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.     

Immunogenicity of tumor peptides: importance of peptide length and stability of peptide/MHC class II complex

Nonameric P815AB, a cytotoxic-T-lymphocyte-defined minimal core peptide encoded by the murine mastocytoma gene P1A, fails to initiate CD4⁺ cell-dependent reactivity in vivo to class-I-restricted epitopes when mice are administered peptide-pulsed dendritic cells. Effective immunization requires T helper effects, such as those mediated by coimmunization with class-II-restricted (helper) peptides or by the use of recombinant interleukin-12 (rIL-12). Although P815AB does possess class-II-restricted epitopes, they are likely suboptimal, resulting in poor affinity and/or stability of MHC/P815AB complexes and inadequate activation of the antigen-presenting cell function of dendritic cells. The present study has examined a series of longer, P815AB-centered peptides (11–14 amino acids in length, all P1A-encoded) for their ability to initiate CD4⁺ and CD8⁺ cell-mediated responses to the nonamer in vivo, their ability to bind class II MHC in vitro, and their ability to assemble class II molecules stably. By means of a class-I-restricted skin test assay in mice receiving peptide-pulsed dendritic cells, we found that a 12-mer and a 13-mer effectively immunized against the core P815AB peptide, and that this correlated with IL-2 production in vitro by CD4⁺ cells in response to the nonamer. In vitro studies, involving affinity-purified class II molecules, showed that the capacity to assemble class II molecules stably, more than the affinity for class II MHC, correlated with the ability of the different P815AB peptides to prime the host to the core peptide seen by the T cells. Received: 25 February 1999 / Accepted: 14 April 1999     

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     

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.     

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.     

T cell-induced secretion of MHC class II-peptide complexes on B cell exosomes

Antigen-specific interactions between B cells and T cells are essential for the generation of an efficient immune response. Since this requires peptide–MHC class II complexes (pMHC-II) on the B cell to interact with TCR on antigen-specific T cells, we have examined the mechanisms regulating the persistence, loss, and secretion of specific pMHC-II complexes on activated B cells. Using a mAb that recognizes specific pMHC-II, we found that activated B cells degrade approximately 50% of pMHC-II every day and release 12% of these pMHC-II from the cell on small membrane vesicles termed exosomes. These exosomes directly stimulate primed, but not naïve, CD4 T cells. Interestingly, engagement of antigen-loaded B cells with specific CD4 T cells stimulates exosome release in a manner that can be mimicked by pMHC-II crosslinking. Biochemical studies revealed that the pMHC-II released on exosomes was previously expressed on the plasma membrane of the B cells, suggesting that regulated exosome release from activated B cells is a mechanism to allow pMHC-II to escape intracellular degradation and decorate secondary lymphoid organs with membrane-associated pMHC-II complexes.     

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.     

MHC II类分子表达调控的研究进展

MHCII类分子提呈经过加工的抗原给CD4 T淋巴细胞 ,在诱发免疫反应中起重要作用。MHCII类分子不正常表达会引起严重的免疫缺陷疾病 ,如裸淋巴细胞综合征 (BLS)等。目前已识别出四种不同的MHCII调控基因。这些基因分别编码RFXANK、RFX5、RFXAP和CIITA。其中 ,前三个是RFX复合物的亚基 ,RFX是一种结合于所有MHCII类基因启动子上的泛式表达的因子。CIITA是MHCII类分子表达的主要调控因子 ,其严密调控的表达模式决定了MHCII类分子表达的细胞特异性 ,及能否被诱导且在何种水平上表达。本文着重介绍近年来国内外对MHCII类分子表达及其调控研究的新进展     

Dendritic cells in glomerulonephritis

Renal biopsies (n = 45) from patients with various forms of glomerulonephritis (GN), comprising mesangial IgA-GN (n = 25), focal glomerular sclerosis (n = 13) and acute GN (n = 7), were examined by double staining immunocytochemistry (APAAP, streptavidinperoxidase) using unconjugated monoclonal antibodies (Ab) against (i) the CD1b antigen expressed on dendritic cells (DCs), (ii) the invariant chain (Ii), and (iii) biotin-conjugated Ab against HLA-DR. In normal control kidneys (n = 7) without interstitial inflammation, CD1b-positive DCs were not detected. Glomerular endothelial cells and a few cells in mesangial areas showed double staining with the Ab against HLA-DR in Ii. In GN without active interstitial inflammation (n = 9), CD1b-positive DCs were not found. In biopsies with interstitial inflammation (n = 36) CDlb-positive DCs were found interspersed among other inflammatory cells. In seven of the biopsies showing IgA-GN DCs were seen in the vicinity of those glomeruli that exhibited either crescents or glomerular sclerosis with splitting of Bowman’s capsule. In proximal tubular epithelial cells de novo expression of HLA-DR/Ii-chain was only seen when DCs were present. We conclude that in different forms of GN: (i) CDlb-positive DCs play an important role in the development of interstitial inflammation, and (ii) their presence may be related to the de novo coexpression of HLA-DR/Ii in tubular epithelial cells, possibly mediated through the production of interferon γ and other cytokines. Supported by the Deutsche Forschungsgemeinschaft (Wa 698/2-1)     

Peptide motif for the rat MHC class II molecule RT1.Da: similarities to the multiple sclerosis-associated HLA-DRB1*1501 molecule

Experimental autoimmune encephalomyelitis induced with myelin proteins in DA and LEW.1AV1 rats is a model of multiple sclerosis (MS). It reproduces major aspects of this detrimental disease of the central nervous system. MS is associated with the HLA-DRB1*1501, DRB5*0101, and DQB1*0602 haplotype. DA and LEW.1AV1 rats share the RT1^av1 haplotype. So far, no MHC class II peptide motif of RT1.D^a molecules has been described. Sequence alignment of the chain of the rat MHC class II molecule RT1.D^a with human HLA class II molecules revealed strong similarity in the peptide-binding groove of RT1.D^a and HLA-DRB1*1501. According to the putative peptide-binding pockets of RT1.D^a, after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.D^a. To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.D^a molecules of lymphoid tissue of DA rats and subsequently analyzed by ESI tandem mass spectrometry. In addition, we performed binding studies with combinatorial nonapeptide libraries to purified RT1.D^a molecules. Based on these studies we could define a peptide-binding motif for RT1.D^a characterized by aliphatic amino acid residues (L, I, V, M) and of F for the peptide pocket P1, aromatic residues (F, Y, W) for P4, basic residues (K, R) for P6, aliphatic residues (I, L, V) for P7, and aromatic residues (F, Y, W) and L for P9. Both methods revealed similar binding characteristics for peptides to RT1.D^a. This data will allow epitope predictions for analysis of peptides, relevant for experimental autoimmune diseases.     

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.     

CLIP-region mediated interaction of Invariant chain with MHC class I molecules

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.     

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.     

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.     

Cathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells

Before a class II molecule can be loaded with antigenic material and reach the surface to engage CD4+ T cells, its chaperone, the class II-associated invariant chain (Ii), is degraded in a stepwise fashion by proteases in endocytic compartments. We have dissected the role of cathepsin S (CatS) in the trafficking and maturation of class II molecules by combining the use of dendritic cells (DC) from CatS(-/-) mice with a new active site-directed probe for direct visualization of active CatS. Our data demonstrate that CatS is active along the entire endocytic route, and that cleavage of the lysosomal sorting signal of Ii by CatS can occur there in mature DC. Genetic disruption of CatS dramatically reduces the flow of class II molecules to the cell surface. In CatS(-/-) DC, the bulk of major histocompatibility complex (MHC) class II molecules is retained in late endocytic compartments, although paradoxically, surface expression of class II is largely unaffected. The greatly diminished but continuous flow of class II molecules to the cell surface, in conjunction with their long half-life, can account for the latter observation. We conclude that in DC, CatS is a major determinant in the regulation of intracellular trafficking of MHC class II molecules.     

Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.