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.     

A gamma-chain complex forms a functional receptor on cloned human lymphocytes with natural killer-like activity

We have recently derived from human fetal blood (25 wks) a series of cloned cell lines that were selected for their ability to kill the conventional natural killer (NK) target cell K562. It was found that a fraction of these clones express CD3 proteins but not the monomorphic Ti alpha beta determinant recognized by WT31 antibody. One interleukin-2-dependent CD3+ WT31- clone, termed F6C7, was used for immunization of mice to generate monoclonal antibodies directed at a potentially novel recognition receptor. It was shown that F6C7 cells, which transcribe Ti beta but not Ti alpha genes, surface-express a clonotypic structure, termed NKFi. Immunoprecipitations performed with anti-NKFi monoclonal antibody (mAb) indicated that the corresponding molecule is resolved in SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of relative molecular mass approximately 85,000 (Mr approximately 85K). After reduction, a major band was detected at 44K and a faint band was present at 41K. The present study was designed to characterize this structure. It was found that NKFi represents either two 44K disulphide-linked gamma (TCR) chains, or possibly one gamma chain associated to an additional undetected molecule, and that the 41K material corresponds to a partially glycosylated fraction of the gamma protein. Anti-NKFi mAb both induces a specific autocrine proliferative response and blocks cytotoxic function, demonstrating that gamma chains serve as functional receptor structures on subpopulations of normal human lymphocytes.     

Resident pulmonary lymphocytes expressing the gamma/delta T-cell receptor

The biological role of cells bearing the gamma delta T-cell antigen receptor (TCR) is as yet unclear. Although there are indications that some gamma delta+ cells can mediate cytotoxicity, their antigen-related functions have not yet been defined. In the mouse, gamma delta+ cells constitute 1-3% of T cells in lymphoid organs. Intestinal intraepithelial lymphocytes (IELs) and dendritic epidermal cells (DECs) also appear to carry the gamma delta TCR. The strategic locations of DECs and IELs have led to the suggestion that gamma delta+ cells could constitute a first line of defence in the vicinity of large surfaces of contact with the environment. We report here that an estimated 8-20% of resident pulmonary lymphocytes (RPLs) are CD3+ alpha beta TCR-, and presumably gamma delta TCR+. Furthermore, mice exposed to aerosols containing a Mycobacterium tuberculosis extract have an increased number of activated CD3+ alpha beta-TCR- pulmonary T cells which can be propagated in vitro.     

The CD2 antigen associates with the T-cell antigen receptor CD3 antigen complex on the surface of human T lymphocytes

T lymphocytes can be activated by various stimuli directed either against the T-cell antigen receptor-CD3 antigen complex (Ti-CD3) or the CD2 molecule; see ref. 1 for a review. Activation signals generated by antigen binding to the antigen-specific alpha/beta heterodimer (Ti) are thought to be transduced via the invariant CD3 gamma, epsilon and delta chains, and the associated zeta and eta subunits. The physiological role of the interaction of CD2 with its homologous cell-surface associated ligand LFA-3 remains to be fully elucidated. It has been suggested that CD2 regulates an antigen-independent pathway of activation or that signals delivered via CD2 are an integral part of the antigen-specific pathway. Several recent studies have indicated a requirement for the Ti-CD3 complex in CD2 signalling. Thus, mutant T-cell lines expressing CD2, but not Ti-CD3, on the cell surface cannot be activated via the CD2 molecules. Functional interaction between the Ti-CD3 complex and the CD2 antigen suggests that these T-lymphocyte cell-surface structures are physically associated. Here we use a digitonin-based solubilization procedure to explore this possibility and show that 40% of the cell-surface CD2 molecules can be specifically co-precipitated in association with the Ti-CD3 complex.     

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.     

Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures

The receptors found on most T lymphocytes bind to antigen presented on major histocompatibility complex proteins and consist of dimers of alpha- and beta-polypeptides associated with the invariant CD3 complex. A fully competent immune system requires a diverse array of T-cell antigen receptors (TCRs) with different specificities. This diversity is generated by rearrangement of TCR alpha- and beta-chain gene segments within the thymus where the receptors are first expressed. Any cells carrying self-reactive receptors must be eliminated, suppressed or inactivated so that destructive autoimmunity is avoided. Recently, compelling evidence has shown that one process involved in producing such self-tolerance is clonal deletion of autoreactive cells within the thymus by an as-yet-undefined mechanism. Here we show that engaging the CD3/TCR complex of immature mouse thymocytes with anti-CD3 antibodies produces DNA degradation and cell death through the endogenous pathway of apoptosis. Activation of this process in immature T cells by the binding of the TCR to self-antigens may therefore be the mechanism which produces clonal deletion and consequently self-tolerance.     

Thy-1-mediated T-cell activation requires co-expression of CD3/Ti complex

In addition to monoclonal antibodies against the CD3 (T3)-T-cell antigen receptor (CD3/Ti) complex, several other monoclonals directed towards distinct cell surface structures on human (CD2 (T11) and Tp44) and murine (Thy-1, TAP, and Ly-6) T lymphocytes are capable of activating T cells. It has been proposed that such structures may function as alternative pathways of stimulation. To examine directly whether any relationship exists between Thy-1-dependent activation phenomena and T-cell activation mediated through the CD3/Ti complex, we have transfected several CD3/Ti- variants of the human T-cell line Jurkat with the murine Thy-1.2 gene. Our data indicate that in CD3/Ti-, Thy-1.2+ transfectants, monoclonal antibodies against Thy-1.2 can induce a rise in cytoplasmic free calcium ([Ca2+]i), but fail to stimulate interleukin-2 (IL-2) production. The only defect in these variant cell lines responsible for the inability to produce IL-2 in response to Thy-1 stimulation was in the expression of the CD3/Ti complex, because replacement of defective Ti alpha- or beta-chain genes reconstributed both surface expression of CD3/Ti and responsiveness to Thy-1 in the IL-2 production assay.     

Diversity of gamma delta T-cell receptors on murine intestinal intra-epithelial lymphocytes

The search for the genes encoding the T-cell receptor (TCR) alpha- and beta-subunits revealed a third gene gamma which shares with the alpha- and beta-genes several properties including somatic rearrangement. This gene, together with a fourth rearranging gene delta, encodes a second type of T-cell receptor, TCR gamma delta. Although TCR gamma delta-bearing T cells constitute a relatively minor subpopulation in the thymus and in peripheral lymphoid organs, they are the major lymphocytes of epidermis (dendritic epidermal cells or DEC) and of intestinal epithelium (intestinal intraepithelial lymphocytes or IEL) in mice, suggesting that at least some gamma delta T cells are important in the surveillance of a variety of epithelia. It was recently reported, however, that the TCR gamma delta on DEC has essentially no structural diversity, implying that the putative ligand is monomorphic. As this finding, if generally applicable, poses severe restrictions on the origin of the ligand, we investigated the diversity of the TCR on the second major epithelium-associated gamma delta T cells, namely IEL from mice. We report here that by contrast with the DEC gamma delta, the IEL gamma delta TCR are structurally diverse.     

Thymic major histocompatibility complex antigens and the alpha beta T-cell receptor determine the CD4/CD8 phenotype of T cells

T-cell receptors and T-cell subsets were analysed in T-cell receptor transgenic mice expressing alpha and beta T-cell receptor genes isolated from a male-specific, H-2Db-restricted CD4-8+ T-cell clone. The results indicate that the specific interaction of the T-cell receptor on immature thymocytes with thymic major histocompatibility complex antigens determines the differentiation of CD4+8+ thymocytes into either CD4+8- or CD4-8+ mature T cells.     

Expression of functional acetylcholine receptor from cloned cDNAs

The cloned cDNAs encoding the four subunits of the Torpedo californica acetylcholine receptor, each carried by a simian virus 40 vector, direct the synthesis of the functional receptor in a combined expression system consisting of COS monkey cells and Xenopus oocytes. Our results suggest that all four subunits are required to elicit a normal nicotinic response to acetylcholine, whereas only the alpha-subunit is indispensable for alpha-bungarotoxin binding activity.     

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.     

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.     

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.     

CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.     

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.     

Structure of a cannabinoid receptor and functional expression of the cloned cDNA

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.     

An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor.

Isolated polypeptide chains of the T-cell antigen receptor complex are degraded or retained in the endoplasmic reticulum (ER). Assembly of the multisubunit complex allows the individual chains to escape retention in the ER and to be expressed on the cell surface. We engineered a series of deletions in the CD3 epsilon subunit of the human T-cell receptor in order to find the sequences responsible for its retention in the ER. Deletion of amino acids 171 to 180 in the cytosolic tail resulted in the cell-surface expression of the isolated chain. This sequence also promotes retention when it is appended to CD4, a plasma membrane protein. Mutagenesis of the 10-amino-acid CD3 epsilon sequence established that the tyrosine and serine residues are important for ER retention. This and other ER retention signals must be hidden when a complete T-cell receptor complex is assembled in order to allow its expression on the cell surface.     

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.