Empty MHC class I molecules come out in the cold

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.     

Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.     

Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.

Virus-infected cells can be eliminated by cytotoxic T lymphocytes (CTL), which recognize virus-derived peptides bound to major histocompatibility complex (MHC) class I molecules on the cell surface. Until now, this notion has relied on overwhelming but indirect evidence, as the existence of naturally processed viral peptides has not been previously reported. Here we show that such peptides can be extracted from virus-infected cells by acid elution. Both the naturally processed H-2-Db-restricted and H-2-Kd-restricted peptides from influenza nucleoprotein are smaller than the corresponding synthetic peptides, which have first been used to determine the respective CTL epitopes. As with minor histocompatibility antigens, occurrence of viral peptides seems to be heavily dependent on MHC class I molecules, because infected H-2d cells do not contain the H-2-Db-restricted peptide, and infected H-2b cells do not contain the H-2-Kd-restricted peptide. Our data provide direct experimental proof for the above notion on MHC-associated viral peptides on virus-infected cells.     

HLA-A2 peptides can regulate cytolysis by human allogeneic T lymphocytes

The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.     

Physical association between MHC class I molecules and immunogenic peptides

Antigenic peptides are presented to T lymphocytes by major histocompatibility complex (MHC) molecules. The binding of peptides to MHC class II molecules has been demonstrated directly, and is found to correlate with the ability of specific class II alleles to restrict the T-cell response to specific peptides. By comparison, a direct demonstration of a physical association between antigenic peptides and MHC class I molecules has proved difficult. A recent report shows that it is possible, however, and the three-dimensional structure of a class I MHC molecule illustrates the site where such binding must occur. Here we describe a simple assay which measures the binding of radiolabelled MHC class I molecules to peptides bound to a solid phase support. We find that class I molecules bind specifically to peptides known to be antigenic for class I-restricted cytotoxic T lymphocytes. Peptides which are recognized by cytotoxic T lymphocytes bind not only to the restricting MHC class I molecule but also to other class I molecules. Our results suggest that quantitative differences in the peptide/MHC class I interaction may influence the-pattern of MHC restriction observed in vivo.     

HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides.

The mutant human cell line T2 is defective in antigen presentation in the context of class I major histocompatibility complex (MHC) molecules, and also in that transfected T2 cells show poor surface expression of exogenous human class I (HLA) alleles. Both defects are thought to lie in the transport of antigenic peptides derived from cytosolic proteins into the endoplasmic reticulum (ER), as peptide-deficient class I molecules might be expected to be either unstable or retained in the ER. The products of several mouse class I (H-2) genes, and the endogenous gene HLA-A2 do, however, reach the surface of T2 cells at reasonable levels although they are non-functional. We report here that, as expected, poorly surface-expressed HLA molecules do not significantly bind endogenous peptides. Surprisingly, H-2 molecules expressed in T2 also lack associated peptides, arguing that 'empty' complexes of mouse class I glycoproteins with human beta 2-microglobulin are neither retained in the ER nor unstable. HLA-A2 molecules, however, do bind high levels of a limited set of endogenous peptides. We have sequenced three of these peptides and find that two, a 9-mer and an 11-mer, are derived from a putative signal sequence (of IP-30, an interferon-gamma-inducible protein), whereas a third, a 13-mer, is of unknown origin. The unusual length of two of the peptides argues that the 9-mers normally associated with HLA-A2 molecules may be generated before their transport from the cytosol rather than in a pre-Golgi compartment. To our knowledge, this is the first report of the isolation of a fragment of a eukaryotic signal peptide generated in vivo.     

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.     

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.     

Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2

Both human and murine cytotoxic T cells (CTL) elicited in response to infection with influenza A viruses have been shown to be specific for internal viral proteins, such as the matrix and nucleoprotein. Individual CTL epitopes have been identified in the nucleoprotein by successfully substituting short synthetic peptides for the intact virus in the preparation of target cells in cytotoxicity assays. The defined peptide epitopes have each been recognized by CTL in association with individual class I major histocompatibility complex (MHC) proteins, H-2Db, H-2Kk, H-2Kd (Taylor, P. et al., unpublished data) and HLA-B37. A logical strategy to investigate the molecular details of the interaction between antigen and MHC class I proteins would be to define an epitope recognized by the MHC class I molecule HLA-A2. This is because the amino-acid sequence is known, several variants of A2 have been characterized and the protein has been purified and crystallized. Here we describe a peptide derived from the influenza matrix protein that is recognized by human CTL in association with the HLA-A2 molecule.     

Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line.

T lymphocytes recognize antigens as peptide fragments associated with molecules encoded by the major histocompatibility complex (MHC) and expressed on the surface of antigen-presenting cells. In the thymus, T cells bearing alpha beta receptors that react with the MHC molecules expressed by radioresistant stromal elements are positively selected for maturation. In (A x B-->A) bone marrow chimaeras, T cells restricted to the MHC-A haplotype are positively selected, whereas MHC-B-reactive thymocytes are not. We investigated whether the introduction of particular thymic stromal elements bearing MHC-B molecules could alter the fate of B-reactive T cells in these (A x B-->A) chimaeras. Thymic epithelial cell (TEC) lines expressing H-2b were introduced by intrathymic injection into (H-2b/s-->H2s) bone marrow chimaeras and we measured their ability to generate H-2b-restricted cytotoxic T-lymphocytes (CTLs). We report here that one TEC line, 427.1, was able positively to select CTLs specific for influenza and vesicular stomatitis virus antigens in association with class I H-2b molecules. In addition, line 427.1 can process cytoplasmic proteins for presentation to H-2Kb- and H-2Db-restricted CTLs. Thus, a TEC line capable of normal class I MHC antigen processing and presentation in vitro can induce positive selection after intrathymic injection.     

Peptide binding to empty HLA-B27 molecules of viable human cells

Intracellular binding of antigenic peptides by polymorphic class I major histocompatibility complex molecules creates the ligands recognized by receptors of CD8+ T cells. Previously described in vitro assays of peptide binding to class I molecules have been limited by either the low proportion of accessible binding sites or the lack of allelic specificity. Here we describe a system in which the human class I molecule HLA-B27 binds considerable amounts of an influenza peptide with precise allelic discrimination. Binding requires viable cells, is stimulated by gamma-interferon and is inhibited by brefeldin A. Our results are consistent with the presence of fairly stable 'empty' HLA-B27 molecules at the cell surface. By contrast, analysis of the binding of a second influenza peptide indicates that empty HLA-Aw68 molecules are relatively short-lived. We speculate that HLA-B27 might bind extracellular peptides in vivo and that this property could underlie its association with autoimmune disease.     

Specificity of peptide presentation by a set of hybrid mouse class I MHC molecules

The class I molecules of the major histocompatibility complex (H-2 in mouse, HLA in man) are membrane proteins composed of a polymorphic heavy chain associated with beta-2-microglobulin. Recent studies suggest that class I molecules present peptides derived from processed antigens to the receptor of cytolytic T cells. In particular, in the H-2d haplotype, synthetic HLA peptides can be recognized on Kd-bearing target cells by Kd-restricted cytolytic T cells specific for HLA. Here we analyse the specificity of presentation of two HLA peptides by a set of chimaeric Kd/Dd molecules to four different cytolytic T-cell clones. We identify two distinct regions within the second external (alpha 2) domain of Kd that contribute to its specificity as a restriction element. Our results indicate that the binding of an immunogenic peptide by a class I molecule is not always sufficient for its recognition by the T-cell antigen receptor. This suggests that the major histocompatibility complex restriction element either interacts with the T-cell antigen receptor or induces the recognized conformation of the peptide.     

A gene in the human major histocompatibility complex class II region controlling the class I antigen presentation pathway

Major histocompatibility complex (MHC) class I molecules export peptides to the cell surface for surveillance by cytotoxic T lymphocytes. Intracellular peptide binding is critical for the proper assembly and transport of class I molecules. This mechanism is impaired as a result of a non-functional peptide supply factor gene (PSF) in several human mutant cell lines with genomic lesions in the MHC. We have now identified PSF in the MHC class II region by deletion mapping in mutants and chromosome-walking. PSF is homologous to mammalian and bacterial ATP-dependent transport proteins, suggesting that it operates in the intracellular transport of peptides.     

A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.     

Direct binding of influenza peptides to class I HLA molecules

Activation of T lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class I or class II major histocompatibility complex (MHC) glycoproteins. The direct binding of peptides to class II molecules has been demonstrated using equilibrium dialysis, gel filtration and fluorescence energy transfer at planar membranes, and its specificity compared to that of T-cell activation. In contrast, direct binding of peptides to class I molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of HLA-A2 (ref. 9) persuasively argue for its occurrence and importance. Here we describe a gel filtration assay from which we derive direct evidence for selective binding of an influenza matrix peptide to HLA-A2 and for binding of an influenza nucleoprotein peptide to HLA-B37. These two peptides have previously been shown to act respectively as targets for certain HLA-A2 or HLA-B37 restricted influenza-specific cytotoxic T lymphocytes (CTL). In addition we demonstrate binding to some, but not all, HLA allospecificities that cannot present these peptides to CTL. We estimate that less than 0.3% of the HLA molecules present in any given purified preparation were able to bind the added peptides.     

Cytolytic T-lymphocyte response to isolated class I H-2 proteins and influenza peptides

T cells recognize antigenic peptides in the context of major histocompatibility complex (MHC) proteins. Peptide binding to class II MHC proteins, and T-cell recognition of these complexes at the functional level has been demonstrated. Although considerable evidence suggests that class I-restricted cytotoxic T lymphocytes (CTL) recognize class I-peptide complexes, this has not yet been directly demonstrated. Chen and Parham have recently detected a low level of direct binding of radiolabelled influenza peptides to class I HLA proteins, but the relevance of this binding to T-cell recognition remains uncertain. We report here that purified class I proteins pulsed with influenza peptides can trigger antigen-specific, TCR-mediated degranulation by CTL. Effective pulsing depends on both peptide concentration and time, and can occur within 60 minutes. These results provide strong support for the formation of an antigenic complex that is recognized by CTL in which peptide antigens are bound to isolated class I proteins.     

Peptide selection by MHC class I molecules.

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.     

Cellular peptide composition governed by major histocompatibility complex class I molecules

Major histocompatibility complex (MHC) class I molecules present peptides derived from cellular proteins to cytotoxic T lymphocytes (CTLs), which check these peptides for abnormal features. How such peptides arise in the cell is not known. Here we show that the MHC molecules themselves are substantially involved in determining which peptides occur intracellularly: normal mouse spleen cells identical at all genes but MHC class I express different patterns of peptides derived from cellular non-MHC proteins. We suggest several models to explain this influence of MHC class I molecules on cellular peptide composition.     

Irreversible association of peptides with class II MHC molecules in living cells.

Functional, morphological and biochemical evidence indicates that class II major histocompatibility complex (MHC) molecules associate with processed peptides during biosynthesis. Peptide/MHC complexes in living cells have been reported to be less stable than similar complexes generated in vitro, which has led to the suggestion that there may be a peptide exchange mechanism operating in vivo. Although this could increase the capacity for binding incoming antigens, it would reduce the efficacy of processed antigenic peptides by exchanging these for self peptides. Here we measure the half-life of peptide/class II complexes in human antigen-presenting cells and find that it is very similar to the half-life of class II molecules themselves, indicating that peptides are bound irreversibly under physiological conditions. Thus class II MHC retains long-term 'memory' of past encounters with antigen to maximize the opportunity for T cell/antigen-presenting cell interaction.     

A kinetic intermediate in the reaction of an antigenic peptide and I-Ek

Helper T cells are triggered by molecular complexes of antigenic peptides and cell surface glycoproteins of the MHC (gene products of the major histocompatibility complex) on antigen-presenting cells. There is now a lot of evidence that the complexes between isolated class II MHC molecules and selected peptides have long half-lives of approximately one day. The reported equilibrium binding constants between antigenic peptides and class II MHC molecules however, are only micromolar, suggesting that the association rate constants are very low. The only reported association rate constant is for a chicken ovalbumin peptide (OVA323-339) binding to I-Ad, and is indeed remarkably low, about 1 litre per mole per second. Prompted by these unusual data, we have used the pigeon cytochrome-c peptide pCytc(88-104) and I-E reconstituted in planar lipid bilayers on glass slides to investigate further the kinetics of peptide-MHC reactions. We report the formation of two IEk-pCytc peptide complexes. One complex has slow apparent association and dissociation kinetics, very similar to those reported previously for the chicken ovalbumin peptide and I-Ad. The second complex forms and dissociates about a hundred times more rapidly. The short-lived complex shows peptide-MHC specificity and is a kinetic intermediate in the formation of the long-lived complex; the long-lived complex is recognized by specific T-helper cells.