Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20K T3/T-cell receptor glycoprotein

The antigen receptor on the surface of human T lymphocytes, which consists of a heterodimer of relative molecular mass (Mr) 90,000 (90K) (alpha- and beta-chains), is associated with the T3 antigen (gamma = 25K, delta = 20K and epsilon = 20K). A working model for the mode of action of the T3/T-cell receptor complex is that the clonotypic alpha- and beta-chains are involved in the recognition and binding of antigen in the context of polymorphic major histocompatibility complex (MHC) gene products on the surface of target cells. Antigen binding by the clonotypic receptor probably results in conformational changes in this structure which are recognized by and subsequently trigger the associated T3 complex to transmit signals into the cell, resulting in a proliferative response. The similarity in structure between murine and human clonotypic antigen receptors suggests that such a mechanism of recognition and activation also exists in mouse T lymphocytes, but so far there has been no evidence for the existence of a murine T3 complex. Here we demonstrate the existence of a T3 delta-chain mRNA in murine T lymphocytes. Our sequence data strongly suggest that this mouse mRNA codes for a complete T3 delta polypeptide chain and reveal some interesting properties of the protein.     

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.     

A T3-like protein complex associated with the antigen receptor on murine T cells

Antigen recognition by human T lymphocytes and initiation of T-cell activation are mediated by a group of integral membrane proteins, the T-cell antigen receptor (TCR) and the T3 complex. The polypeptides which comprise T3 (a gamma-chain of relative molecular mass (Mr) 25,000 (25K), and delta and epsilon chains of 20K each) are physically associated with the TCR chains. Surface expression of the complex requires the presence of all the component T3 and TCR proteins. In contrast to the human system, murine T3 has not been identified using antibodies. Here we describe a murine T3-like protein complex. It appears to be more complicated than human T3, containing three monomeric glycoproteins (21-28K), two of which have N-linked carbohydrate side chains and a novel family of TCR-associated homo- and heterodimers. The 28K protein is identified as the murine T3 delta-chain. The 21K protein is phosphorylated on cell activation with concanavalin A (Con A).     

Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.     

Two forms of the T-cell receptor gamma protein found on peripheral blood cytotoxic T lymphocytes

The T-cell receptor (TCR) gamma polypeptide is expressed associated with CD3 (T3) on the surface of normal human peripheral blood lymphocytes. These cells function as non-MHC-restricted cytotoxic T lymphocytes (CTL)and thus may play an important role in host immune defence. The TCR gamma polypeptide occurs as a dimer in at least two molecular forms based on the absence or presence of disulphide linkage. These forms use TCR gamma polypeptides with strikingly different peptide backbone sizes.     

Activation of cytolytic T lymphocyte and natural killer cell function through the T11 sheep erythrocyte binding protein

The T11 sheep erythrocyte binding glycoprotein [relative molecular mass (Mr)50,000(50K)] is expressed throughout human T-lymphocyte ontogeny and appears to play an important physiological role in T-cell activation. Thus, the treatment of T cells with certain monoclonal anti-T11 antibodies results in antigen-independent polyclonal T-cell activation as assessed by proliferation and lymphokine secretion. In addition, the majority of thymocytes that have not yet acquired the T3-Ti antigen/major histocompatibility complex (MHC) receptor can be activated to express interleukin-2 (IL-2) receptors through this T11 structure. We show here that the triggering of cytolytic T (Tc) cells via T11 causes an antigen-independent activation of the cytolytic mechanism as evidenced by the induction of nonspecific cytolytic activity. Furthermore, T11+T3-Ti- natural killer (NK) cell clones can also be induced to lyse NK-cell-resistant targets by treatment with anti-T11 monoclonal antibodies directed at defined T11 epitopes. These results indicate that T11 triggering can activate cytotoxic lymphocytes to express their functional programmes in the absence of specific antigen recognition via the T3-Ti complex and provide further evidence for the notion that certain NK cells and T lymphocytes are related.     

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.     

Abnormal tyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders

The study of human autoimmune diseases has benefited greatly from analysis of animal models. Mice that are homozygous for either the lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mutant genes develop a disease characterized by massive lymphadenopathy and autoantibody formation. With age, the lymphoid organs in these mice are replaced with a greatly expanded population of abnormal lymphocytes. Recent work has shown that these cells are likely to be in the T-cell lineage. They rearrange and transcribe the genes for the alpha and beta subunits of the T-cell receptor (TCR) and a third, T-cell receptor-like gene, T gamma. As determined by immunofluorescence with anti-receptor antibodies the cells also express TCR on the cell surface. The murine T-cell receptor consists of the alpha and beta chains, derived from the rearranged alpha and beta genes, in non-covalent association with seven other chains; the delta chain, of relative molecular mass (Mr) 26,000 (26K), the epsilon chain (25K), a glycosylated 21K chain (gp21) which is probably the homologue of the gamma chain of T3 (CD3), a 16K homodimer (zeta) and a 21K dimer (p21). This multichain complex is thought to be the murine analogue of the human T3 complex. After activation of normal T cells by antigen or lectin, p21 is phosphorylated on tyrosine residues and gp21 is phosphorylated on serine residues. In contrast, in the gld and lpr cells, p21 is phosphorylated even in the absence of antigen or lectin, whereas gp21 is not phosphorylated.     

Peptide sequences of T-cell receptor delta and gamma chains are identical to predicted X and gamma proteins

Although most mature peripheral T lymphocytes express a major histocompatibility complex restricted, CD3-associated, antigen receptor (TCR) which has been well characterized, some T cells carry a different CD3-associated heterodimer on their surface. One of the two disulphide-linked chains of this putative second receptor, which in mice has relative molecular mass (Mr) 35,000 (35K), has been identified as a product of the group of gamma genes. The other chain, termed delta (Mr 45K in mice), is not as well characterized. Although gamma/delta-bearing cells are a minor subset among peripheral T lymphocytes, they are the only CD3+ cells in the thymus early in ontogeny. Taking advantage of these kinetics, we have generated gamma/delta-bearing hybridomas, using a new TCR alpha chain-negative variant of the AKR thymoma BW5147 as tumour parent, fetal thymocytes as normal cell partners, and an anti-CD3 monoclonal antibody (mAb) as screening reagent. Gamma and delta chains from one of these hybrids have been purified and partially sequenced. The sequences obtained indicate that delta is indeed identical to the polypeptide encoded by the recently described gene X, as suggested by Chien et al.     

Identification of a putative second T-cell receptor

Framework monoclonal antibodies have identified a population of human lymphocytes that express the T3 glycoprotein but not the T-cell receptor (TCR) alpha- and beta-subunits. Chemical crosslinking experiments reveal that these lymphocytes express novel T3-associated polypeptides, one of which appears to be the product of the T gamma gene. The other polypeptide may represent a fourth TCR subunit, designated T delta.     

Identification of antigen receptor-associated structures on murine T cells

The specific antigen receptor on human and murine T lymphocytes is a heterodimer of relative molecular mass (Mr) 80,000-90,000 (80-90K) composed of two 40-50K disulphide-linked glycoprotein subunits. Peptide map analysis of the alpha- and beta-chains of receptor isolated from distinct tumour cell lines suggests the presence of both constant and variable regions. Unlike the antigen receptor on B lymphocytes (that is, surface immunoglobulin), the human T-cell antigen receptor seems to be non-covalently associated with another invariant structure recognized by monoclonal antibodies to the cell-surface antigens T3 and Leu 4 (refs 4, 5, 9, 12). Meuer et al. have demonstrated comodulation of the T3 structure and T-cell antigen receptor using anti-clonotypic and anti-T3 monoclonal antibodies. Furthermore, immunoprecipitation with anti-T3 weakly co-precipitates a small amount of the 80-90K heterodimer in certain conditions. The murine homologue of the Leu 4/T3 structure has not been identified, although Gunter et al. have suggested that Thy-1 may be the counterpart of Leu 4/T3 (ref. 13). Here we describe a Leu 4/T3-like structure, distinct from Thy-1, associated with the T-cell receptor of a murine T-lymphoma cell line.     

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.     

The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.     

Presence of Ti (WT31) negative T lymphocytes in normal blood and thymus

The antigen receptor expressed on most T lymphocytes is a disulphide-linked heterodimer (Ti) that is composed of alpha-chain and beta-chain subunits. On the surface of human T lymphocytes, Ti is non-covalently associated with three invariant proteins, designated CD3-gamma, -delta, and -epsilon. It has been suggested that Ti is obligatory for CD3 expression. But a T leukaemia cell line, IL-2 (interleukin 2) dependent T-cell clones established from fetal blood and IL-2 dependent cell lines established from immunodeficiency patients with bare lymphocyte syndrome and ectodermal dysplasia syndrome have recently been shown to express CD3, but not Ti (detected due to monoclonal antibody WT31). These lymphocytes may express the product of the T-cell antigen receptor gamma (TCR-gamma) gene, rather than the alpha/beta heterodimer, in association with CD3. Preliminary studies suggested that T cells expressing CD3 but lacking Ti are present in low frequency in normal lymphoid tissues. Here we show that in normal blood and thymus CD3+, WT31-T cells express neither CD4 nor CD8. The low frequency (less than 0.2-0.9% of total thymocytes) of CD3+, WT31- cells in the thymus suggests that this population does not represent a major stage of thymic development and may be a distinct lineage of T cells.     

CD3-negative natural killer cells express zeta TCR as part of a novel molecular complex

Natural killer (NK) cells are large granular lymphocytes capable of killing tumour cells in a non-MHC restricted manner. NK cells do not express cell-surface CD3, or any known target recognition structure analogous to the T cell antigen receptor (TCR) heterodimers (alpha beta or gamma delta). Consistent with their lack of expression of a CD3-TCR complex, NK cells do not require prior sensitization or antigen presentation by accessory cells to specifically recognize their tumour targets. Although NK cells do not express CD3-TCR, they do express CD2, the target of an alternative activation pathway which is functional in both T cells and NK cells. In T cells, this alternative activation pathway utilizes some component of the CD3-TCR complex as a transducer molecule that is required for mitogenesis. The fact that NK cells are activated by this alternative pathway suggested that they might express a related subunit of the CD3-TCR complex capable of transducing the CD2-mediated signal. Here we show that human NK cells express the zeta-chain of the TCR complex in association with additional structures not included in CD3-TCR.     

Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody

The specificity of cytotoxic T lymphocytes (Tc) cells is conferred by an antigen-specific receptor, Ti, which in humans is physically associated with an invariant cell-surface glycoprotein, T3. Monoclonal antibodies specific for either T3 and Ti are able to elicit a variety of T-cell responses such as lymphokine production, mitogenesis and cytotoxicity. For example, human Tc cells lyse anti-T3-expressing hybridoma cells, but not cells of other specificity, presumably because anti-T3 on the hybridoma cells binds to T3 on the Tc cells and triggers lysis. Here, we have adapted approaches used in a different cytotoxic effector system, antibody-dependent cellular cytotoxicity (ADCC), to alter the specificity of Tc cell. Studies of ADCC showed that heteroaggregates containing anti-Fc receptor (Fc gamma R) antibody cross-linked to a second antibody bind to Fc gamma R on ADCC effectors and cause them to kill target cells bearing antigen recognized by the second antibody. The present studies use anti-T3-containing heteroaggregates to re-target human Tc cells to cells for which we have appropriate antibodies, including xenogeneic tumour cells and chicken erythrocytes. These results extend previous observations on the role of T3 in triggering cytotoxicity and suggest that effector cell re-targeting could be used for in vivo treatment of neoplasms and other pathogens that express distinctive surface antigens.     

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.     

A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors

Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen.     

Delta is the Cx-gene product in the gamma/delta antigen receptor of dendritic epidermal cells

Most T cells bear an antigen receptor that is a protein of a disulphide-linked heterodimer composed of an alpha chain and a beta chain associated with the non-polymorphic CD3 (T3) complex. A small subpopulation of thymic and peripheral T cells, as well as Thy-1+dendritic epidermal cells (dEC), express an alternative CD3-associated dimeric receptor composed of the product of the T-cell antigen receptor (TCR) gamma gene and a fourth chain, designated delta. Recently a new murine TCR constant-region gene, designated Cx, has been cloned and proposed as a candidate for the C delta gene. We have previously demonstrated that murine Thy-1+ dEC cell lines express a CD3-associated disulphide-linked heterodimer composed of a relative molecular mass Mr 41,000 (41K) gamma chain and a 50K delta chain. We have further analysed the receptor of one of these cloned dEC lines, 7-17.1, by endoglycosidase treatment of the isolated gamma and delta chains. The gamma chain was found to contain two N-linked oligosaccharide residues, consistent with the expression of a chain encoded by the V gamma 3 and C gamma 1 gene segments. The delta chain contains at least three N-linked oligosaccharides and has a core size of 38K. Northern blot analysis indicated the presence of abundant Cx messenger RNA in 7-17.1 cells. Immunoprecipitation with two antisera to peptides comprising distinct regions of the Cx sequence indicates that the delta chain is encoded by the Cx gene.