MHC-restricted Immune Response Research Articles

MHC class II complexes sample intermediate states along the peptide exchange pathway.

The presentation of peptide-MHCII complexes (pMHCIIs) for surveillance by T cells is a well-known immunological concept in vertebrates, yet the conformational dynamics of antigen exchange remain elusive. By combining NMR-detected H/D exchange with Markov modelling analysis of an aggregate of 275 microseconds molecular dynamics simulations, we reveal that a stable pMHCII spontaneously samples intermediate conformations relevant for peptide exchange. More specifically, we observe two major peptide exchange pathways: the kinetic stability of a pMHCII's ground state defines its propensity for intrinsic peptide exchange, while the population of a rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on our model shift the exchange behaviour towards the HLA-DM-catalysed pathway and further allow us to conceptualize how allelic variation can shape an individual's MHC restricted immune response.

The reversal of the metastatic phenotype by gene transfer.

When studying the function of MHC-restricted immune responses in controlling metastatic growth we discovered that highly metastatic clones of mouse tumours express the H-2D but lack expression of the H-2K gene of the MHC system. The de novo expression of the H-2K antigen, after H-2K gene transfection, resulted in the reversal of a metastatic to a non-metastatic phenotype. This reversal was causally related to the acquisition of H-2K-restricted immunogenic properties. Immunization with H-2K-transfected cells, after surgical removal of the local tumour, abolished or significantly reduced the growth of metastases. We subsequently observed that H-2K expression is correlated with expression of the c-fos oncogenes. Transfection of H-2K-negative cells with v-fos or c-fos genes resulted in the expression of H-2K. Our studies suggest that one of the main functions of the c-fos proto-oncogenes is control of the expression of the MHC genes. Searching for additional molecular properties which characterize the metastatic phenotype, we observed that the metastatic clones of each of our lung-metastasizing tumours expresses an fms-related oncogene. This was correlated with a membrane-bound tyrosine kinase, which has the properties of growth factor receptors. We examine the possibility that our fms-like gene codes for this protein kinase, which represents a receptor for a local growth factor that controls metastatic growth in the lung.

Enhanced ability of dendritic cells to stimulate innate and adaptive immunity on short-term incubation with zoledronic acid

AbstractVγ9/Vδ2 (γδ) T cells play a major role in innate immunity against microbes, stressed, and tumor cells. They represent less than 5% of peripheral blood lymphocytes but can be activated and expanded in vitro by aminobisphosphonates (ABP)–treated monocytes. The aim of this work was to determine whether ABP-treated dendritic cells (DCs) can also activate γδ T cells and regulate immune responses mediated by conventional αβ T cells. Highly purified immature (iDC) and mature DC (mDC) were generated from peripheral blood monocytes of healthy donors and incubated with zoledronic acid (Zol) for 24 hours. Zol-treated iDC and mDC retained their immunostimulatory properties and induced the vigorous expansion of central memory and effector memory γδ T cells. γδ T cells displayed antitumor activity and appropriate cell surface antigens to target secondary lymphoid organs and exert costimulatory activity. Antigen-specific MHC-restricted immune responses, mediated by conventional αβ T cells, were improved by the concurrent γδ T-cell activation. In conclusion, large numbers of γδ T cells with effector and costimulatory activities are rapidly generated by Zol-treated iDC/mDC. This strategy is worthy of further investigation to improve adoptive cell therapy and vaccine interventions against tumors and infections.

Dendritic Cells Can Be Primed to Concurrently Activate Both Unconventional Gamma/delta and Conventional alpha/beta T Cells.

Vgamma9/Vdelta2 (gamma/delta) T cells represent the major subset of unconventional T cells circulating in the peripheral blood. Gamma/delta T cells play a major role in immune defenses against microbes, stressed cells and tumor cells. This property is based on their capability to naturally recognize phosphoantigens (pAgs), which are produced via the mevalonate (Mev) or the DOXP pathway in mammalian and nonmammalian cells, and induced self-ligands, which are de novo expressed or upregulated on the surface of stressed or tumor cells. Interestingly, gamma/delta T cells can also be activated by aminobisphosphonates (ABP)-treated monocytes. We have previously shown that ABP specifically target the Mev pathway of monocytes and induce the accumulation of phosphorylated Mev metabolites naturally recognized by gamma/delta T cells. The aim of this work was to determine whether ABP-treated dendritic cells (DC) can also activate gamma/delta T cells and whether this activation, if any, is detrimental or beneficial to the generation of antigen (Ag)-specific MHC-restricted immune responses mediated by conventional alpha/beta T cells. To this end, we have generated highly purified immature (iDC) and mature DC (mDC) from peripheral blood monocytes of healthy donors and incubated with zoledronic acid (Zol) for 24 hours. Zol is the most potent ABP currently available for clinical use. Zol treatment did not affect the phenotype and immunostimulatory properties of iDC and mDC. Zol-treated iDC and mDC induced a rapid and vigorous expansion of central memory and effector memory gamma/delta T cells. Zol-treated iDC were more potent inducers of gamma/delta T-cell activation than mDC and monocytes. Activated gamma/delta T cells displayed antitumor activity and expressed on the cell surface the appropriate antigen repertoire to target secondary lymphoid organs and exert costimulatory activity on conventional alpha/beta T cells. Indeed, an in vitro model showed that antigen-specific MHC-restricted immune responses againt the influenza matrix peptide were significantly improved by the concurrent activation of gamma/delta T cells. This is the first report showing that: 1) DC can simultaneously be primed to activate both gamma/delta and alpha/beta T cells; 2) the former act as cellular adjuvants for the development of adaptive immune responses. In conclusion, large numbers of gamma/delta T cells with effector and costimulatory activities can rapidly be generated by Zol-treated iDC/mDC. This strategy is worth of further investigation to improve adoptive cell therapy and vaccine interventions against tumors and infections.

Breaking the non-responsiveness of C57BL/6 mice to the malarial antigen EB200--the role of carrier and adjuvant molecules.

Poor immunogenicity and major histocompatibility complex (MHC) restriction of immune responses to certain recombinant proteins or synthetic peptides impose problems in developing effective vaccines. EB200 is one of the vaccine candidate antigens from Plasmodium falciparum, which induces MHC-restricted immune responses in mice of different haplotypes. A way of overcoming this problem is to conjugate the antigen to an immunogenic protein carrier and to use optimal adjuvant substances. We have investigated the carrier effect of glutathione-S-transferase (GST) in CBA and C57BL/6 mice which are high and low responder to EB200, respectively. Our results reveal that the MHC restriction in C57BL/6 mice was broken by the use of GST as a carrier. Studies on the B-cell repertoires in both strains of mice immunized with GST-EB200 by preparing hybridoma cell lines indicate that the B-cell repertoires were similar in both CBA and C57BL/6 mice. However, the antibody affinity and the magnitude of the response were still lower in the low-responder C57BL/6 mice compared with that in CBA even when cholera toxin (CT) was used as adjuvant. To improve the response, the efficacy of various adjuvant substances like alum and Hsp 70 from Trypanosoma cruzi and the combination of various adjuvants was analysed. CT and Hsp 70 together act synergistically and markedly improve the immunogenicity of EB200 by increasing antibody affinity and the magnitude of the responses in C57BL/6 mice, which may be explained by the complementary effect of adjuvants. These results are of importance in the design of efficient vaccines.

Phenotypic and functional analysis of bovine gammadelta lymphocytes.

The studies have provided the first comprehensive comparison of the factors regulating activation and proliferation of WC1+ and WC1- gammadelta T cells. The investigation has shown that accessory molecules essential for activation and function of WC1+ and WC1- gammadelta T cells and the sources and roles of cytokines in activation of gammadelta T cells through the T cell receptor (TCR). The study has also shown that the role of cytokines in activation and function of gammadelta T cells activated indirectly through cytokines secreted by ab T cells, accessory cells and antigen presenting cells (APC). Cytokines were differentially produced by subpopulations of gammadelta T cells under different conditions of activation. The investigation obtained in this study has revealed that factors account for activation and proliferation of gammadelta T cells in cultures designed to study MHC-restricted responses to antigens. Evidence obtained here has shown there is biological relevance to activation under these culture conditions that points to potential regulatory and effector functions of gammadelta T cells. The investigations have also provided the information needed to begin identifying and characterizing antigens recognized by the TCR repertoires of WC1+ and WC1- gammadelta T cells. Finally, the investigations have provided the information needed to begin analysis of the mechanisms by which gammadelta T cells modulate MHC restricted immune responses to pathogens and derived vaccines.

MHC restricted immune response in totally resected adenocarcinoma of the pancreas: [formula omitted], G. Schwall, M. Bohrer, M. Schwab, A. Philipp, M.V. Singer. Depts. of Medicine, Pathology & Surgery, University of Heidelberg, Klinikum Manaheim, Germany

MHC restricted immune response in totally resected adenocarcinoma of the pancreas: [formula omitted], G. Schwall, M. Bohrer, M. Schwab, A. Philipp, M.V. Singer. Depts. of Medicine, Pathology & Surgery, University of Heidelberg, Klinikum Manaheim, Germany

Clonal V alpha 12.1+ T cell expansions in the peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) represents a heterogenous disease characterized by chronic polyarthritis. Most patients with adult RA inherit HLA-DR4 or -DR1 major histocompatibility complex (MHC) genes. While the molecular basis for this genetic predisposition is unknown, the major function of these MHC-encoded molecules is to present peptides to T lymphocytes. It is hypothesized that an endogenous or environmental antigen initiates a MHC-restricted immune response mediated by T lymphocytes, which is followed by a chronic inflammatory reaction involving many cell types. In chronic RA, previous or ongoing antigenic activation might result in detectable skewing of the peripheral alpha/beta T cell receptor (TCR) repertoire. Here we demonstrate a marked expansion of V alpha 12.1-bearing CD8+ T cells in the peripheral blood (mean, 22%; range, 10-43%) of > 15% of RA patients. A major proportion of these patients shared HLA-DQ2 in addition to the expected high frequency DR1 and DR4 alleles. Detailed molecular analysis in three of the RA patients with elevated V alpha 12.1+ T cells identified repeated TCR alpha chain sequences consistent with clonal V alpha 12.1+,CD8+ T cell expansion. In addition to shared TCR V alpha 12.1 germline gene usage among unrelated subjects, a conserved J alpha motif was also detected. Together, these results suggest an antigen-driven mechanism of T cell expansion in these patients and may offer a new approach in examining specific antigen that stimulate T cells in RA.

Profound alteration in an alpha beta T-cell antigen receptor repertoire due to polymorphism in the first complementarity-determining region of the beta chain.

Amino acid residues that are critical in maintaining the framework structure of immunoglobulin heavy- and light-chain variable (V) regions are strongly conserved in the V alpha and V beta proteins of the alpha beta T-cell antigen receptor (TCR alpha beta). Consequently, it has been proposed that TCR alpha beta has a conformation similar to that of an immunoglobulin Fab fragment and that the regions of the TCR homologous to the three immunoglobulin complementarity-determining regions (CDRs 1, 2, and 3) bind to the peptide antigen-major histocompatibility complex (MHC) molecule ligand. A single amino acid substitution in the predicted CDR1 of the V beta 3 protein of certain mouse strains dramatically altered TCR alpha beta usage in an antigen-specific MHC-restricted immune response but did not abrogate V beta 3 specificity for the superantigens minor lymphocyte stimulatory locus (Mls)c and staphylococcal enterotoxin A (SEA). The results confirm the importance of the V beta CDR1 in antigen-MHC molecule recognition, supporting the Fab-like structural model of TCR alpha beta, and provide further evidence that conventional antigen-MHC recognition and superantigen recognition are mediated by distinct regions of the TCR beta chain. They also suggest that allelic polymorphism may be a significant source of diversity in the TCR repertoire.

Macrophages as accessory cells for class I MHC-restricted immune responses.

Ag do not elicit T lymphocyte responses unless they are presented in conjunction with MHC molecules on the surface of an appropriate APC. In the case of CD4+ T lymphocytes dendritic cells can deliver all signals required for complete induction as can macrophages and (activated) B cells. The function of CTL also depends on the presence of specialized accessory cells. Here we show that these accessory cells can behave like scavenger cells. They use foreign Ag in the form of cellular debris as immunogen. They are also crucial for CTL induction because in vivo depletion of phagocytotic cells completely inhibits CTL responses. In these animals CTL activity could be restored by transfer of macrophages. All of the reappearing CTL used MHC restriction elements expressed by the infused macrophages. These experiments suggest that a cognate interaction between macrophages and CTL precursors initiates class I MHC-restricted immune responses.

A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes.

BALB/3T3 cells infected with a retroviral vector encoding the influenza virus nucleoprotein (NP) gene are efficiently lysed by CTL generated in BALB/c mice (H-2d background). Cells transduced with a mutant form of NP which contains a frameshift mutation at its NH2 terminus (NPm) do not express biochemically detectable levels of protein but nevertheless present Ag to CTL with high efficiency. Cold target inhibition studies indicate that the same CTL epitope(s) are recognized in cells harboring NP or NPm. L929 cells transduced with the NPm gene also present Ag efficiently to CTL raised in C3H mice (H-2k background). Cells engineered to express 5- to 15-fold lower levels of wild-type NP were not capable of presenting Ag to CTL, arguing against the notion that CTL are able to lyse cells expressing very low levels of Ag which might have resulted from suppression of the frameshift mutation in NPm. Implications to the mechanism of epitope generation in class I MHC-restricted immune responses are considered.

Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency.

CD8 molecules expressed on the surface of a subset of T cells participate in the selection of class I MHC antigen-restricted T cells in the thymus, and in MHC-restricted immune responses of mature class I MHC antigen-restricted T cells. Here we describe an immune-deficient patient with lack of CD8+ peripheral blood cells. The patient presented with Pneumocystis carinii pneumonia and was unable to reject an allogeneic skin graft, but had normal primary and secondary antibody responses. Examination of the patient's thymus revealed that the loss of CD8+ cells occurred during intrathymic differentiation: the patient's immature cortical thymocytes included both CD4+ and CD8+ cells while the mature medullary cells expressed the CD4 but not the CD8 protein on their surface. Northern blot and polymerase chain reaction analyses revealed the presence of CD8 alpha and beta mRNA in the patient's thymus but not in the peripheral blood. Both class I MHC antigen expression and the expressed TCR V beta repertoire are normal in this patient. These data are consistent with an impaired selection of CD8+ cells in the patient's thymus and support the role of the CD8 surface protein in thymic selection previously characterized in genetically manipulated and inbred mice.

Transplantable Marek's disease lymphomas. III. Induction of MHC‐restricted tumor immunity by lymphoblastoid cells in F1 hosts

F1 chickens, which were a cross between birds of the related inbred lines G-B1 and G-B2, were immunized with in vitro cultured virus-non-producer lymphoblastoid cells that were either syngeneic or allogeneic with the challenge tumor cells. The lymphoblastoid cells were derived from Marek's disease herpesvirus (MDV)-induced transplantable tumors. Previous findings showed that such immunization of G-B1 and G-B2 chickens prevented early mortality caused by the tumors. Lymphoblastoid cells syngeneic with the tumors were more effective than allogeneic cells, suggesting that an MHC-restricted immune response was induced, or, alternatively, that immune elimination of allogeneic cells prevented them from initiating strong immunity to MDV-associated tumor antigens. Immune elimination of the immunizing cells should not occur in F1 birds heterozygous for the 2 different parental MHC haplotypes (B6 and B13). Early mortality among F1 chickens immunized with lymphoblastoid cells that were syngeneic with the challenge tumor cells was significantly lower than for non-immunized control chickens or for birds immunized with lymphoblastoid cells that were allogeneic with the challenge tumor cells. Our results suggest that MDV-induced tumor antigens may be recognized by the host as altered-self MHC antigens.

Restricted or absent immune responses in human populations to Plasmodium falciparum gamete antigens that are targets of malaria transmission-blocking antibodies.

We have studied the antibodies to sexual stage antigens of Plasmodium falciparum in human sera from Papua New Guinea where intense transmission of P. falciparum occurs as well as the less prevalent P. malariae and P. vivax. In extracts of gametes of P. falciparum we have studied the reactivity of serum antibodies with antigens labeled with 125I on the surface of the gametes as well as intracellular gamete antigens. A prominent 27-kD sexual stage-specific intracellular protein was recognized more or less in proportion to the general antibody response to gamete proteins. The response to the gamete surface proteins, however, was quite unrepresentative of the general antibody response to the intracellular gamete proteins. No antibodies were detected against Pfs25, a 21-kD protein expressed on zygotes and ookinetes of P. falciparum and known to be a sensitive target of malaria transmission-blocking antibodies. The antibody response to two other target antigens of transmission-blocking antibodies on the surface of gametes of P. falciparum, a 230- and a 48- and 45-kD protein doublet, was very variable and independent of the response to the internal protein antigens. Several possibilities are discussed that may account for the variable response to these gamete surface antigens in individuals with otherwise good antibody responses to internal sexual stage proteins. Among these is the possibility that there is MHC restriction of the immune response to the gamete surface antigens in the human population. This interpretation accords well with evidence for MHC-restricted immune response to the same P. falciparum gamete surface antigens in studies with H-2 congenic mice (24).

Immunoelectron microscopic observations on the inflammatory infiltrates and HLA antigens in hepatitis B and non-A, non-B.

The present knowledge of the inflammatory reaction occurring in situ during hepatitis B favors a T cell-dependent MHC-restricted immune response. However, the reports in the literature are primarily based on the application of monoclonal antibodies directed at different lymphocyte subsets which discern only lymphocytic phenotypes and do not reflect the actual situation adequately. Therefore, we investigated the liver biopsies of patients with hepatitis B (28 patients) and non-A, non-B (21 patients) by immunoelectron microscopy with monoclonal antibodies directed at lymphocyte subtypes (pan-B, pan-T, T8, T4 and NKH1) and at activation epitopes (IL-2 receptor, TA1 and T11/3) as well, in order to determine the phenotype in association with the activation status of the lymphocytes that are in close contact with hepatocytes; thus, establishing an effector-target cell relationship on the ultrastructural level. We were able to confirm the central role of T8 lymphocytes being the predominant type of lymphocytes in close contact with liver cells in the space of Disse. A certain percentage of these cells expressed "activation" markers as IL-2 receptor, TA1 and T11/3. In acute hepatitis, the NK lymphocytes made up a fifth of all lymphocytes, whereas their number dropped below 10% in the chronic stage. There was a vague correlation between the inflammatory activity of the disease and the expression of HLA antigens (both classes I and II) on inflammatory cells and also on hepatocytes. The results did not show significant differences between hepatitis B and non-A, non-B.(ABSTRACT TRUNCATED AT 250 WORDS)