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.     

Intestinal intraepithelial lymphocytes are a distinct set of gamma delta T cells

Lymphocytes are most reliably subdivided on the basis of their receptors for antigen at the cell surface. Three subtypes of lymphocytes are well defined: B cells that bear surface immunoglobulin and make antibody, CD4+T cells with CD3 alpha beta receptors specific for antigen associated with class II major histocompatibility complex molecules, and CD8+T cells with CD3 alpha beta receptors specific for antigen associated with class I MHC molecules. These T cells are responsible for known forms of cell-mediated immunity. The discovery of a third rearranging T-cell specific gene called gamma (refs 1 and 2) has revealed the presence of a new class of T cells bearing a new receptor type, CD3 gamma delta (refs 3-7). To date, neither the function nor the specificity of cells bearing this receptor has been determined. Because gamma delta T cells are the main lymphocyte of epidermis, it was proposed that such cells could be important in surveillance of all epithelia. We have isolated intraepithelial lymphocytes from murine small intestine, and shown that they predominantly or exclusively express CD3 gamma delta receptors. Unlike the epidermal lymphocytes, these cells also express CD8, and they use a different V lambda gene to form their receptor. This strongly suggests that gamma delta T cells home in a very specific manner to epithelia, where they presumably mediate their function.     

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.     

T-cell receptor delta-chain can substitute for alpha to form a beta delta heterodimer

Specific monoclonal antibodies have made possible the identification of two T-cell antigen receptor (TCR) heterodimers, alpha beta TCR and gamma delta TCR. Formation of these receptors is largely separated by the preferential pairing of alpha-TCR with beta and gamma-TCR with delta, the sequential rearrangement and expression of the TCR loci during thymic development and the deletion of the delta-loci either prior to or concomitant with alpha-rearrangement in alpha beta TCR cells. Here we show that delta-TCR can substitute for alpha in pairing with beta to form a beta delta heterodimer. This receptor is expressed on the cell surface of the T-leukaemia cell line DND41 as analysed with beta- and delta-specific monoclonal antibodies. We suggest that a variety of factors including, for example, the deletion of the delta-TCR loci, can now be understood as exclusion mechanisms operating to prevent not only the formation of gamma delta receptors, but also of beta delta T-cell receptors, thereby promoting the numerically dominant alpha beta TCR lineage. Nevertheless, some developing T-cells that do not rearrange the alpha-loci may express the beta delta TCR as described here.     

A new subunit of the human T-cell antigen receptor complex

The T-cell antigen receptor binds antigen in association with a cell surface molecule encoded by the major histocompatibility complex (MHC). MHC restricted recognition of antigen by this receptor leads to the complex pattern of programmed gene expression that characterizes T-cell activation. The eventual understanding of human T-cell function will require the complete elucidation of the structure of the human T-cell antigen receptor. On human T cells, clonally determined, disulphide-linked alpha and beta chains of the receptor are non-covalently and stoichiometrically associated with three additional polypeptides known as the T3 complex. These receptor subunits are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20K (gamma and delta) and a non-glycosylated 20K protein (epsilon). Our studies of murine T cells show that the mouse T-cell antigen receptor consists of at least seven distinct polypeptide chains. In addition to clonotypic alpha and beta chains, the murine complex consists of glycoproteins of 26K and 21K and endoglycosaminidase F (endo F)-insensitive polypeptides of 25K, 21K and 16K. The latter, which we have termed zeta (zeta), exists as a homodimer within the complex. The 26K component (gp26) has been shown to be the murine analogue of the human delta chain. Other cross species homologies remain to be established, however none of the described human receptor components appear similar to the murine zeta polypeptide. We report here the use of an antiserum raised against the murine zeta subunit to identify a previously unrecognized component of the human T-cell antigen receptor. This human protein is T-cell specific and biochemically similar to the murine zeta polypeptide.     

Analysis of specificity for antigen, Mls, and allogenic MHC by transfer of T-cell receptor alpha- and beta-chain genes

The majority of peripheral T lymphocytes bear cell-surface antigen receptors comprised of a disulphide-linked alpha beta dimer. In an immune response, this receptor endows T cells with specificities for foreign antigenic protein fragments bound to cell surface glycoproteins encoded in the major histocompatibility complex (MHC). At a high frequency (greater than 1%), the same population of T lymphocytes responds to allogeneic MHC glycoproteins, or to differences at other genetic loci termed Mls, in conjunction with MHC. The alpha beta-antigen receptor has been implicated in alloreactivity and Mls reactivity. In fact, many monoclonal T-cell lines recognize a foreign protein fragment bound to self-MHC molecules and, in addition, recognize allogeneic MHC glycoproteins, an Mls-encoded determinant, or both. For at least one T-cell clone, a monoclonal antibody directed against the alpha beta antigen receptor has been shown to block activation induced by either antigen-bound self-MHC or by allogeneic MHC. However, it remains to be demonstrated directly that a single alpha beta receptor can mediate antigen specificity, alloreactivity and Mls reactivity, a prerequisite to understanding the structural basis of these high-frequency cross-reactivities. To address this issue we have performed transfers of receptor chain genes from a multiple-reactive T-cell clone into an unrelated host T lymphocyte. We now demonstrate definitively that the genes encoding a single alpha beta-receptor chain pair can transfer the recognition of self-MHC molecules complexed with fragments of antigen, allogeneic MHC molecules, and an Mls-encoded determinant (presumably in conjunction with MHC). In this case the transfer of antigen specificity and alloreactivity requires a specific alpha beta-receptor chain combination, whereas Mls reactivity can be transferred with the beta-chain gene alone into a recipient expressing a randomly selected alpha-chain.     

Predictable acquisition of a new MHC recognition specificity following expression of a transfected T-cell receptor beta-chain gene

Activation of mature T lymphocytes requires specific corecognition of antigen together with membrane-associated glycoprotein products of the major histocompatibility complex (MHC). This dual specificity is determined by a single receptor structure consisting of a clone-specific alpha beta heterodimer. Because both the alpha and beta subunits possess unique combining-site-containing V regions, it remains an open issue as to what contribution each of the two chains of the receptor makes to the antigen versus MHC recognition specificities of the complete dimer present on any given T cell or in the T-cell pool as a whole. In the present work, we have used DNA-mediated gene transfer to express a new alpha or beta chain in a recipient murine T-cell hybridoma possessing a related antigen but distinct MHC specificity compared to the receptor-gene donor. Our results demonstrate that a beta-gene transfected hybridoma expresses new receptors with a predictable hybrid specificity, establishing that the beta chain has the predominant role in MHC molecule recognition in this model.     

Predominant use of a V alpha gene segment in mouse T-cell receptors for cytochrome c

The T-cell receptor is a cell surface heterodimer consisting of an alpha and a beta chain that binds foreign antigen in the context of a cell surface molecule encoded by the major histocompatibility complex (MHC), thus restricting the T-cell response to the surface of antigen presenting cells. The variable (V) domain of the receptor binds antigen and MHC molecules and is composed of distinct regions encoded by separate gene elements--variable (V alpha and V beta), diversity (D beta) and joining (J alpha and J beta)--rearranged and joined during T-cell differentiation to generate contiguous V alpha and V beta genes. T-helper cells, which facilitate T and B cell responses, bind antigen in the context of a class II MHC molecule. The helper T-cell response to cytochrome c in mice is a well-defined model for studying the T-cell response to restricted antigen and MHC determinants. Only mice expressing certain class II molecules can respond to this antigen (Ek alpha Ek beta, Ek alpha Eb beta, Ev alpha Ev beta and Ek alpha Es beta). Most T cells appear to recognize the C-terminal peptide of cytochrome c (residues 81-104 in pigeon cytochrome c). We have raised helper T cells to pigeon cytochrome c or its C-terminal peptide analogues in four different MHC congenic strains of mice encoding each of the four responding class II molecules. We have isolated and sequenced seven V alpha genes and six V beta genes and analysed seven additional helper T cells by Northern blot to compare the structure of the V alpha and V beta gene segments with their antigen and MHC specificities. We have added five examples taken from the literature. These data show that a single V alpha gene segment is responsible for a large part of the response of mice to cytochrome c but there is no simple correlation of MHC restriction with gene segment use.     

Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes

The vast majority of mature T lymphocytes in the peripheral blood and lymphoid organs use the CD3-associated alpha, beta T-cell receptor (TCR) heterodimer for antigen recognition. A second class of TCRs consists of disulphide-linked gamma and delta proteins that are also CD3-associated. A subset of early CD3+ fetal and adult CD4- 8- thymocytes express gamma, delta TCRs before alpha, beta TCRs are detectable. In addition, a minor (1-5%) subpopulation of peripheral T lymphocytes, and some spleen cells from nude mice express gamma, delta TCRs. Notably, dendritic epidermal cells have also been shown to express gamma, delta TCRs. All of these populations lack CD4 and CD8 molecules. We now report that most mature T cells residing in the murine intestinal epithelium express CD3-associated TCRs composed of gamma-chains disulphide-linked to a protein resembling the delta-chain. The striking feature of these intraepithelial lymphocytes (IEL) was that they were exclusively CD4-8+. In addition, approximately half of CD3-bearing IEL lacked detectable Thy-1 on the cell surface, which is unprecedented for murine T cells. In contrast to other CD8+ peripheral T cells, freshly isolated IEL could be induced to display cytolytic activity by engaging the CD3 molecule, indicating that activation had occurred in vivo. Thus, CD8+ IEL are a phenotypically diverse and anatomically restricted population of lymphocytes that use gamma-chain containing heterodimers for antigen recognition.     

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.     

Developmental regulation of T-cell receptor gene expression

In contrast to B cells or their antibody products, T lymphocytes have a dual specificity, for both the eliciting foreign antigen and for polymorphic determinants on cell surface glycoproteins encoded in the major histocompatibility complex (MHC restriction). The recent identification of T-cell receptor glycoproteins as well as the genes encoding T-cell receptor subunits will help to elucidate whether MHC proteins and foreign antigens are recognized by two T-cell receptors or by a single receptor. An important feature of MHC restriction is that it appears to be largely acquired by a differentiating T-cell population under the influence of MHC antigens expressed in the thymus, suggesting that precursor T cells are selected on the basis of their reactivity with MHC determinants expressed in the host thymus. To understand this process of 'thymus education', knowledge of the developmental regulation of T-cell receptor gene expression is necessary. Here we report that whereas messenger RNAs encoding the beta-and gamma-subunits are relatively abundant in immature thymocytes, alpha mRNA levels are very low. Interestingly, whereas alpha mRNA levels increase during further development and beta mRNA levels stay roughly constant, gamma mRNA falls to very low levels in mature T cells, suggesting a role for the gamma gene in T-cell differentiation.     

Importance of IL-2 receptors in intra-thymic generation of cells expressing T-cell receptors

During development, lymphoid stem cells migrate into the thymic rudiment where they proliferate, rearrange their antigen receptor genes and become differentiated into functionally mature T cells. At present, the regulation of these processes is poorly understood, although recent studies have shown that early fetal and adult immature thymocytes express receptors for the T-cell growth factor, interleukin-2 (IL-2). We now present direct evidence that IL-2 receptors have a function in intra-thymic development by demonstrating that proliferation and the generation of cells expressing the T-cell antigen receptor (alpha beta TCR), which is responsible for the recognition of antigens in the context of MHC, are inhibited when antibodies to IL-2 receptors are added to fetal thymus organ cultures. The inhibition is specific in that it does not affect pre-thymic stem cells and can be partially reversed by addition of exogenous recombinant IL-2.     

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.     

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.     

Genomic organization of the genes encoding mouse T-cell receptor alpha-chain

The vertebrate immune system uses two kinds of antigen-specific receptors, the immunoglobulin molecules of B cells and the antigen receptors of T cells. T-cell receptors are formed by a combination of two different polypeptide chains, alpha and beta (refs 1-3). Three related gene families are expressed in T cells, those encoding the T-cell receptor, alpha and beta, and a third, gamma (refs 4-6), whose function is unknown. Each of these polypeptide chains can be divided into variable (V) and constant (C) regions. The V beta regions are encoded by V beta, diversity (D beta) and joining (J beta) gene segments that rearrange in the differentiating T cell to generate V beta genes. The V gamma regions are encoded by V gamma, J gamma and, possibly, D gamma gene segments. Studies of alpha complementary DNA clones suggest that alpha-polypeptides have V alpha and C alpha regions and are encoded by V alpha and J alpha gene segments and a C alpha gene. Elsewhere in this issue we demonstrate that 18 of 19 J alpha sequences examined are distinct, indicating that the J alpha gene segment repertoire is much larger than those of the immunoglobulin (4-5) or beta (14) gene families. Here we report the germline structures of one V alpha and six J alpha mouse gene segments and demonstrate that the structures of the V alpha and J alpha gene segments and the alpha-recognition sequences for DNA rearrangement are similar to those of their immunoglobulin and beta-chain counterparts. We also show that the J alpha gene-segment organization is strikingly different from that of the other immunoglobulin and rearranging T-cell gene families. Eighteen J alpha gene segments map over 60 kilobases (kb) of DNA 5' to the C alpha gene.     

The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes

T lymphocytes recognize foreign molecules using the T-cell receptor (TCR), a disulphide-linked heterodimer closely associated with the CD3 polypeptide complex on the cell surface. The TCR alpha beta heterodimers seem largely responsible for the recognition properties of both helper (TH) and cytotoxic (TC) T cells. Recently, a second CD3-associated T-cell receptor heterodimer, gamma delta, has been described. Cells bearing the gamma delta receptor appear before those bearing alpha beta during thymic ontogeny and persist as a minor component (1-10%) of mature peripheral T cells. Their function is unknown. As there are a limited number of functional TCR V gamma gene segments, the size and potential diversity of the V delta repertoire is important for the number of different antigens that may be recognized by gamma delta heterodimers. The delta-chain locus is located 75 kilobases (kb) 5' to the TCR C alpha coding region, raising the possibility that the alpha and delta V-region repertoires may overlap. Also, analysis of rearrangements at the delta-chain locus in developing thymocytes shows distinct fetal and adult patterns indicating that there may be differences between the fetal and adult V delta repertoires. To address these questions, we have characterized a large number of delta-containing complementary DNA clones from adult double-negative thymocytes (CD4-8-), an immature population that is enriched for gamma delta-bearing cells. We find that a limited number of V delta sequences are used, showing little overlap with known adult V alpha s and differing significantly from fetal V delta s. But as two D elements may participate simultaneously in V delta gene assembly, and random nucleotides may be added at any one of three junctional points, the potential number of different delta chains that can be made in the adult thymus is very large (approximately 10(13)).     

CD1b restricts the response of human CD4-8- T lymphocytes to a microbial antigen.

Molecules encoded by the human CD1 locus on chromosome 1 (ref. 33) are recognized by selected CD4-8- T-cell clones expressing either alpha beta or gamma delta T-cell antigen receptors. The known structural resemblance of CD1 molecules to antigen-presenting molecules encoded by major histocompatibility complex (MHC) genes on human chromosome 6 (refs 3, 4, 34, 35), suggested that CD1 may represent a family of antigen-presenting molecules separate from those encoded in the MHC. Here we report that the proliferative and cytotoxic responses of human CD4-8- alpha beta TCR+ T cells specific for Mycobacterium tuberculosis can be restricted by CD1b, one of the four identified protein products of the CD1 locus. The responses of these T cells to M. tuberculosis seemed not to involve MHC encoded molecules, but were absolutely dependent on the expression of CD1b by the antigen-presenting cell and involved an antigen processing requirement similar to that seen in MHC class II-restricted antigen presentation. These results provide, to our knowledge, the first direct evidence for the proposed antigen-presenting function of CD1 molecules and suggest that the CD1 family plays a role in cell-mediated immunity to microbial pathogens.