Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells

The first step in the induction of immune responses, whether humoral or cell mediated, requires the interaction between antigen-presenting cells and T lymphocytes restricted at the major histocompatibility complex (MHC). These cells invariably express MHC class II molecules (HLA-D region in man and Ia in mouse) which are recognized by T cells of the helper/inducer subset in association with antigen fragments. Interestingly, in certain pathological conditions, for example in autoimmune diseases such as thyroiditis and diabetic insulitis, class II molecules may be expressed on epithelial cells that normally do not express them. We speculated that these cells may be able to present their surface autoantigens to T cells, and that this process may be crucial to the induction and maintenance of autoimmunity. A critical test of this hypothesis would be to determine whether epithelial cells bearing MHC class II molecules (class II+ cells) can present antigen to T cells. We report here that class II+ thyroid follicular epithelial cells (thyrocytes) can indeed present viral peptide antigens to cloned human T cells.     

SB-restricted presentation of influenza and herpes simplex virus antigens to human T-lymphocyte clones

The HLA-D region of the human major histocompatibility complex (MHC) has been shown to be homologous to the murine I region in terms of both structure and function. Both regions encode class II MHC molecules which restrict T-lymphocyte interactions with antigen-presenting cells. We have recently described the MHC restriction and antigen specificities of human T-lymphocyte clones directed at strain A influenza virus. The majority of T-lymphocyte clones recognized antigen in the context of cell surface interaction products encoded by HLA-D/DR genes. However, a few clones recognized antigen presented by cells histoincompatible for D/DR antigens. We report here that some of these clones recognized viral antigens in association with antigens encoded by genes identical with or closely linked to the recently described secondary B-cell (SB) locus of the MHC. This is the first report that SB-restricted antigen recognition may form an integral part of normal, human immune responses.     

Processing of a minimal antigenic peptide alters its interaction with MHC molecules

Before their recognition by T lymphocytes, protein antigens generally require processing by antigen-presenting cells. In a poorly understood series of events, the protein antigen is internalized, transformed and re-expressed on the surface of the antigen-presenting cell in association with gene products of the major histocompatibility complex (MHC). Small peptides derived from the native protein can be recognized in the absence of antigen processing, suggesting that processing involves proteolytic degradation. These peptides are thought to mimic the naturally produced peptide fragment. We describe here a synthetic peptide antigen of this type which does not require processing but which is nevertheless further processed by splenic antigen-presenting cells. Interestingly, this processing event specifically alters the interaction of the peptide with the class II MHC (Ia) molecule, markedly affecting both its potency as an antigen in vitro and its immunogenicity in vivo (IR gene control).     

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.     

In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine

Cytotoxic T lymphocytes (CTL) constitute an essential part of the immune response against viral infections. Such CTL recognize peptides derived from viral proteins together with major histocompatibility complex (MHC) class I molecules on the surface of infected cells, and usually require in vivo priming with infectious virus. Here we report that synthetic viral peptides covalently linked to tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can efficiently prime influenza-virus-specific CTL in vivo. These lipopeptides are able to induce the same high-affinity CTL as does the infectious virus. Our data are not only relevant to vaccine development, but also have a bearing on basic immune processes leading to the transition of virgin T cells to activated effector cells in vivo, and to antigen presentation by MHC class I molecules.     

Interaction of peptide antigens and class II major histocompatibility complex antigens

T lymphocytes require a foreign antigen to be presented on a cell surface in association with a self-transplantation antigen before they can recognize it effectively. This phenomenon is known as major histocompatibility complex (MHC) restriction. It is not clear how an incalculably large number of foreign proteins form unique complexes with a very limited number of MHC molecules. We studied the recognition properties of T cells specific for a peptide derived from bacteriophage lambda cI protein. Analogues of this peptide, as well as peptides derived from other unrelated antigens which can be presented in the context of the same MHC molecule, can competitively inhibit activation of these T cells by the cI peptide. Furthermore, these unrelated antigens can stimulate cI-specific T cells if certain specific amino-acid residues are replaced. Here we suggest a model in which all antigens give rise to peptides that can bind to the same site on the MHC molecule. T-cell recognition of this site (which is presumed to be polymorphic) with or without antigen bound can explain self-selection in the thymus and MHC restriction.     

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.     

Identification of classical minor histocompatibility antigen as cell-derived peptide.

Histocompatibility antigens expressed on tissue grafted between individuals are recognized by host T cells, which reject the graft. The major histocompatibility complex (MHC) antigens have been identified on the molecular level, whereas the molecules representing the remaining ones, the minor histocompatibility antigens, are unknown, apart from some exceptions. The cytotoxic T lymphocyte (CTL) response against minor histocompatibility antigens shares many aspects with that against virus-infected cells. Virus-specific CTL recognize peptides derived from viral proteins produced in the infected cell. These peptides are presented by MHC class I molecules, as indicated by functional and crystallographic data. By analogy, minor histocompatibility antigens have been postulated to be peptides derived from normal cellular proteins presented by MHC class I molecules. Here we report that peptides derived from normal cellular proteins can indeed be recognized by CTL raised in the classical minor histoincompatible mouse strain combination, C57BL/6 against BALB.B. Thus, we have proven the above postulate, and isolated one of the minor histocompatibility molecules elusive for several decades.     

Responses to the H-2Kba mutant involve recognition of syngeneic Ia molecules

Conventional antigens appear to be recognized by T lymphocytes only when associated with major histocompatibility complex (MHC) antigens. Using antigen-specific proliferation as a model for helper T lymphocytes, it has been demonstrated that Ly1+T cells recognize antigen presented in association with syngeneic Ia molecules. In contrast to responses to conventional antigens, however, a large number of studies have suggested that the stimulation of alloreactive Ly1+T cells, and helper T cells specific for allogeneic cytotoxic T lymphocyte (CTL) responses, involve the direct recognition of Ia alloantigens. For the generation of optimal allogeneic CTL activity it has been proposed that Ly1+T cells recognize allo-Ia antigens directly and provide help to pre-CTLs that respond to allo-H-2K and/or D determinants. Thus, the B6.C.H-2bm1 mutant (bm1, formerly referred to as Hz1), which is believed to consist of a substitution of two amino acids in the H-2Kb antigen, has presented a paradox, for it can stimulate strong mixed lymphocyte culture (MLC), graft versus host and CTL responses by T cells of H-2b haplotype mice in the apparent absence of any alloantigenic differences in the I region. We now present evidence that the stimulation of proliferative and helper T cells by the mutant B6.C.H-2bm1 results from the H-2Kba antigen being recognized in the context of syngeneic Ia determinants. Thus responses to both conventional antigens and allogeneic MHC gene products may proceed via the recognition of antigen in the context of self Ia molecules.     

Essential requirement for major histocompatibility complex recognition in T-cell tolerance induction

The induction of T-cell responses involves the recognition of extrinsic antigen in association with antigens of the major histocompatibility complex (MHC), in mice and man, with different T cells recognizing antigen in association with either class I (H-2K/D, HLA-A, B, C) or class II (Ia, HLA-D/DR) MHC antigens. However, the requirement of MHC recognition in the induction of immunological tolerance remains ill defined. With human T helper clones recognizing synthetic peptides of influenza haemagglutinin (HA-1), we have investigated the nature of antigen-induced stimulation, and antigen-induced antigen-specific unresponsiveness, immunological tolerance. Tolerance is not due to cell death, as the cells remain responsive to interleukin-2 and is associated with the loss of T3 antigen from the cell surface. Using monoclonal antibodies to the non-polymorphic regions of human class II antigens to inhibit the induction of T-cell tolerance we report here that induction of tolerance requires the recognition of MHC antigens.     

Truncation variants of peptides isolated from MHC class II molecules suggest sequence motifs.

T cells recognize foreign protein antigens in the form of peptide fragments bound tightly to the outer aspect of molecules encoded by the major histocompatibility complex (MHC). Most of the amino-acid differences that distinguish MHC allelic variants line the peptide-binding cleft, and different allelic forms of MHC molecules bind distinct peptides. It has been demonstrated that peptide-binding to MHC class I involves anchor residues in certain positions and that antigenic peptides associated with MHC class I exhibit allele-specific structural motifs. We have previously reported an analysis of MHC class II-associated peptide sequences. Here we extend this analysis and show that certain amino-acid residues occur at particular positions in the sequence of peptides binding to a given MHC class II molecule. These sequence motifs require the amino terminus to be shifted one or two positions to obtain alignment; such shifts occur naturally for a single peptide sequence without qualitatively altering CD4 T-cell recognition.     

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.     

T-cell-mediated association of peptide antigen and major histocompatibility complex protein detected by energy transfer in an evanescent wave-field

Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.     

Antigenic peptides recognized by T lymphocytes from AIDS viral envelope-immune humans

T-lymphocyte immunity is likely to be an important component of the immune defence against the AIDS virus, because helper T cells are necessary for the antibody response as well as the cytotoxic response. We have previously predicted two antigenic sites of the viral envelope protein gp120 likely to be recognized by T lymphocytes, based on their ability to fold as amphipathic helices, and have demonstrated that these are recognized by T cells of mice immunized with gp120 (ref. 1). A peptide corresponding to one of these sites can also be induce immunity in mice to the whole gp120 protein. Because many clinically healthy seropositive blood donors have already lost their T-cell proliferative response to specific antigen, we tested the response to these synthetic peptides of lymphocytes from 14 healthy human volunteers who had been immunized with a recombinant vaccinia virus containing the AIDS viral envelope gene and boosted with a recombinant fragment. Eight of the 14 responded to one peptide, and four to the other peptide, not included in the boost. These antigenic sites recognized by human T cells may be useful components of a vaccine against AIDS. We also found a correlation between boosting with antigen-antibody complexes (compared to free antigen) and higher stimulation indices, suggesting a more effective method of immunization.     

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.     

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.     

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.     

Sequence similarity analysis of non-self CTL epitopes and mouse proteins using sequence alignment

This research analyzed amino acid sequence similarity between non-self T cell epitopes recognized by mouse antibodies and mouse proteins. Using sequence alignment,we found that only 8 of 1 108 epitopes are highly similar to mouse protein sequences. The result shows that non-self T cell epitopes are not similar or have little similarity to mouse protein sequences. Furthermore,reviewing the related literature,we also found that the eight epitopes would trigger immune responses in some particular environment,which are ignored by T cells in normal condition. The result suggests that no or low-similarity peptide vaccines can reduce the chance of collateral cross-reactions and enhance the antigen-specific immune response to vaccine.     

Striking similarities between antigen receptor J pieces and sequence in the second chain of the murine CD8 antigen

The CD8 antigen is a marker for T-lymphocyte subsets that is absent from helper T cells but expressed on cytotoxic T cells which recognize foreign determinants in association with class I major histocompatibility complex (MHC) antigens. It has been suggested that CD8 plays some part in recognition by CD8+ cytotoxic T cells since anti-CD8 antibodies can block their functions and the human CD8 antigen contains a domain with clear similarities to immunoglobulin and T-cell receptor (TCR) variable-region (V) domains. Human CD8 antigen is thought to be a homodimer but in the mouse and rat the equivalent antigens (alternatively called Lyt2,3 and OX8) are heterodimeric. Rat CD8 contains two chains of relative molecular mass 32,000 (32K) and 37K: the 32K chain is the rat homologue of human CD8 and mouse Lyt2. We describe here the molecular cloning of the rat 37K chain using an oligonucleotide probe predicted from peptide sequence. The full protein sequence is derived from the complementary DNA and matches limited peptide sequence for mouse Lyt3. The new sequence is more like immunoglobulin and T-cell receptor V domains than other T-cell antigens and includes a patch that is almost identical to some joining (J) piece sequences. This suggests that the CD8 and receptor heterodimers may have evolved directly from a common ancestor.