A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes

Peptides that are antigenic for T lymphocytes are ligands for two receptors, the class I or II glycoproteins that are encoded by genes in the major histocompatibility complex, and the idiotypic alpha/beta chain T-cell antigen receptor. That a peptide must bind to an MHC molecule to interact with a T-cell antigen receptor is the molecular basis of the MHC restriction of antigen-recognition by T lymphocytes. In such a trimolecular interaction the amino-acid sequence of the peptide must specify the contact with both receptors: agretope residues bind to the MHC receptor and epitope residues bind to the T-cell antigen receptor. From a compilation of known antigenic peptides, two algorithms have been proposed to predict antigenic sites in proteins. One algorithm uses linear motifs in the sequence, whereas the other considers peptide conformation and predicts antigenicity for amphipathic alpha-helices. We report here that a systematic delimitation of an antigenic site precisely identifies a predicted pentapeptide motif as the minimal antigenic determinant presented by a class I MHC molecule and recognized by a cytolytic T lymphocyte clone.     

Rejection of transplantable AKR leukaemia cells following MHC DNA-mediated cell transformation

Major histocompatibility complex (MHC) class I molecules can function as specific target antigens in T-cell-mediated cytotoxity. In addition, T cells can kill target cells through non-MHC antigens, for example, virally infected cells, if the target and effector cells express the same MHC class I antigens. Consequently, quantitative and/or qualitative variations in the expression of the H-2/HLA antigens on the target cells could interfere with MHC-restricted immune reactions. We have reported that the AKR leukaemia cell line K36.16, a subline of K36 (ref. 3), on which the H-2Kk antigen cannot be detected, is resistant to T-cell lysis and grows very easily in AKR mice. Other AKR tumour cell lines, like 369, which have a relatively large amount of H-2Kk on their surface, are easily killed by T cells in vitro and require a much larger inoculum to grow in vivo. Monoclonal antibodies against H-2Kk, but not against H-2Dk, prevented the killing by T cells. This suggests that some tumour cells grow in vivo because tumour-associated antigen(s) cannot be recognized efficiently by the host's immune system, due to the absence of MHC molecules which would function as restriction elements for T-cell cytotoxicity. We have tested this hypothesis by introducing the H-2Kk gene into the H-2Kk-deficient AKR tumour cell line K36.16 and have now demonstrated directly the biological relevance of H-2Kk antigen expression in the regulation of the in vivo growth of this tumour cell line.     

Positive and negative selection of an antigen receptor on T cells in transgenic mice

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.     

Progression of autoimmune diabetes driven by avidity maturation of a T-cell population

For unknown reasons, autoimmune diseases such as type 1 diabetes develop after prolonged periods of inflammation of mononuclear cells in target tissues. Here we show that progression of pancreatic islet inflammation to overt diabetes in nonobese diabetic (NOD) mice is driven by the 'avidity maturation' of a prevailing, pancreatic beta-cell-specific T-lymphocyte population carrying the CD8 antigen. This T-lymphocyte population recognizes two related peptides (NRP and NRP-A7) in the context of H-2Kd class I molecules of the major histocompatibility complex (MHC). As pre-diabetic NOD mice age, their islet-associated CD8+ T lymphocytes contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/H-2Kd tetramers with increased specificity, increased avidity and longer half-lives. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunts the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by selectively deleting those clonotypes expressing T-cell receptors with the highest affinity and lowest dissociation rates for peptide-MHC binding. This inhibits the local production of T cells that are cytotoxic to beta cells, and halts the progression from severe insulitis to diabetes. We conclude that avidity maturation of pathogenic T-cell populations may be the key event in the progression of benign inflammation to overt disease in autoimmunity.     

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.     

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.     

Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis

Antigenic peptides are presented to CD8+T lymphocytes by class I major histocompatibility complex (MHC) molecules. Peptides specifically bind to purified class I molecules in vitro, and to class I molecules on cells at nonphysiological temperatures. We report here the kinetic and equilibrium parameters for the binding of radiolabelled influenza nucleoprotein peptides (NP-Y365-380 and shorter homologues) to the murine H-2Db molecule on intact, viable cells at 37 degrees C. In contrast to earlier reports, we show that peptide binding is rapid and reversible, with dissociation constants ranging from nanomolar to micromolar, suggestive of typical ligand-receptor interactions. Only 10% of cell-surface Db molecules can bind these peptides. To address the relationship between peptide binding and T-cell recognition of the antigen-MHC complex, we determined the minimum number of complexes required to sensitize a target cell for lysis by class I-restricted cytotoxic T-lymphocytes. Our data indicate that EL4 thymoma cells (H-2b) can be sensitized for lysis by cytotoxic T-lymphocytes when as few as 200 class I-peptide complexes (less than 0.08% of surface Db molecules) are present per cell.     

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.     

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.     

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.     

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.     

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.     

Transfection of the CD8 gene enhances T-cell recognition

Antibodies against CD8 or CD4 antigens can prevent T-cell functions induced by T-cell targets. As CD8 or CD4 antibodies can also initiate negative signals in T cells in the absence of appropriate targets it is not clear whether CD8 and CD4 molecules are directly involved in the interaction of T cells with their targets. In previous experiments we have introduced the T-cell receptor alpha- and beta-chain genes from a CD8-positive cytolytic T cell specific for the antigen fluorescein (FL) and the H-2D molecule of the major histocompatibility complex (MHC) into a CD8-negative recipient cell. The CD8-positive donor cell lysed both FL-conjugated fibroblasts and lymphoblasts, which express relatively high and low amounts of H-2D molecules, respectively. In contrast the CD8-negative transfectant lysed FL-conjugated fibroblasts only. Here we show that recognition of FL-conjugated lymphoblasts by the transfectant is enhanced by supertransfecting it with the CD8 gene.     

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.     

Binding of immunogenic peptides to Ia histocompatibility molecules

Most cellular interactions essential for the development of an immune response involve the membrane glycoproteins encoded in the major histocompatibility gene complex. The products of the I region, the class II histocompatibility molecules (Ia molecules), are essential for accessory cells such as macrophages to present polypeptide antigens to helper T cells. This interaction, antigen presentation, is needed for T-cell recognition of the antigen and its consequent activation. How the Ia molecules regulate the immune response during antigen presentation is not known, although it is commonly thought to result from their association with the presented antigen. Recent studies, including the elucidation of the structure of the T-cell receptor, favour recognition of a single structure, an antigen-Ia complex. Here we report attempts to determine whether purified Ia glycoproteins have an affinity for polypeptide antigens presented by intact cells in an Ia-restricted manner. We first identified the epitope of a peptide antigen involved in presentation. Several laboratories have shown that globular proteins are altered (processed) in intracellular vesicles of the antigen-presenting cell before antigen presentation. A major component of the T-cell response is directed toward determinants found in the unfolded or denatured molecule, and our laboratory has shown that the determinant of the hen-egg lysozyme protein (HEL), presented in H-2k mice to T cells, is a sequence of only 10 amino acids. This portion resides in an area of the native molecule partially buried inside the molecule, in a beta-sheet conformation. To be presented, intact or native HEL must first be processed in acidic intracellular vesicles. Having isolated the peptide responsible for T-cell recognition of HEL, we sought a physical association of this peptide with purified, detergent-solubilized I-Ak molecules from B-hybridoma cells. We have found such an association, which may explain the role of the Ia glycoproteins in cellular interactions.     

Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.     

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.     

Functionally distinct subsites on a class II major histocompatibility complex molecule

Mature T lymphocytes are activated by recognition of the combination of foreign protein antigen and membrane products of the major histocompatibility complex (MHC). Studies of peptide antigen binding to detergent-solubilized class II MHC molecules (Ia) have established that peptide-Ia interaction occurs in the absence of the T-cell receptor and varies according to allele-specific features of Ia molecules. The residues of immunogenic peptides thus contribute to two largely independent functions--the control of association with Ia molecules and the determination of the specificity of T-cell receptor binding. Two analogous and potentially independent functional sites have been postulated for Ia molecules--a region that controls binding to peptides and a region that interacts with T-cell receptors. Here we present evidence from functional analysis of recombinant class II molecules that these two postulated functional regions of Ia molecules do exist and can be independently manipulated, consistent with our recent demonstration of the segmental nature of Ia molecule structure-function relationships.     

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.     

HLA-restricted recognition of viral antigens in HLA transgenic mice

Cytotoxic T lymphocytes (CTL) recognize antigen in the context of the class-I products of the major histocompatibility complex (MHC). The extensive polymorphism of class-I molecules is thought to be linked to their capacity to present a large variety of foreign antigens. Whether a single T-cell receptor (TCR) recognizes two separate epitopes (the foreign antigen and an epitope on MHC molecules), or a single epitope resulting from the combination of a foreign antigen and an MHC molecule, has not yet been resolved. In view of the differences between species in primary structure of histocompatibility antigens, it might be predicted that the TCR repertoire would evolve in concert with the diversity of MHC antigens. The mouse and human TCR repertoire would be optimally adapted to engage in productive interactions only with mouse (H-2) and human (HLA) MHC antigens respectively, especially if the more conserved features of histocompatibility antigens, in addition to foreign antigen, were seen by the TCR. Alternatively, only the most variable segments of MHC antigens might be engaged in antigen presentation and thus in interaction with the TCR. In that case, interaction between MHC plus antigen and the TCR might not necessarily be limited by species-specific features. By analysis of the T-cell response against virus-infected cells in HLA-B27/human beta 2-microglobulin double transgenic mice, we report here that the mouse T-cell repertoire is perfectly capable of using the human HLA-B27 antigen as a restriction element.