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.     

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

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

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

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

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

Human gamma delta+ T cells respond to mycobacterial heat-shock protein

Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.     

Major histocompatibility complex-linked specificity of gamma delta receptor-bearing T lymphocytes

Several recent studies have identified a distinct subset of CD3(T3)+CD4-CD8-T lymphocytes that express a CD3-associated heterodimer made up of the protein encoded by the T-cell receptor (TCR) gamma-gene and a second glycoprotein termed TCR delta (refs 1-4). TCR gamma delta is expressed on CD3+ thymocytes during fetal ontogeny before the appearance of TCR alpha-beta (alpha beta) (refs 5-7), on CD3+CD4-CD8- adult thymocytes, and on a subset (1-10%) of CD3+ cells in adult peripheral lymphoid organs and the peripheral blood. TCR gamma delta-expressing T cells probably represent a distinct mature T-cell lineage with the capacity to proliferate in response to receptor-mediated signals, and to display non-major histocompatibility complex (MHC)-restricted cytolysis. Critical to understanding the function of this T-cell subset is the identification of the ligand(s) recognized by TCR gamma delta. Here we describe an alloreactive CD3+CD4-CD8-TCR gamma delta-expressing, TCR alpha beta-negative, T-cell line that manifests MHC-linked recognition specificity for both proliferation and cytotoxicity. Our results suggest that T cells expressing TCR gamma delta are capable of self-non-self MHC discrimination and that they can undergo MHC-influenced selection during differentiation like TCR alpha beta-expressing T cells.     

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.     

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

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

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

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

Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen

Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

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.     

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.     

Positive selection of CD4-CD8+ T cells in the thymus of normal mice

The diversification of the repertoire of T-cell antigen receptor (TCR) specificities is influenced by at least two selection processes which occur in the thymus. One of these, termed 'negative selection', is required to install a state of tolerance to self-antigens in the T-cell repertoire and is often achieved by clonal deletion. The second type of selection operating in the thymus results in preferential differentiation of T cells that have restriction specificity for thymic major histocompatibility complex glycoproteins, but the mechanisms leading to this selective process are not yet clear. One model used to describe this 'positive selection' proposes that only those T cells with sufficient avidity for the MHC glycoproteins expressed in the thymus are allowed to acquire functional competence. Here we directly investigate the generation of TCR specificities by following the fate of developing V beta 17+ CD4-CD8+ T cells under conditions where one of the main class I-MHC molecules, either H-2K or H-2D, was specifically blocked by in vitro monoclonal antibody treatment. The results show that development of V beta 17+ CD4-CD8+ T cells in the SJL H-2s mouse strain is selectively abrogated by blocking class I-Ks molecules but is unaffected by blocking class I-Ds molecules. These data directly demonstrate that generation of CD4-CD8+ T cells expressing a particular TCR V beta segment can be correlated with the expression of a particular class I-MHC molecule, thereby providing evidence for positive selection.     

Inefficient positive selection of T cells directed by haematopoietic cells.

Intrathymic differentiation of alpha beta TCR+ T cells depends on positive selection of CD4+CD8+ thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows the maturation of only those T cells capable of restricted antigen recognition in the context of the hosts' MHC alleles. Studies of normal or T-cell receptor-transgenic mice engrafted with MHC-different bone marrow or thymuses support the conclusion that positive selection is directed by MHC molecules expressed on non-haematopoietic cells, presumably thymic epithelial cells. Here we, present contrary evidence that class I MHC molecules expressed by haematopoietic cell types direct positive selection of CD8+ T cells, though at a reduced rate compared with positive selection directed by thymic epithelial cells. The identity of cell types that direct positive selection bears directly on mechanistic models of the process, including the idea that thymic epithelial cell MHC molecules uniquely present specialized peptides that mediate positive selection, and the notion that thymic epithelial cells express unique differentiation-inducing cell surface molecules.     

Cell-cell adhesion mediated by CD8 and MHC class I molecules

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.     

A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family

Recent studies have demonstrated that CD3 is expressed on a subset of thymocytes with a CD4-CD8- (double negative) phenotype. At least some of these cells bear the CD3-associated gamma delta T-cell receptor (TCR gamma delta). Here we describe a second subset of double negative thymocytes which expresses CD3-associated alpha beta receptors (TCR alpha beta). Surprisingly, these cells express predominantly the products of a single V beta gene family (V beta 8). These CD4-CD8-, TCR alpha beta+ cells appear relatively late in ontogeny (between birth and day 5 of life) and thus are unlikely to be the precursors to the TCR alpha beta-bearing cells (CD4+CD8- and CD4-CD8+) already present at birth. They can be selectively expanded in vitro by stimulation with a monoclonal antibody to V beta 8 (F23.1) in the presence of interleukin I (IL-1). We propose that this cell type is a unique T-cell population distinguishable from typical TCR alpha beta+ T cells by its CD4-CD8- phenotype and a restricted TCR V beta repertoire. Analysis of the unique phenotype of these cells suggests that they may represent the normal counterpart of the defective CD4-CD8- T cells found in the lpr autoimmune mouse.     

Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes

The CD8 (Lyt 2) molecule is a phenotypic marker for T lymphocytes that recognize and react with major histocompatibility complex (MHC) class I molecules. Antibody blocking experiments and gene transfection studies indicate that CD8 binds to a determinant on MHC class I molecules on the target cells, facilitating interaction between effector T lymphocytes and the target cell. The CD8 molecule may also be involved in transmembrane signalling during T-cell activation. The existence of CD8- cytotoxic T lymphocytes (CTL) and class I-reactive CTL that are not inhibited by antibody to CD8 suggests that at least some CTL do not require the CD8 molecule to interact with and lyse target cells. We have recently demonstrated that cells transfected with an H-2Dd gene that carries a mutation at residue 227 are not killed by primary CTL8. Here we show that although this mutation abrogates recognition by primary CTL, it does not affect recognition by CD8-independent CTL, suggesting that residue 227 of class I molecules might contribute to a determinant that is the ligand of the CD8 molecule.     

T-cell receptors of human suppressor cells

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.