Crystal structure of MHC class II I-Ab in complex with a human CLIP peptide: prediction of an I-Ab peptide-binding motif

Association between the class II major histocompatibility complex (MHC) and the class II invariant chain-associated peptide (CLIP) occurs naturally as an intermediate step in the MHC class II processing pathway. Here, we report the crystal structure of the murine class II MHC molecule I-A(b) in complex with human CLIP at 2.15A resolution. The structure of I-A(b) accounts, via the peptide-binding groove's unique physicochemistry, for the distinct peptide repertoire bound by this allele. CLIP adopts a similar conformation to peptides bound by other I-A alleles, reinforcing the notion that CLIP is presented as a conventional peptide antigen. When compared to the related HLA-DR3/CLIP complex structure, the CLIP peptide displays a slightly different conformation and distinct interaction pattern with residues in I-A(b). In addition, after examining the published sequences of peptides presented by I-A(b), we discuss the possibility of predicting peptide alignment in the I-A(b) binding groove using a simple scoring matrix.     

Contribution of a single hydrogen bond between betaHis81 of MHC class II I-E(k) and the bound peptide to the pH-dependent thermal stability

To determine the energetic contribution of the hydrogen bond between betaHis81 of the major histocompatibility complex class II (MHC II) molecule, I-E(k), and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond-disrupted mutant, I-E(k)-Hb betaH81Y, in which the betaHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I-E(k)-Hb betaH81Y mutant was lower than that of the I-E(k)-Hb wild-type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I-E(k)-Hb betaH81Y mutant as compared with that of the I-E(k)-Hb wild-type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by betaHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.     

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide: MHC class II complexes in B cells

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-A^b and an antigenic peptide E52–68 covalently linked to its -chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, E52–68:I-A^b complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.     

A probabilistic meta-predictor for the MHC class II binding peptides

Several computational methods for the prediction of major histocompatibility complex (MHC) class II binding peptides embodying different strengths and weaknesses have been developed. To provide reliable prediction, it is important to design a system that enables the integration of outcomes from various predictors. The construction of a meta-predictor of this type based on a probabilistic approach is introduced in this paper. The design permits the easy incorporation of results obtained from any number of individual predictors. It is demonstrated that this integrated method outperforms six state-of-the-art individual predictors based on computational studies using MHC class II peptides from 13 HLA alleles and three mouse MHC alleles obtained from the Immune Epitope Database and Analysis Resource. It is concluded that this integrative approach provides a clearly enhanced reliability of prediction. Moreover, this computational framework can be directly extended to MHC class I binding predictions.     

Endocytic recycling is required for the presentation of an exogenous peptide via MHC class II molecules

Exogenous antigenic peptides captured and presented in the context of major histocompatibility (MHC) class II molecules on APC, have been employed as potent vaccine reagents capable of activating cellular immune responses. Binding and presentation of select peptide via surface class II molecules has been reported. Here, a role for endocytosis and early endosomes in the presentation of exogenous peptides via MHC class II molecules is described. T cell recognition of a 14 amino acid human serum albumin-derived peptide in the context of HLA-DR4 was observed only with metabolically active APC. The delayed kinetics and temperature dependence of functional peptide presentation via APC, were consistent with a requirement for peptide internalization to early endosomal compartments prior to T cell recognition. Ablating endocytosis by exposing cells to inhibitors of ATP production completely blocked the display of functional peptide:class II complexes on the surface of the APC. Presentation of the peptide was also found to be sensitive to primaquine, a drug that perturbs the recycling of transport vesicles containing endocytic receptors and mature class II complexes. Functional presentation of the endocytosed peptide was dependent upon these mature class II complexes, as inhibitor studies ruled out a requirement for newly synthesized class II molecules. N-terminal processing of the endocytosed peptide was observed upon trafficking through endosomal compartments and linked to the formation of functional peptide:class II complexes. These findings establish a novel mechanism for regulating class II-restricted peptide presentation via the endocytic pathway.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.     

Regulation of MHC class I and II gene transcription: differences and similarities

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8⁺ (cytotoxic) and CD4⁺ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes. Received: 12 January 1998     

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

Summary. High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes ( Pvu II, Hind III, Bg /II, and Bam HI), electrophoresed for 18 or 45h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II β-chain probe (β2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B halotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

The immune function of MHC class II molecules mutated in the putative superdimer interface

Analysis of the crystal structure of human class II (HLA-DR1) molecules suggests that the heterodimer may be further ordered as a dimer of heterodimers (superdimer), leading to the hypothesis that T cell receptor dimerisation is a mechanism for initiating signaling events preceding T cell activation. The interface between pairs of molecules is stabilised by both salt bridges, polar and hydrophobic interactions. The residues that form the superdimer interface occur in three areas distinct from the antigen-binding groove. They can be defined as follows: region 1, - contacts in the helix of the 1 domain; region 2, - contacts near the 1/2 domain junction and region 3; - contacts in the 2/2 domains adjacent to the plasma membrane. To determine whether salt bridges and polar interactions formed within these regions are involved in the immune function of the murine MHC class II molecule, I-A^b, appropriate residues in both the and chain were identified and mutated to uncharged alanine. Cell lines transfected with different combinations of mutated and chains were generated and tested for MHC class II expression, peptide binding capabilities, and ability to present antigenic peptide to an OVA-specific T cell hybridoma. With the exception of two residues in region 2, the substitutions tested did not modulate MHC class II expression, or peptide binding function. When tested for ability to present peptide to an antigen-specific T cell hybridoma, with the exception of mutations in region 2, the substitutions did not appear to abrogate the ability of I-A^b to stimulate the T cells. These results suggest that mutation of residues in region 2 of the putative superdimer interface have a gross effect on the ability of I-A^b to be expressed on the cell surface. However, abrogation of salt bridges in region 1 and 3 do not influence I-A^b cell surface expression, peptide binding or ability to stimulate antigen-specific T cells.     

Diversification of porcine MHC class II genes: evidence for selective advantage

The major histocompatibility complex (MHC) is an immunological gene-dense region of high diversity in mammalian species. Sus scrofa was domesticated by at least six independent events over Eurasia during the Holocene period. It has been hypothesized that the level and distribution of MHC variation in pig populations reflect genetic selection and environmental influences. In an effort to define the complexity of MHC polymorphisms and the role of selection in the generation of class II gene diversity (DQB, DRB1, and pseudogene ΨDRB3), DNA from globally distributed unrelated domestic pigs of European and Asian origins and a Suidae out-group was analyzed. The number of pseudogene alleles identified (ΨDRB3 33) was greater than those found in the expressed genes (DQB 20 and DRB1 23) but the level of observed heterozygosity (ΨDRB3 0.452, DQB 0.732, and DRB1 0.767) and sequence diversity (ΨDRB3 0.029, DQB 0.062, and DRB1 0.074) were significantly lower in the pseudogene, respectively. The substitution ratios reflected an excess of d _N (DQB 1.476, DRB1 1.724, and ΨDRB3 0.508) and the persistence of expressed gene alleles suggesting the influence of balancing selection, while the pseudogene was undergoing purifying selection. The lack of a clear MHC phylogeographic tree, coupled with close genetic distances observed between the European and Asian populations (DQB 0.047 and DRB1 0.063) suggested that unlike observations using mtDNA, the MHC diversity lacks phylogeographic structure and appears to be globally uniform. Taken together, these results suggest that, despite regional differences in selective breeding and environments, no skewing of MHC diversity has occurred. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

MHC class II expression and antigen presentation by human endometrial cells

It has been demonstrated previously that mixed cell suspensions from the female reproductive tract consisting of human epithelial and stromal cells were capable of presenting foreign antigen to autologous T cells. There have been, however, no reported studies examining antigen presentation by isolated epithelial cells from the human female reproductive tract. It is now shown that freshly isolated epithelial cells from the uterine endometrium constitutively express MHC class II antigen and that class II was upregulated on cultured epithelium by interferon gamma (IFNγ). Using a highly purified preparation, it was demonstrated that these epithelial cells were able to process and present tetanus toxoid recall antigen driving autologous T cell proliferation. Cells isolated from the basolateral sub-epithelium stroma were also potent antigen presenting cells in this model system. Thus, isolated endometrial epithelial cells were able to directly process and present antigen to T cells and may be responsible for the transcytosis and delivery of antigen to professional antigen presenting cells found in the sub-epithelial stroma.     

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha-and beta-chain cDNA probes

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.     

Presentation of antigenic peptides by products of the major histocompatibility complex

Molecules encoded by the major histocompatibility complex (MHC) are polymorphic integral membrane proteins adapted to the presentation of peptide fragments of foreign antigens to antigen-specific T-cells. The diversity of infectious agents to which an immune response must be mounted poses a unique problem for receptor–ligand interactions; how can proteins whose polymorphism is necessarily limited bind an array of peptides almost infinite in its complexity? Both MHC class I and class II determinants have achieved this goal by harnessing a limited number of peptide side chains to anchor the epitope in place while exploiting conserved features of peptide structure, independent of their primary sequence. While class I molecules interact predominantly with the N- and C-termini of peptides, class II determinants form an extensive hydrogen bonding network along the length of the peptide backbone. Such a strategy ensures high-affinity binding, while selectively exposing the unique features of each ligand for recognition by the T-cell receptor. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

MHC class II polymorphism is associated with a canine SLE-related disease complex

Nova Scotia duck tolling retrievers are predisposed to a SLE-related disease complex including immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis–arteritis (SRMA). IMRD involves symptoms that resemble those seen in systemic autoimmune rheumatic diseases, such as systemic lupus erythematosus, SLE, or SLE-related diseases, in humans. This disease complex involves persistent lameness, stiffness, mainly after resting, and palpable pain from several joints of extremities. The majority of affected dogs display antinuclear autoantibody (ANA)-reactivity. SRMA is manifested in young dogs with high fever and neck stiffness and can be treated with corticosteroids. We have investigated the possible role of MHC class II as a genetic risk factor in IMRD and SRMA etiology. We performed sequence-based typing of the DLA-DRB1, -DQA1, and -DQB1 class II loci in a total of 176 dogs including 51 IMRD (33 ANA-positive), 49 SRMA cases, and 78 healthy controls (two dogs were both IMRD- and SRMA-affected). Homozygosity for the risk haplotype DRB1*00601/DQA1*005011/DQB1*02001 increased the risk for IMRD (OR?=?4.9; ANA-positive IMRD: OR?=?7.2) compared with all other genotypes. There was a general heterozygote advantage, homozygotes had OR?=?4.4 (ANA-positive IMRD: OR?=?8.9) compared with all heterozygotes. The risk haplotype contains the five amino acid epitope RARAA, known as the shared epitope for rheumatoid arthritis. No association was observed for SRMA. We conclude that DLA class II is a highly significant genetic risk factor for ANA-positive IMRD. The results indicate narrow diversity of DLA II haplotypes and identify an IMRD-related risk haplotype, which becomes highly significant in homozygous dogs.     

The selection of tumor variants with altered expression of classical and nonclassical MHC class I molecules: implications for tumor immune escape

Tumor immune escape variants can be identified in human and experimental tumors. A variety of different strategies are used by tumor cells to avoid recognition by different immune effector mechanisms. Among these escape routes, alteration of MHC class I cell surface expression is one of the mechanisms most widely used by tumor cells. In this review we focus our attention on the T-cell immune selection of MHC class I–deficient tumor variants. Different altered MHC class I phenotypes that originate from multiple molecular mechanisms can be identified in human tumors. MHC-deficient tumor clones can escape T-cell immune responses, but are in theory more susceptible to NK-cell–mediated lysis. In this context, we also review the controversial issue of the aberrant expression of nonclassical HLA class I molecules, particularly HLA-G, in tumors. This expression may be relevant in tumor cells that have lost the capacity to interact with NK inhibitory receptors—namely, those tumor cells with no HLA-B or HLA-C expression. Most published studies have not analyzed these possibilities and do not provide information about the complete HLA-A, HLA-B, or HLA-C molecule profiles of the tumors studied. In contrast, HLA-E has been reported to be expressed in some tumor cell lines with very low HLA-A, HLA-B, and HLA-C expression, suggesting that HLA-E may indeed, in some cases, play a role by inhibiting NK lysis of cells that otherwise would be destroyed by NK cells. Finally, we provide evidence that the status of the immune system in the tumor-bearing animal is capable of defining the MHC profile of the tumor cells. In other words, MHC class I–negative metastatic colonies are produced in immunocompetent animals, and MHC class I–positive colonies in T-cell immunodeficient individuals.This article forms part of the Symposium in Writing Tumor escape from the immune response, published in Vol. 53.