Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.     

Identical RT1 class II molecules are expressed by rat RT1m and RT1c haplotypes

The RT1m haplotype of MNR rats has been suggested to be a recombinant RT1 haplotype inheriting RT1.A (class I) alleles from RT1a (DA) and RT1.B (class II) alleles from RT1c (AUG). Additional serologic and biochemical assays, however, have suggested that RT1m and RT1c share a single identical RT1.B molecule, although differing in the expression of the second RT1.B molecule. To resolve this contradiction, RT1.B class II molecules, comparable to I-A and I-E molecules in mice, expressed by the RT1c and RT1m haplotypes were immunoprecipitated by cross-reactive mouse anti-Ia antibodies and were compared by two-dimensional gel electrophoresis and by high pressure liquid chromatographic separation of tryptic peptides. Respective subunits expressed by the two haplotypes co-migrate on two-dimensional gels and have identical tryptic peptide maps. The results at the protein level were confirmed at the DNA level by Southern blot analysis of MNR and AUG genomic DNA. Identical restriction fragments associated with the RT1m and RT1c haplotypes hybridized with each of the DC1 beta, DR alpha, and DR beta cDNA probes. The results at both the protein and DNA levels suggest that the RT1m and RT1c haplotypes share identical expressed alleles at the RT1.Ba, RT1.Bb, RT1.Bc, and RT1.Bd loci.     

PCR/oligonucleotide probe typing of HLA class II alleles in a Filipino population reveals an unusual distribution of HLA haplotypes.

We have analyzed the distribution of HLA class II alleles and haplotypes in a Filipino population by PCR amplification of the DRB1, DQB1, and DPB1 second-exon sequences from buccal swabs obtained from 124 family members and 53 unrelated individuals. The amplified DNA was typed by using nonradioactive sequence-specific oligonucleotide probes. Twenty-two different DRB1 alleles, including the novel Filipino *1105, and 46 different DRB1/DQB1 haplotypes, including the unusual DRB1*0405-DQB1*0503, were identified. An unusually high frequency (f = .383) of DPB1*0101, a rare allele in other Asian populations, was also observed. In addition, an unusual distribution of DRB1 alleles and haplotypes was seen in this population, with DR2 (f = .415) and DRB1*1502-DQB1*0502 (f = .233) present at high frequencies. This distribution of DRB1 alleles differs from the typical HLA population distribution, in which the allele frequencies are more evenly balanced. The distribution of HLA class II alleles and haplotypes in this Filipino population is different from that of other Asian and Pacific groups: of those populations studied to date; the Indonesian population is the most similar. DRB1*1502-DQB1*0502 was in strong linkage disequilibrium (D'' = .41) with DPB1*0101 (f = .126, for the extended haplotype), which is consistent with selection for this DR, DQ, DP haplotype being responsible for the high frequency of these three class II alleles in this population.     

Opposing effects of HLA class I molecules in tuning autoreactive CD8+ T cells in multiple sclerosis

The major known genetic risk factors in multiple sclerosis reside in the major histocompatibility complex (MHC) region. Although there is strong evidence implicating MHC class II alleles and CD4(+) T cells in multiple sclerosis pathogenesis, possible contributions from MHC class I genes and CD8(+) T cells are controversial. We have generated humanized mice expressing the multiple sclerosis-associated MHC class I alleles HLA-A(*)0301 (encoding human leukocyte antigen-A3 (HLA-A3)) and HLA-A(*)0201 (encoding HLA-A2) and a myelin-specific autoreactive T cell receptor (TCR) derived from a CD8(+) T cell clone from an individual with multiple sclerosis to study mechanisms of disease susceptibility. We demonstrate roles for HLA-A3-restricted CD8(+) T cells in induction of multiple sclerosis-like disease and for CD4(+) T cells in its progression, and we also define a possible mechanism for HLA-A(*)0201-mediated protection. To our knowledge, these data provide the first direct evidence incriminating MHC class I genes and CD8(+) T cells in the pathogenesis of human multiple sclerosis and reveal a network of MHC interactions that shape the risk of multiple sclerosis.     

Activation of murine T cells by toxic shock syndrome toxin-1. The toxin-binding structures expressed on murine accessory cells are MHC class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding structures present on murine lymphoid tissues were investigated by using 125I-TSST-1. T-depleted C57BL/6 spleen cells incubated with TSST-1 for 3 h at 0 degree C were mitogenic to splenic T cells, indicating that the former cells bind and present TSST-1 to T cells. TSST-1-binding activity was observed in C57BL/6 splenic B cells and L cells transfected with I-Ab genes, but not in splenic T cells and control L cells. Scatchard plot analysis showed that these B cells and transfectants bound TSST-1 with similar binding affinity. SDS-PAGE analysis showed that lysates of C57BL/6 spleen cells and the I-Ab-positive transfectants contain a single band which bound TSST-1 and comigrated with I-Ab heterodimers. TSST-1-binding activity observed clearly in C57BL/6. BALB/c, and C3H/HeN spleen cells and L cells transfected with I-Ab or I-Ak genes was not reduced by paraformaldehyde fixation. Binding of 125I-TSST-1 to the three spleen cells was markedly reduced by anti-I-A antibodies, but not by anti-I-E antibodies. C57BL/6, C3H/HeN, and (C3H/HeN x C57BL/6) F1 T cells were activated by TSST-1 to proliferate and produce IL-2 in the presence of FT6.2 cells, LT1-30-3 cells and either of them, respectively, but not in the presence of control L cells. These results indicate that I-A molecules function as the structures via that accessory cells directly bind TSST-1 on the cell surface and present a triggering signal of TSST-1 to T cells.     

Possible association between IL-2 receptors and class I HLA molecules on T cells

In the process of evaluating murine hybridomas for an antibody to the beta-subunit of the IL-2R (p70) we identified an antibody that immunoprecipitated a 55- to 57-kDa complex from cross-linked lysates. We demonstrate that this complex is composed of IL-2 (15.5 kDa) cross-linked to the H chain of HLA class I (40 to 42 kDa), suggesting a molecular interaction between HLA class I molecules and IL-2R. Although the exact role of this association remains to be determined, the specific cross-linking of IL-2 to HLA class I Ag is intriguing in view of published claims for a role of HLA class I in OKT3-induced lymphocyte proliferation and in NK cell lytic activity.     

Regulation of class II MHC molecules on human endothelial cells. Effects of IFN and dexamethasone

Human vascular endothelial cells normally do not express class II MHC molecules in culture. IFN-gamma has been shown to induce expression of class I and class II MHC molecules on endothelial cell cultures from umbilical cord. We could detect these Ag by FACS analysis when endothelial cells were cultured for 3 days in the presence of 200 to 1000 U/ml of rIFN-gamma. Among the class II MHC molecules, HLA-DR and -DP but not -DQ were consistently induced. Addition of rIFN-alpha-D/A to IFN-gamma-treated cells inhibited the expression of class II MHC but not class I MHC molecules. Furthermore, the inhibition was more pronounced when IFN-alpha-D/A was added before or simultaneously as IFN-gamma. Natural IFN-alpha also exhibited similar inhibition and its suppressive effect was abolished in the presence of anti-IFN-alpha antibody. On the contrary, dexamethasone, a known inhibitor of class II MHC molecules on murine macrophages, showed a slight enhancing effect on class II MHC Ag. These results suggest an immunoregulatory role for IFN-alpha on non-lymphoid cells and that controlling elements for expression of class II MHC molecules may be different on various cell types as well as species.     

Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.     

Physical association between MHC class I and class II molecules detected on the cell surface by flow cytometric energy transfer

The physical association of HLA class I and class II Ag in the membranes of PGF and JY lymphoblastoid cell lines was studied using flow cytometric energy transfer. This technique measures the proximity of cell surface molecules in the nm range and provides a distribution histogram of the average proximity of molecules on each cell of a population. HLA Ag were labeled with mAb conjugated to fluorescein, serving as donor, or tetramethylrhodamine, serving as acceptor molecules. Significant fluorescence energy transfer was detected between various combinations of class I and class II molecules indicating that these molecules are within 10 nanometers of each other. Specifically, energy transfer was observed between class I molecules and DR, DQ, or DP class II HLA molecules. In addition, energy transfer between all combinations of DR, DQ, and DP molecules was observed. No transfer was observed among class I molecules or among DR or among DP molecules. Among DQ molecules, subpopulations transferred fluorescence energy to each other. The close contact measured between class I and class II Ag correlates with previous reports of cocapping and may reflect an immunologically significant interaction or the reported tendency of class I Ag to associate with other cell surface receptors, including growth factor receptors. The energy transfer between fluorescent antibodies to class II Ag suggests the existence of heterodimers formed from the different locus products, as well as possible quaternary surface interactions between alpha/beta complexes from separate loci.     

Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.     

Evidence for specific association between class I major histocompatibility antigens and the CD8 molecules of human suppressor/cytotoxic cells

Human T lymphocytes, metabolically labeled with 35S-cysteine and 35S-methionine, were reacted with the homobifunctional cross-linking reagent, dithiobis (succinimidyl propionate) (DSP). When detergent lysates from these cells were immunoprecipitated with a monoclonal antibody reactive with the CD8 antigen, a radiolabeled protein of approximately 44 kd was coprecipitated with the CD8 molecule. Immunoprecipitates from detergent lysates prepared without prior chemical cross-linking contained only the 33 kd CD8 molecule. Similar results were obtained when T lymphocytes or a cytotoxic T cell clone (T4T8Cl) were radiolabeled with 32P-orthophosphoric acid. The 44 kd CD8-associated protein was identified as the heavy chain of the class I major histocompatibility antigen by depletion in preclearing experiments with anti-class I MHC antibody and by peptide mapping. Further analyses indicated that the CD8-class I MHC association is due, in part at least, to disulfide bonding, which may be susceptible to cleavage during processing of cell lysates.     

Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells.

MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen-presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195-212 of the human acetylcholine receptor (AChR) alpha-subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA-DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195-212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195-212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195-212 was monitored using phycoerythrin-avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195-212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti-I-A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC.     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

B cells induce tolerance by presenting endogenous peptide-IgG on MHC class II molecules via an IFN-gamma-inducible lysosomal thiol reductase-dependent pathway

We have previously demonstrated that splenic B cells, transduced with peptide-IgG fusion proteins, are efficient tolerogenic APCs in vivo. Specific hyporesponsiveness to epitopes encoded in the peptide-IgG fusion protein has been achieved to over one dozen Ags, and clinical efficacy has been established in animal models for several autoimmune diseases and hemophilia. Previous studies also demonstrated that tolerance in this system requires MHC class II expression by the transduced B cells. Yet, the mechanisms of this B cell tolerogenic processing pathway remain unclear. In this study, we show that MHC class II molecules on tolerogenic B cells present epitopes derived from endogenous, but not exogenous (secreted), peptide-IgG fusion protein. These class II epitopes from the IgG fusion protein are processed in lysosomes/endosomes in an IFN-gamma-inducible lysosomal thiol reductase-dependent manner. We suggest that the MHC class II presentation of endogenously produced fusion protein epitopes represents a novel mechanism for tolerance induced by peptide-IgG-transduced B cells. An understanding of this process might provide insights into central and peripheral tolerance induced by other professional and nonprofessional APCs.     

Induction of HLA class II molecules on human T cells: relationship to immunoregulation and the pathogenesis of AIDS.

Human T cells express HLA class II molecules upon activation. The factors that regulate the induction of expression of these molecules are for the most part unknown. Here we report preliminary results indicating that tumor necrosis factor-alpha (TNF-alpha) regulates the induction of cell-surface HLA-DR, DO, and DP molecules in human T cells stimulated with PHA. In contrast, recombinant interferon-gamma (rIFN-gamma), recombinant interleukin-1 alpha (rIL-1 alpha), or rIL-4 appear to have no effect on class II expression. The role of class II molecules on activated T cells is discussed in relationship to immunoregulation and the progression of HIV infection. Three non-mutually exclusive hypotheses are discussed. In the first hypothesis, we consider the role of these class II molecules in antigen presentation of endogenously synthesized HIV envelope by CD4+ cells. The second is a clonal inactivation of virus-specific helper T cells that might occur as a consequence of a direct T cell to T cell interaction and a bypass of the "accessory signal" normally delivered by antigen-presenting cells such as macrophages. The third is a molecular mimicry between HIV envelope proteins and HLA class II molecules, which may lead to the development of autoimmunity against CD4+ T-cell-expressing class II molecules.     

Monomeric class I molecules mediate TCR/CD3 epsilon/CD8 interaction on the surface of T cells.

Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using fluorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3epsilon is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular activation.     

Effect of isotypic and allotypic variations of MHC class II molecules on staphylococcal enterotoxin presentation to murine T cells.

Staphylococcal enterotoxins (SE) are known to be potent T cell activators, stimulating +/- proliferation and lymphokine production. These toxins have recently have been termed "superantigens" because of their ability to bind directly to class II molecules forming a ligand that interacts with particular V beta gene elements within the TCR complex. This interaction between SE and MHC class II molecules plays a central role in toxin-induced mitogenesis. In the present study we have examined the effect of polymorphism on the ability of MHC class II molecules to bind and present SE. Through the use of H-2 congenic mouse strains, it was possible to look directly at haplotype differences within the MHC and their effect on SE presentation to a panel of responsive V beta-bearing T cells. The results demonstrate that toxin presentation by class II-bearing accessory cells to murine T cells is greatly affected by polymorphisms within the H-2 complex. Toxin-pulsed accessory cells obtained from mice of an H-2k and H-2u haplotype were found to be less efficient in activating a variety of T cell clones and hybridomas. However, one T cell clone responded similarly to the enterotoxins presented on all H-2 haplotypes, suggesting that differences in responses of T cells are not simply a function of the degree of binding of these toxins to various class II molecules. Neutralization analysis with monoclonal anti-class II antibodies demonstrates that both I-A and I-E molecules play a significant role in SEA and SEB presentation to murine T cells. These results suggest that the differential activation of T cells by a particular enterotoxin may reflect a difference in recognition of an SE:class II ligand by a surface T cell receptor complex.