerance during autoimmunity.

    PubMed

    Challa, Dilip K; Mi, Wentao; Lo, Su-Tang; Ober, Raimund J; Ward, E Sally

    2016-08-01