PKC-theta is required for TCR-induced NF-kappaB activation in mature but not immature T lymphocytes

Productive interaction of a T lymphocyte with an antigen-presenting cell results in the clustering of the T-cell antigen receptor (TCR) and the recruitment of a large signalling complex to the site of cell-cell contact. Subsequent signal transduction resulting in cytokine gene expression requires the activation of one or more of the multiple isoenzymes of serine/threonine-specific protein kinase C (PKC). Among the several PKC isoenzymes expressed in T cells, PKC-theta is unique in being rapidly recruited to the site of TCR clustering. Here we show that PKC-theta is essential for TCR-mediated T-cell activation, but is dispensable during TCR-dependent thymocyte development. TCR-initiated NF-kappaB activation was absent from PKC-theta(-/-) mature T lymphocytes, but was intact in thymocytes. Activation of NF-kappaB by tumour-necrosis factor alpha and interleukin-1 was unaffected in the mutant mice. Although studies in T-cell lines had suggested that PKC-theta regulates activation of the JNK signalling pathway, induction of JNK was normal in T cells from mutant mice. These results indicate that PKC-theta functions in a unique pathway that links the TCR signalling complex to the activation of NF-kappaB in mature T lymphocytes.     

Cbl-b regulates the CD28 dependence of T-cell activation

Whereas co-stimulation of the T-cell antigen receptor (TCR) and CD28 triggers T-cell activation, stimulation of the TCR alone may result in an anergic state or T-cell deletion, both possible mechanisms of tolerance induction. Here we show that T cells that are deficient in the adaptor molecule Cbl-b (ref. 3) do not require CD28 engagement for interleukin-2 production, and that the Cbl-b-null mutation (Cbl-b(-/-)) fully restores T-cell-dependent antibody responses in CD28-/- mice. The main TCR signalling pathways, such as tyrosine kinases Zap-70 and Lck, Ras/mitogen-activated kinases, phospholipase Cgamma-1 and Ca2+ mobilization, were not affected in Cbl-b(-/-) T cells. In contrast, the activation of Vav, a guanine nucleotide exchange factor for Rac1/Rho/CDC42, was significantly enhanced. Our findings indicate that Cbl-b may influence the CD28 dependence of T-cell activation by selectively suppressing TCR-mediated Vav activation. Mice deficient in Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis, suggesting that the dysregulation of signalling pathways modulated by Cbl-b may also contribute to human autoimmune diseases such as multiple sclerosis.     

Structure of a human gammadelta T-cell antigen receptor.

T-cell antigen receptors composed of gamma and delta polypeptide chains (gammadelta TCRs) can directly recognize antigens in the form of intact proteins or non-peptide compounds, unlike alphabeta TCRs, which recognize antigens bound to major histocompatibility complex molecules (MHC). About 5% of peripheral blood T cells bear gammadelta TCRs, most of which recognize non-peptide phosphorylated antigens. Here we describe the 3.1 A resolution structure of a human gammadelta TCR from a T-cell clone that is phosphoantigen-reactive. The orientation of the variable (V) and constant (C) regions of the gammadelta TCR is unique when compared with alphabeta TCRs or antibodies, and results from an unusually small angle between the Vgamma and Cgamma domains. The complementarity-determining regions (CDRs) of the V domains exhibit a chemically reasonable binding site for phosphorylated antigens, providing a possible explanation for the canonical usage of the Vgamma9 and Vdelta2 gene segments by phosphoantigen-reactive receptors. Although the gammadelta TCR V domains are similar in overall structure to those of alphabeta TCRs, gammadelta TCR C domains are markedly different. Structural differences in Cgamma and Cdelta, and in the location of the disulphide bond between them, may enable gammadelta TCRs to form different recognition/signalling complexes than alphabeta TCRs.     

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.     

Association of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha beta T-cell receptor.

The membrane glycoprotein CD4 enhances antigen-mediated activation of T cells restricted by class II molecules of the major histocompatibility complex (MHC). This positive function has been attributed to the protein tyrosine kinase p56lck (ref. 4), which is noncovalently associated with the cytoplasmic portion of CD4, and is activated on CD4 aggregation. Antigen presentation by MHC class II molecules coaggregates CD4 and the T-cell antigen receptor (TCR alpha beta-CD3). Thus, the mutual specificity of CD4 and TCR alpha beta for the MHC-antigen complex results in the juxtaposition of p56lck and TCR alpha beta-CD3. In contrast, anti-CD4 antibodies can abrogate antigen-induced, as well as anti-TCR-induced T-cell activation, indicating that CD4 might also transduce negative signals. The molecular basis for this opposing function remains unclear. Here we show that the CD4-p56lck complex prohibits the induction of activation signals through the TCR-CD3 complex when not specifically included in the signalling process. This negative effect does not require anti-CD4 treatment, indicating that the induction of distinct negative signals is probably not involved. Rather, the results demonstrate that the CD4-p56lck complex provides prerequisite signals for antigen-receptor-induced T-cell growth and thus characterize a molecular mechanism for functional constraints imposed on T-cell activation by the MHC.     

Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes

Major histocompatibility complex (MHC) class I and II molecules are highly polymorphic proteins that bind and present foreign peptides to the clonally distributed alphabeta receptors (TCR) of T lymphocytes. As a population, the immature T lymphocytes generated in the thymus express a very diverse set of TCR specificities. A process of positive selection filters this broad repertoire to optimize peripheral T cells for antigen recognition in the context of available MHC products. Only those precursor T cells whose TCRs generate an adequate but not excessive signalling response to self-peptides bound to the expressed MHC proteins undergo successful maturation. Here we show that post-thymic self-recognition facilitates the antigen reactivity of mature T cells. Both experimental and physiological interruption of T-cell contact with self-peptide MHC ligands leads to a rapid decline in signalling and response sensitivity to foreign stimuli. Because the adaptive immune system must be recruited early in an infectious process when antigen is limiting, these findings suggest that positive selection ensures predictable T-cell recognition of available self-ligands, which in turn promotes efficient responses to pathogens.     

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.     

Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells

T lymphocytes are found not only as recirculating cells in the lymphoid system, but also as immobile cells in certain epithelia. T-cell antigen receptors (TCR) of both alpha/beta and gamma/delta-heterodimer subtypes can exhibit an extremely high degree of diversity. The diversity of alpha/beta TCRs derives from the use of a large number of variable (V) gene segments, as well as junctional diversity generated during rearrangement of these segments, whereas the diversity of gamma/delta TCRs derives largely from junctional elements, with a smaller contribution from a limited number of V gene segments. Many T cells in the epidermal and intestinal epithelia of mice express TCR composed of gamma/delta heterodimers. We demonstrate here that gamma/delta TCRs of T cells in both these tissues are restricted in V gene usage, with different elements predominating. The TCR junctional diversity of epidermal T cells, however, is extremely limited, whereas that of intestinal T cells is extremely diverse. The distinctive features of these two populations suggest that they develop or are selected differently for particular tissue-specific functions.     

Immunization with a synthetic T-cell receptor V-region peptide protects against experimental autoimmune encephalomyelitis

T cells expressing the alpha beta T-cell receptor (TCR) for antigen can elicit anti-idiotypic antibodies specific for the TCR that regulate T-cell function. Defined sequences of the TCR, however, have not been used to elicit specific antibodies and the role of cellular immunity directed against TCR determinants has not been studied. We immunized Lewis rats with a synthetic peptide representing a hypervariable region of the TCR V beta 8 molecule. Subsequent induction of experimental autoimmune encephalomyelitis, a paralytic disease of the central nervous system mediated primarily by V beta 8+ T cells specific for myelin basic protein was prevented. T cells specific for the TCR V beta 8 peptide conferred passive protection against the disease to naive rats, apparently by shifting the predominant T-cell response away from the major encephalitogenic epitope of basic protein. This is the first report demonstrating the use of a synthetic TCR V-region peptide to induce specific regulatory immunity and has important implications for the regulation of human disease characterized by common TCR V-gene usage.     

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.     

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.     

Intrathymic deletion of self-reactive cells prevented by neonatal anti-CD4 antibody treatment

T-cell differentiation in the thymus involves the coordinate expression of genes encoding the alpha and beta chains of the major histocompatibility complex-restricted heterodimeric antigen receptor (TCR) complex, as well as other functionally important molecules such as CD4 and CD8. The repertoire of TCR expressed by T cells is generally thought to be influenced by positive and/or negative selection events occurring when TCRs on developing T cells interact with self-antigens and major histocompatibility complex components. Using a model system in which specific antigen-reactive cells can be monitored by virtue of their preferential expression of certain TCR beta-chain variable (V beta) domains, it has been shown that self-reactive T cells are clonally deleted during development. We report here that clonal deletion of V+ beta 6 cells in Mlsa mice can be prevented by in vivo neonatal administration of monoclonal antibodies directed against CD4. Furthermore, as anti-CD4 monoclonal antibody treatment resulted in the reappearance of V+ beta 6 cells in the mature CD8+ T-cell subset, it is likely that clonal deletion acts on the CD4+CD8+ thymocyte subset and that this subset is an intermediate stage in the differentiation pathway of both CD4+ and CD8+ T-cell lineages.     

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.     

ICOS co-stimulatory receptor is essential for T-cell activation and function

T-lymphocyte activation and immune function are regulated by co-stimulatory molecules. CD28, a receptor for B7 gene products, has a chief role in initiating T-cell immune responses. CTLA4, which binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses. The inducible co-stimulatory molecule (ICOS), a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand B7H/B7RP-1 is expressed on B cells and in non-immune tissues after injection of lipopolysaccharide into animals. To understand the role of ICOS in T-cell activation and function, we generated and analysed ICOS-deficient mice. Here we show that T-cell activation and proliferation are defective in the absence of ICOS. In addition, ICOS -/- T cells fail to produce interleukin-4 when differentiated in vitro or when primed in vivo. ICOS is required for humoral immune responses after immunization with several antigens. ICOS-/- mice showed greatly enhanced susceptibility to experimental autoimmune encephalomyelitis, indicating that ICOS has a protective role in inflammatory autoimmune diseases.     

Participation of CD4 coreceptor molecules in T-cell repertoire selection.

During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection.     

Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin

Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR). Calcineurin and Ras are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.     

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.     

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.     

Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway

Stimulation of T lymphocytes through their antigen receptor (T-cell receptor; TCR) results in the activation of a tyrosine kinase and the generation of phosphatidyl inositol (PtdIns)-derived second messengers. Several reports have indicated that CD45, a haematopoietic cell-specific surface glycoprotein with tyrosine phosphatase activity in its cytoplasmic domain, is important in lymphocyte activation. To examine the possibility that CD45 might influence proximal signal transduction events through the TCR, we have isolated a variant of the human T-cell leukaemic line, HPB-ALL, which fails to express this phosphatase. Unlike cells expressing CD45, stimulation of the TCR in the CD45-negative cell does not result in PtdIns-derived second messengers. Reconstitution of CD45 expression restored early signalling events through the TCR. To localize the site of CD45 action, the human muscarinic type 1 receptor, which also activates the PtdIns second messenger pathway, was transfected into the CD45-negative cell. Although stimulation of the TCR failed to generate PtdIns-derived second messengers, there was normal activity of the PtdIns pathway when human muscarinic receptor type 1 was stimulated, despite the absence of CD45. These data indicate that CD45 influences a cellular component that is essential for effective coupling of the TCR to the PtdIns second messenger pathway.     

Residues of the variable region of the T-cell-receptor beta-chain that interact with S. aureus toxin superantigens

The alpha beta T-cell antigen receptor (TCR) recognizes antigenic peptides in the context of self major histocompatibility complex (MHC) molecules. The specificity of recognition of MHC plus antigen is generally determined by a combination of the variable elements of alpha- and beta-chains of the TCR. Several types of antigen, however, have been identified that, when bound to MHC molecules, stimulate T cells bearing particular variable-region beta-chain (V beta) elements irrespective of the other variable components of the TCR. These have been termed 'superantigens', and here we are concerned with one type of superantigen, the toxins produced by Staphylococcus aureus. T cells have been found that bear closely related members of the same V beta family but respond differently to S. aureus toxins; in particular, cells bearing the human V beta 13.2 element respond to toxin SEC2, whereas cells bearing human V beta 13.1 do not. We have now defined the residues of the V beta element responsible for this difference, and find that they reside in a region thought to lie on the side of the TCR molecule, away from the conventional antigen/MHC-binding site. The evolutionary conservation of this site may be due to its having an important role in some function of the TCR other than the binding of conventional antigen plus MHC.