Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b(-/-) lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b(-/-) T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-gamma or tumour necrosis factor-alpha. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.     

Negative regulation of transforming growth factor-beta by the proteoglycan decorin

Decorin is a small chondroitin-dermatan sulphate proteoglycan consisting of a core protein and a single glycosaminoglycan chain. Eighty per cent of the core protein consists of 10 repeats of a leucin-rich sequence of 24 amino acids. Similar repeats have been found in two other proteoglycans, biglycan and fibromodulin, and in several other proteins including Drosophila morphogenetic proteins. Expression of high levels of decorin in Chinese hamster ovary cells has a dramatic effect on their morphology and growth properties. We now report that this effect is due at least in part to the ability of decorin to bind transforming growth factor-beta, an autocrine factor that stimulates the growth of Chinese hamster ovary cells. As transforming growth factor-beta induces synthesis of decorin in many cell types, our results suggest that decorin may be a component of a feedback system regulating cell growth.     

Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen.

Immunization with myelin basic protein (MBP) induces experimental allergic encephalomyelitis (EAE), a prototype of CD4+ T-cell mediated autoimmune disease. In rodents, MBP-reactive T-cell clones are specific for a single, dominant determinant on MBP and use a highly restricted number of T-cell receptor genes. Accordingly, EAE has been prevented by various receptor-specific treatments, suggesting similar strategies may be useful for therapy of human autoimmune disease. Here we report that in (SJL x B10.PL)F1 mice, immune dominance of a single determinant, MBP:Ac1-11, is confined to the inductive phase of EAE. In mice with chronic EAE, several additional determinants of MBP in peptides 35-47, 81-100 and 121-140 recall proliferative responses. Most importantly, reactivity to the latter determinants was also detected after induction of EAE with MBP peptide Ac1-11 alone; this demonstrates priming by endogenous MBP determinants. Thus, determinants of MBP that are cryptic after primary immunization can become immunogenic in the course of EAE. Diversification of the autoreactive T-cell repertoire due to 'determinant spreading' has major implications for the pathogenesis of, and the therapeutic approach to, T-cell driven autoimmune disease.     

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.     

Stimulation of p21ras upon T-cell activation

External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.     

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.     

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.     

Stimulation of the phosphatidylinositol pathway can induce T-cell activation

The T-cell antigen receptor (TCR) regulates two signal transduction pathways: the phosphatidylinositol (PtdIns) and tyrosine kinase pathways. Stimulation of T cells with antigen or anti-TCR monoclonal antibodies induces an increase in inositol phosphates and diacylglycerol, the second messengers responsible for the mobilization of cytoplasmic free calcium and activation of protein kinase C-4. The TCR also activates a tyrosine kinase that is not intrinsic to the TCR. The relationship between these two signal transduction pathways and their contribution to later T-cell responses is unclear. Studies using variants of a murine hybridoma suggested that the PtdIns pathway might not be necessary for or be involved in regulating interleukin-2 (IL-2) production. To address the relationship between later T-cell responses and the early biochemical signals, we investigated the ability of a heterologous receptor with defined signal transduction function to induce T-cell activation. The human muscarinic subtype-1 receptor (HM1), which elicits PtdIns metabolism in neuronal cells through a G protein-coupled mechanism, also functionally activates this pathway when expressed in the T-cell line Jurkat-derived host, J-HM1-2.2 (ref.8). We show here that stimulation of HM1 alone induced IL-2 production and IL-2 receptor alpha chain expression. HM1 does not induce the tyrosine kinase pathway, suggesting that this pathway does not directly influence later T cell-activation responses. Instead, our studies indicate that activation of the PtdIns pathway is probably sufficient to induce later T-cell responses.     

pH-sensitive activation of the intracellular-pH regulation system in squid axons by ATP-gamma-S

The regulation of intracellular pH (pHi) is essential for normal cell function, and controlled changes in pHi may play a central role in cell activation. Sodium-dependent Cl-HCO3 exchange is the dominant mechanism of pHi regulation in the invertebrate cells examined, and also occurs in mammalian cells. The transporter extrudes acid from the cell by exchanging extracellular Na+ and HCO3- (ref. 9) (or a related species) for intracellular Cl- (refs 3, 4). It is blocked by the stilbene derivatives DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate, ref. 10) and SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonate, ref. 3), and has a stoichiometry of two intracellular H+ neutralized for each Na+ taken up and each Cl- extruded by the axon. Because the inwardly-directed Na+ concentration gradient is sufficiently large to energize both the HCO3- influx and Cl- efflux, this electroneutral exchanger could be a classic secondary active transporter, thermodynamically independent of ATP hydrolysis. However, at least in the squid axon, the exchanger has an absolute requirement for ATP (ref. 3). Thus, a major unresolved issue is whether this Na-dependent Cl-HCO3 exchanger stoichiometrically hydrolyses ATP (the pump hypothesis), or whether ATP activates the transporter by a mechanism such as phosphorylation or simple binding (the activation hypothesis). We have now explored the role of ATP in pHi regulation by dialysing axons with the ATP analogue ATP-gamma-S. In many systems, ATP-gamma-S is an acceptable substrate for protein kinases, whereas the resulting thiophosphorylated proteins are not as readily hydrolysed by phosphatases as are phosphorylated proteins. Our results rule out the pump hypothesis, and show that the basis of the axon's ATP requirement is the pH-dependent activation (by, for instance, phosphorylation or ATP binding) of the exchanger itself, or of an essential activator.     

Pleiotropic loss of activation pathways in a T-cell receptor alpha-chain deletion variant of a cytolytic T-cell clone

Untransformed T-cell clones maintained in culture are dependent on signals transmitted through their antigen receptors (Ti; alpha and beta chains associated with the CD3 molecules) for growth and effector function. For cytolytic T cells (CTL), Ti stimulation also activates the killing machinery and induces synthesis of gamma interferon (IFN-gamma) messenger RNA and IFN-gamma secretion. The Thy-1 molecule, expressed on all murine cells of the T-cell lineage, has been suggested to function in transmembrane signalling, based on the ability of some anti-Thy-1 monoclonal antibodies (mAb) to activate T cells. Recently, it was suggested that Thy-1 could function as a signal-transduction molecule when expressed in B-cell lymphomas after transfection of the gene, leading to speculation that the molecule was part of an activation pathway independent of the Ti/CD3 structures. Here we report the immunoselection of a variant CTL clone which has lost expression of mRNA for the alpha-chain of the Ti. The Ti- variant was defective in lectin-mediated activation whether measured by increase in intracytoplasmic Ca2+, CTL effector function or IFN-gamma synthesis. The variant, which expressed normal levels of Thy-1, was also unresponsive to Thy-1 mAb activation as measured by IFN-gamma secretion, whereas it responded to calcium ionophore plus phorbol ester. These results indicate that in a non-transformed, functional mature T-cell, Thy-1 mediated signalling is not an alternative to, but might depend on elements associated with the Ti/CD3-mediated T-cell activation pathway.     

Enhancement of T-cell activation by the CD43 molecule whose expression is defective in Wiskott-Aldrich syndrome

CD43 (sialophorin, leukosialin, leukocyte large sialoglycoprotein), a heavily sialylated molecule found on most leukocytes and platelets, was initially identified as a major glycoprotein of mouse, rat and human T cells. CD43 expression is defective on the T cells of males with the Wiskott-Aldrich syndrome, an X chromosome-linked recessive immunodeficiency disorder. Affected males are susceptible to opportunistic infections and do not respond to polysaccharide antigens, reflecting defects in cytotoxic and helper T-cell functions. Anti-CD43 monoclonal antibodies have a modest costimulatory effect on T cells, natural killer cells, B cells and monocytes, and one such antibody has been shown to activate T cells directly. To investigate a possible physiological role for CD43, a complementary DNA encoding the human protein was introduced into an antigen-responsive murine T-cell hybridoma. We observed that CD43 enhances the antigen-specific activation of T cells and that the intracellular domain of CD43, which is hyperphosphorylated during T-cell activation, is required for this function. We also found that antigen-presenting cells can bind specifically to immobilized purified CD43 and that the binding can be inhibited by liposomes containing CD43 as well as by anti-CD43 monoclonal antibodies.