Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding

Class II major histocompatibility complex (MHC) molecules are heterodimeric cell surface glycoproteins which bind and present immunogenic peptides to T lymphocytes. Such peptides are normally derived from protein antigens internalized and proteolytically degraded by the antigen-presenting cell. Class I MHC molecules also bind immunogenic peptides, but these are derived from proteins synthesized within the target cell. Whereas class I molecules seem to bind peptides in the endoplasmic reticulum, class II molecules are thought to bind peptides late in transport. Intracellular class II molecules associate in the endoplasmic reticulum with a third glycoprotein, the invariant (I) chain, which is proteolytically removed before cell surface expression of the alpha beta class II heterodimer. It has been suggested that the I chain prevents peptides from associating with class II molecules early in transport. Preventing such binding until the class II molecules enter an endosomal compartment could maintain the functional dichotomy between class I and class II MHC molecules. We have examined the ability of I chain-associated HLA-DR5 molecules to bind a well characterized influenza haemagglutinin-derived peptide (HAp). The results show that whereas mature HLA-DR alpha beta dimers effectively bind this peptide, the I chain-associated form does not.     

Structural and evolutionary analysis of HLA-D-region products

The major histocompatibility complex (MHC)--HLA in man and H-2 in mouse--encodes two classes of cell-surface antigens involved in the immune response. The amino acid sequences have been determined for a number of these molecules. Class I antigens, typified by the HLA-ABC antigens, are composed of a 43,000-molecular weight (MW) glycosylated transmembrane polypeptide with three external domains (alpha 1, alpha 2 and alpha 3), of which the one nearest the membrane (alpha 3) is associated with a 12,000-MW nonglycosylated polypeptide, beta 2-microglobulin. The HLA-D-region or class II antigens, DR, DC and SB, are composed of two glycosylated transmembrane polypeptides, of MWs 34,000 (alpha-chain) and 28,000 (beta-chain). Both chains have two external domains which presumably associate with each other, alpha 2, beta 2 being membrane proximal and alpha 1, beta 1 N-terminal and membrane distal. All four membrane-proximal domains (class I alpha 3, beta 2-microglobulin, class II alpha 2 and beta 2) have amino acid sequences that show significant similarities with immunoglobulin constant-region domains. This, together with the similarly placed internal disulphide bonds, suggests they might have an immunoglobulin-like structure (Fig. 1). We have now used computer graphics techniques to predict a detailed three-dimensional structure for the membrane-proximal domains of the class II antigens (alpha 2 and beta 2) based on the known coordinates of immunoglobulin constant domains (Fig. 2). The transmembrane regions of class II antigens have been modelled as two alpha-helices packed together. The proposed structure accounts for conservation of amino acids and leads to evolutionary predictions.     

Excess beta 2 microglobulin promoting functional peptide association with purified soluble class I MHC molecules

T lymphocytes expressing alpha beta receptors recognize antigenic peptide fragments bound to major histocompatibility complex class I or class II molecules present on the surface membranes of other cells. Peptide fragments are present in the two available HLA crystal structures and recent data indicate that peptide is required for the stable folding of the class I heavy chain and maintenance of its association with the class I light chain, beta 2-microglobulin (beta 2m), at physiological temperature. To explain how the exogenous peptide used to create targets for cytotoxic cells bearing CD8 antigen could associate with apparently peptide-filled extracellular class I molecules, we hypothesized that stable binding of exogenous peptide to mature class I molecules reflects either the replacement of previously bound peptide during the well documented beta 2m exchange process or the loading of 'empty' class I heavy chains dependent on the availability of excess beta 2m. In either case, free beta 2m should enhance peptide/class I binding. Using either isolated soluble class I molecules or living cells, we show here that free purified beta 2m markedly augments the generation of antigenic complexes capable of T-cell stimulation.     

Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Signal for T-cell differentiation to a CD4 cell lineage is delivered by CD4 transmembrane region and/or cytoplasmic tail.

Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4+CD8- and CD4-CD8+ T cells expressing alpha beta T-cell antigen receptors (TCR) develop from immature thymocytes through CD4+CD8+ alpha beta TCR+ intermediates. Experiments using mice transgenic for alpha beta TCR suggest that the specificity of the TCR determines the CD4/CD8 phenotype of mature T cells. These results, however, do not indicate how a T cell differentiates into the CD4 or CD8 lineage. Here we show that the CD4 transmembrane region and/or cytoplasmic tail mediates the delivery of a specific signal that directs differentiation of T cells to a CD4 lineage. We generated transgenic mice expressing a hybrid molecule composed of the CD8 alpha extracellular domains linked to the CD4 transmembrane region and cytoplasmic tail. We predicted that this hybrid molecule would bind to class I MHC proteins through the extracellular domains but deliver the intracellular signals characteristic of CD4. By crossing our transgenic mice with mice expressing a transgenic alpha beta TCR specific for a particular antigen plus class I MHC protein, we were able to express the hybrid molecule in developing thymocytes expressing the class I MHC-restricted TCR. Our results show that the signal transduced by the hybrid molecule results in the differentiation of immature thymocytes expressing a class I-restricted TCR into mature T cells expressing CD4.     

MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8.

Interactions between major histocompatibility complex (MHC) molecules and the CD4 or CD8 coreceptors have a major role in intrathymic T-cell selection. On mature T cells, each of these two glycoproteins is associated with a class-specific bias in MHC molecule recognition by the T-cell receptor. CD4+ T cells respond to antigen in association with MHC class II molecules and CD8+ T cells respond to antigen in association with MHC class I molecules. Physical interaction between the CD4/MHC class II molecules and CD8/MHC class I molecules has been demonstrated by cell adhesion assay, and a binding site for CD8 on class I has been identified. Here we demonstrate that a region of the MHC class II beta-chain beta 2 domain, structurally analogous to the CD8-binding loop in the MHC class I alpha 3 domain, is critical for function with both mouse and human CD4.     

Unexpected expression of a unique mixed-isotype class II MHC molecule by transfected L-cells

Class II (Ia) major histocompatibility complex (MHC) molecules are heterodimeric integral membrane proteins composed of non-covalently linked alpha and beta glycoprotein chains. Studies of both normal cells and L-cell transfectants have shown that neither alpha- nor beta-chains are found on the cell surface alone, and that alpha beta dimers are required for membrane expression. In both mouse and man, several distinct non-allelic alpha and beta genes exist. Analysis of Ia molecules by immunoprecipitation and two-dimensional gel electrophoresis has demonstrated apparently selective association of particular pairs of the various alpha- and beta-chains to form the expressed class II isotypes I-A and I-E (mouse) or DQ, DP and DR (human). Because the various alpha- or beta-chains encoded by distinct loci exist in many allelic forms within a species, such specific pairing suggests a special role for isotypically conserved regions of each chain in the association process. In attempting to localize such putative assembly-controlling regions using the technique of DNA-mediated gene transfer, various combinations of murine alpha and beta genes were introduced into L-cells. Here we report the unexpected observation, following transfection, of mixed-isotype (Ad beta Ea/k alpha) molecules on the L-cell membrane and document that the formation of this pair is strongly influenced by allelic polymorphism of the A beta chain.     

An immunological role for the CD8 beta-chain.

Mature T cells can be functionally divided into two categories distinguished by surface expression of either CD4 or CD8, which in turn corresponds to restriction by and binding to class II or class I major histocompatibility complex proteins, respectively. CD8 can be expressed as a homodimer of the alpha-chain, or as a heterodimer of alpha- and beta-chains on human and mouse T cells, although most peripheral T cells seem to express CD8 alpha beta heterodimers exclusively (reviewed in ref. 9). Functional characterization of CD8 has focused primarily on the effect of the alpha-chain, which enhances or reconstitutes T-cell responses in homodimeric form and may play a specific role in thymic selection. In contrast, no role has been ascribed to CD8 beta or alpha beta heterodimers specifically. Here we show that CD8 alpha beta transfectants produce more interleukin-2 than CD8 alpha transfectants in response to specific stimuli. Increased interleukin-2 is also observed in cells expressing hybrid CD8 beta-alpha molecules (extracellular CD8 beta plus CD8 alpha transmembrane and cytoplasmic regions) on their surface. These results indicate that external portions of CD8 beta could be critical and that they may act independently of CD8 alpha in mediating their augmentation effect.     

A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules

Class II and class I histocompatibility molecules allow T cells to recognize 'processed' polypeptide antigens. The two polypeptide chains of class II molecules, alpha and beta, are each composed of two domains (for review see ref. 6); the N-terminal domains of each, alpha 1 and beta 1, are highly polymorphic and appear responsible for binding peptides at what appears to be a single site and for being recognized by MHC-restricted antigen-specific T cells. Recently, the three-dimensional structure of the foreign antigen binding site of a class I histocompatibility antigen has been described. Because a crystal structure of a class II molecule is not available, we have sought evidence in class II molecules for the structural features observed in the class I binding site by comparing the patterns of conserved and polymorphic residues of twenty-six class I and fifty-four class II amino acid sequences. The hypothetical class II foreign-antigen binding site we present is consistent with mutation experiments and provides a structural framework for proposing peptide binding models to help understand recent peptide binding data.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

Tetrameric cell-surface MHC class I molecules.

Purified major histocompatibility complex (MHC) class I molecules have been studied at high resolution by X-ray crystallography; the structure is a complex of a single heavy chain, a beta 2-microglobulin light chain and a tightly bound peptide moiety. We show here that complete MHC class I molecules are post-translationally assembled into tetramers (made up of four heavy chains and four beta 2-microglobulin units) and that this tetrameric species is expressed on the cell surface. The multivalent tetrameric structure of class I molecules can be reconciled with models of T-cell activation that invoke antigen-receptor crosslinking, as opposed to models that depend on an allosteric change.     

A molecular map of the immune response region from the major histocompatibility complex of the mouse

A stretch of 200 kilobases (kb) of DNA from the I region of the mouse major histocompatibility complex has been cloned and characterized. It contains the genes for the biochemically defined class II proteins E alpha, E beta and A beta. DNA blot analyses suggest that the I region may contain only 6-8 class II genes. Correlation of our molecular map with the genetic map of the I region confines two of the five I subregions, I-J and I-B, to less than 3.4 kb of DNA at the 3' end of the E beta gene where a hotspot for recombination has been observed. Indeed, the I-A and I-E subregions may be contiguous. If so, the I-B and I-J subregions are not encoded in the I region between the I-A and I-E subregions.     

A combination of a particular HLA-DP beta allele and an HLA-DQ heterodimer confers susceptibility to coeliac disease

Coeliac disease is an autoimmune disease of the intestinal mucosa, elicited by ingestion of wheat gluten in genetically susceptible individuals. Susceptibility to coeliac disease has been associated with the serologically defined variants DR3 and DR7 of the class II antigens encoded by the HLA-D region. Three related class II antigens, each consisting of an alpha and a beta glycoprotein chain, have been identified and are designated HLA-DR, HLA-DQ, and HLA-DP. These highly polymorphic transmembrane proteins bind peptides derived from the processing of foreign antigens and present them to T lymphocytes; they also influence the specificity of the mature T-cell repertoire. The role of HLA-DP polymorphism in susceptibility has not been as fully explored as that of the other class II antigens because of the complexity of the primed lymphocyte typing (PLT) method for determining DPw specificities. Here we use a new DNA-based method of HLA-DP typing to analyse the distribution of DP beta alleles in a group of coeliac disease patients and healthy controls. Two specific DP beta alleles (DPB4.2 and DPB3) are increased in the patient population. Comparison of the DP beta sequences suggests that the polymorphic residues at position 69 and at 56 and 57 may be critical in conferring susceptibility. Further, the contribution of the susceptible DP beta alleles appears to be independent of linkage to the previously reported DR3 and DR7 markers for coeliac disease. The distribution of DQ alpha and beta alleles in patients suggests that a specific DQ heterodimer may be responsible for the observed DR associations. Individuals with both this DQ antigen and a specific DP beta allele are at increased risk for coeliac disease.     

Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules.

The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells. CD4+ and CD8+ T cells recognize antigens on major histocompatibility complex (MHC) class II-bearing and class I-bearing target cells respectively. The ability of monoclonal antibodies against CD4 and CD8 to block antigen recognition by T cells, as well as cell-cell adhesion assays, indicate that CD4 and CD8 bind to nonpolymorphic determinants of class II or class I MHC. Here we demonstrate that soluble recombinant HLA-DR4 molecules from insect cells and HLA-DR-derived peptides bind to immobilized recombinant soluble CD4. CD4 binds recombinant soluble DR4 heterodimers, as well as the soluble DR4-beta chain alone. Furthermore, two out of twelve DR4-beta peptides could interact specifically with CD4. These findings show that CD4 interacts with a region of MHC class II molecules analogous to a previously identified loop in class I MHC proteins that binds CD8 (refs 8, 9).     

A minimum of four human class II alpha-chain genes are encoded in the HLA region of chromosome 6

The major histocompatibility complex (MHC) in man, also called the HLA region, is located on the short arm of chromosome 6 and encodes antigens involved in immunological processes. The class II HLA antigens consist of two noncovalently associated polypeptide chains, one of molecular weight 34,000 (alpha) and the other of molecular weight 29,000 (beta). The extensive polymorphism of the beta chain(s) has allowed the genetic mapping of the corresponding beta gene(s) to the HLA-DR region. cDNA clones for the HLA-DR alpha chain have been used to map the non-polymorphic DR alpha-chain gene to chromosome 6 using mouse-human somatic cell hybrids. Similarly, the DR alpha-chain gene has been mapped to the short arm of chromosome 6 centromeric to the HLA-A, -B and -C loci by in situ hybridization experiments. We isolated a cDNA clone that is related to the DR alpha chain and encodes the class II antigen DC alpha chain. We describe here how this DC alpha clone was used to find two or three additional alpha-chain genes by cross-hybridization and how HLA-antigen loss mutants of a human lymphoblastoid cell line (LCL) were used to ascertain that these additional class II antigen alpha-chain genes are also located in the HLA region.     

Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen

Effector T cells are restricted to recognizing antigens associated with major histocompatibility complex (MHC) molecules. Specific recognition is mediated by the alpha beta heterodimer of the T-cell receptor (TCR)/CD3 complex, although other membrane components are involved in T-cell antigen recognition and functions. There has been much controversy in this regard over the part played by the CD4 glycoprotein. It is known that expression of CD4 correlates closely with the cell's ability to recognize antigens bound to class II MHC molecules and that CD4 can bind to class II molecules. Also monoclonal antibodies to CD4 can modify signals generated through the TCR/CD3 complex. It has therefore been proposed that CD4 binds to class II molecules, coaggregates with the TCR-CD3 complex and aids the activation of T cells. But given that TCR can itself impart restriction on the cell, it remains unclear whether the contribution of CD4-derived signals to those generated through the TCR alpha beta-CD3 complex is central to this activation. Here we report that when preceded by ligation of CD4, signalling through TCR alpha beta results in T cell unresponsiveness due to the induction of activation dependent cell death by apoptosis. These results imply that CD4 is critically involved in determining the outcome of signals generated through TCR, and could explain why the induction of effector T cells needs to be MHC-restricted.     

A complex between the MHC class I homologue encoded by human cytomegalovirus and beta 2 microglobulin

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that persists in the host and can cause severe disease in the immunocompromised individual or in the fetus. Analysis of the nucleotide sequence of the virus genome has revealed the presence of an open reading frame whose predicted translation product has homology with the heavy chain of the major histocompatibility complex (MHC) class I molecule of higher eukaryotes, and the observed sequence homology was given additional significance by the independent observation that HCMV virions can bind beta 2 microglobulin (beta 2m), the light chain of the MHC class I molecule. We expressed both the HCMV class I homologue and the human beta 2m gene in recombinant vaccinia viruses. We show that the coexpressed gene products associate, that the transport of beta 2m to the cell surface is dependent on coexpression of the class I homologue and that the viral gene product is therefore functionally related to its cellular counterpart. We observe also that, in HCMV-infected cells, no synthesis of mature cellular class I molecules occurs, while messenger RNA levels remain unaltered, and we speculate that one function of the viral homologue may be to sequester beta 2m, thus preventing the maturation of cellular class I molecules and rendering the infected cell unrecognizable by cytotoxic T cells.     

Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells

Mice homozygous for a beta 2-microglobulin gene disruption do not express any detectable beta 2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of gamma delta, CD4+8+ and CD4+8- T cells, but have no mature CD4-8+ T cells and are defective in CD4-8+ T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor alpha beta+ CD4-8+ T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.