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.     

Cytotoxic T lymphocytes against a soluble protein

Thymus-derived (T) lymphocytes recognize antigen in conjunction with surface glycoproteins encoded by major histocompatibility complex (MHC) genes. Whereas fragments of soluble antigens are presented to T helper lymphocytes (TH), which carry the CD4 antigen, in association with class II MHC molecules, CD8-bearing cytotoxic T lymphocytes (CTL) usually see cellular antigens (for instance virally-encoded proteins) in conjunction with MHC class I molecules. The different modes of antigen presentation may result from separate intracellular transport: vesicles containing class II molecules are thought to fuse with those carrying endocytosed soluble proteins. Class I molecules, in contrast, can only pick up degradation products of intracellular proteins (see refs 7 and 8). This makes biological sense; during an attack of a virus, class I-restricted CTL destroy infected cells and class II-restricted TH guide the humoural response to neutralize virus particles and toxins. But here we provide evidence that CTL specific for ovalbumin fragments can be induced with soluble protein, and that intracellular protein degradation provides epitopes recognized by these CTL. These findings suggest the existence of an antigen presenting cell that takes up soluble material and induces CTL.     

Evidence from in vitro studies that tolerance to self antigens is MHC-restricted

Mature T cells respond to foreign antigens in the context of self major histocompatibility complex (MHC)-encoded products: T helper cells recognize antigen in the context of class II molecules, while cytotoxic T cells (CTL) recognize antigen plus class I molecules. Recent evidence suggests that the MHC-restricted T cell is unable to recognize either the foreign antigen or the self-MHC product alone, but only a complex of the two. Unresponsiveness to self antigens--self tolerance--implies the deletion or suppression of clones of T cells having reactivity to self antigens. Here we demonstrate the presence in normal mice of T cells which recognize self antigens together with allogeneic MHC products. This finding suggests the MHC restriction of T-cell recognition during the entire process of T-cell ontogeny, that is, MHC restriction of self tolerance.     

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.     

Strong xenogeneic HLA response in transgenic mice after introducing an alpha 3 domain into HLA B27.

The pronounced response by mouse T cells to the major histocompatibility complex (MHC) class I antigens of the same species is characterized by a relatively large fraction of responding cells. Responses to MHC class I allelles of other species are, however, generally much weaker. T lymphocytes are positively selected on thymic MHC antigens, resulting in a T-cell repertoire with strong alloreactivity. This has been explained in terms of a mouse T-cell repertoire that is not efficiently selected for recognition of HLA molecules owing to the absence of HLA in mice. Here we show that mice transgenic for HLA mount a T-cell response against allogeneic HLA that is no better than in normal mice. We decided instead to test whether the mouse accessory molecule Lyt-2 on cytotoxic T lymphocytes could interact efficiently with the alpha 3 domain of HLA. To do this, we replaced the alpha 3 domain of HLA-B27 by a murine alpha 3 domain in a gene construct used to produce transgenic mice, and then used the spleen cells from these mice to stimulate normal mouse T cells. Under these conditions cytotoxic T lymphocytes were generated with the same frequency against xenogeneic HLA-B27 determinants as against allogeneic mouse class I antigens. These findings indicate that the normally weak xeno-MHC response is due to the inefficient interaction of the murine Lyt-2 accessory molecule with HLA class I, and not to limitations of the mouse T-cell repertoire.     

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.     

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.     

Recognition of pre-processed endogenous antigen by class I but not class II MHC-restricted T cells

Class I and class II MHC-restricted T lymphocytes recognize non-native forms of antigen. The presentation of antigen to these two classes of T lymphocytes can occur through distinct pathways. Several mechanisms, including differences in antigen processing in different intracellular compartments, have been proposed to account for these pathway differences. Here we describe a T-cell epitope located on the influenza virus haemaglutinin, which is recognized by both class I and class II MHC-restricted cytolytic T lymphocytes (CTL). When expressed de novo in target cells, from a synthetic minigene encoding only the epitope, this pre-processed antigenic site is recognized by class I but not class II MHC-restricted T lymphocytes, even though target cells treated with the exogenously introduced peptide can be recognized by both classes of T cells. Because endogenous expression of the pre-processed antigenic fragment results in differential presentation to class I and class II MHC-restricted CTL, differences between the two different pathways of presentation could lie not at the level of processing but at the level of targeting and/or interaction of processed antigen with MHC.     

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.     

Qa-1 restricted recognition of foreign antigen by a gamma delta T-cell hybridoma

Distinct T-lymphocyte subsets recognize antigens in conjunction with different classes of major histocompatibility complex (MHC) glycoproteins using the T-cell receptor (TCR), a disulphide-linked heterodimer associated with the CD3 complex on the cell surface. In general, class I and class II MHC products provide a context for the recognition of foreign antigens by CD8+ and CD4+ T cells, respectively. This recognition seems to be largely dependent on alpha beta TCR heterodimers, whereas the function of the second gamma delta TCR, present on a minor subpopulation of cells, is still unknown. In the mouse, the existence of six cell-surface MHC class I products (K, D, L, Qa-1, Qa-2 and Tla) has been firmly established by serological, biochemical and genetic evidence. So far, only the most polymorphic of them, K, D and L ('classical' class I) have been reported as restriction elements for T-cell recognition of foreign antigens. The function of the relatively invariant Qa and Tla molecules remains unknown. We have made a T-helper cell hybridoma clone (DGT3) that recognizes synthetic copolymer poly(Glu50Tyr50) in the context of Qa-1 cell surface product, and has a CD4-CD8- phenotype. Our studies indicate that DGT3 cells express the gamma delta TCR on the cell surface, implicating its role in Qa-1-restricted antigen recognition. This is the first evidence that T cells can recognize foreign antigen in association with self Qa product, confirming that Qa molecules not only topologically, but also functionally, belong to the MHC.     

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.     

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.     

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.     

Killing of antigen-reactive B cells by class II-restricted, soluble antigen-specific CD8+ cytolytic T lymphocytes

Cytolytic T lymphocytes (CTLs) are generally thought to recognize cellular antigens presented by class I MHC molecules. A number of studies, however, have revealed responses of considerable magnitude involving both CD8+ and CD4+ CTLs with class II restriction, suggesting that class II-restricted CTLs recognizing exogeneous protein antigens may exist. As class II antigens are normally expressed on limited types of cells such as B cells and macrophages, such CTLs might be expected to exert a suppressive effect on antibody responses. Here we report that stimulation of mouse lymphocytes with a soluble antigen induced CD8+ and CD4+ CTLs specific for the antigen with class II restriction. The specific lysis was far more efficient when target B cells specifically recognized the antigen than when they did not, indicating that the primary targets for these CTLs are probably B cells expressing immunoglobulin receptors reactive for the same antigen molecule. These results suggest that the natural occurrence of such CTLs during immune responses may explain antigen-specific suppression on antibody responses by T cells.     

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 late-differentiation antigen associated with the helper inducer function of human T cells

T lymphocytes possessing helper function produce soluble factors that greatly augment B-cell proliferation and differentiation into antibody-secreting cells. In humans the subset of T lymphocytes bearing the T4 surface antigen comprises most of the cells that display helper activity and recognize class II antigens of the major histocompatibility complex (MHC), while the subset bearing the T8 antigen comprises T cells recognizing class I MHC antigens and exhibiting cytotoxic or suppressor function. Monoclonal antibodies to T4 or T8 greatly inhibit the cognitive and effector function of cells with the corresponding phenotype. This function/phenotype correlation is not absolute, however, for there are many examples of T8-positive clones that recognize MHC class II antigens and have helper activity, as well as of T4-positive clones with suppressor or cytotoxic function. Recently a family of cell-surface neoantigens, which might be relevant to T-cell function and which are present on activated but not on resting T lymphocytes, has been identified in mouse and humans using monoclonal antibodies. Some of these antibodies block the cytolytic activity of alloreactive T-cell clones, suggesting the possible involvement of such molecules in the activation of cytotoxic T-cell clones or in the lytic process itself. We now describe a similar late-differentiation antigen (LDA1) that is expressed by human T lymphocytes only following activation and is recognized by a monoclonal antibody that inhibits the antibody-inducing helper function of T lymphocytes.     

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.     

Class II MHC molecules can use the endogenous pathway of antigen presentation

Models for antigen presentation have divided the world of antigens into two categories, endogenous and exogenous, presented to T cells by class I and class II major histocompatibility complex (MHC) encoded molecules, respectively. Exogenous antigens are though to be taken up into peripheral endosomal compartments where they are processed for binding to class II MHC molecules. Endogenous antigens are either synthesized or efficiently delivered to the cytoplasm before being partially degraded in an as yet undefined way, and complexed with class I MHC molecules. A useful phenotypic distinction between the two pathways has been the sensitivity to weak bases, such as chloroquine, which is a property only of the exogenous pathway. The fungal antibiotic brefeldin A (BFA), which blocks protein transport from the endoplasmic reticulum to the Golgi network, also blocks class I-restricted antigen-presentation, providing us with the corresponding marker of the endogenous pathway. Experiments with influenza virus antigens have supported the view that class II MHC molecules can present exogenous but not endogenous antigen, whereas the observation that class II MHC molecules present measles virus non-membrane antigens by a chloroquine-insensitive pathway suggests that this is not always the case. We show here that influenza A matrix protein can be effectively presented to class II-restricted T cells by two pathways: one of which is chloroquine-sensitive, BFA-insensitive, the other being chloroquine-insensitive and BFA-sensitive. Our results indicate that both class I and class II molecules can complex with antigenic peptides in a pre-Golgi compartment and favour a unified mechanism for MHC-restricted endogenous antigen presentation.     

Domain interactions of H-2 class I antigens alter cytotoxic T-cell recognition sites

H-2 class I antigens appear to direct the recognition of virus-infected and neoplastic transformed cells by cytotoxic T lymphocytes (CTLs). Here, to identify the regions of class I antigens involved in CTL recognition, four hybrid class I genes were constructed in which exons were exchanged between the H-2Kb and H-2Db genes. These class I genes were expressed in mouse L cells and recognition of the hybrid Kb/Db antigens by CTLs and monoclonal antibodies specific for either Kb or Db was investigated. The pattern of CTL and monoclonal antibody recognition obtained indicates three correlations between structure and function of class I antigens. First, most CTL recognition sites and alloantigenic determinants are located on domains 1 and 2 of the antigen molecule. Second, these CTL recognition sites and alloantigenic determinants are not influenced by interaction of domains 1 and 2 with polymorphic regions of domain 3. Third, in contrast, interaction between domains 1 and 2 alters these CTL recognition sites and alloantigenic determinants. The alteration of CTL recognition sites by interaction between domains 1 and 2 suggests that a CTL site may be formed by amino acids from both domains 1 and 2, or that the conformation of amino acids at a CTL site may be altered by interactions between domains 1 and 2. Through these two features, the conformation of CTL recognition sites on H-2 class I antigens may be sensitive to alteration by interaction of either domain 1 or 2 with viral antigens.     

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.