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.     

ICOS is essential for effective T-helper-cell responses

The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-gamma.     

T-cell co-stimulation through B7RP-1 and ICOS

T-cell activation requires co-stimulation through receptors such as CD28 and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine costimulatory receptor-ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-I do not interact with proteins in the CD28-B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1-Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyer's patches. Presensitized mice treated with B7RP-1-Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor-ligand pair that is structurally related to CD28-B7 and is involved in the adaptive immune response.     

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.     

Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses

Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 A resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.     

Structural basis for co-stimulation by the human CTLA-4/B7-2 complex

Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the disulphide-linked homodimeric T-cell surface receptors, CD28 and CTLA-4 (ref. 1). Engagement of CD28 with B7-1 and B7-2 ligands on antigen-presenting cells (APCs) provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response. Given their central function in immune modulation, CTLA-4- and CD28-associated signalling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumour immunity. However, little is known about the cell-surface organization of these receptor/ligand complexes and the structural basis for signal transduction. Here we report the 3.2-A resolution structure of the complex between the disulphide-linked homodimer of human CTLA-4 and the receptor-binding domain of human B7-2. The unusual dimerization properties of both CTLA-4 and B7-2 place their respective ligand-binding sites distal to the dimer interface in each molecule and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal. Direct observation of this CTLA-4/B7-2 network provides a model for the periodic organization of these molecules within the immunological synapse and suggests a distinct mechanism for signalling by dimeric cell-surface receptors.     

Dampening of death pathways by schnurri-2 is essential for T-cell development

Generation of a diverse and self-tolerant T-cell repertoire requires appropriate interpretation of T-cell antigen receptor (TCR) signals by CD4(+?) CD8(+) double-positive thymocytes. Thymocyte cell fate is dictated by the nature of TCR-major-histocompatibility-complex (MHC)-peptide interactions, with signals of higher strength leading to death (negative selection) and signals of intermediate strength leading to differentiation (positive selection). Molecules that regulate T-cell development by modulating TCR signal strength have been described but components that specifically define the boundaries between positive and negative selection remain unknown. Here we show in mice that repression of TCR-induced death pathways is critical for proper interpretation of positive selecting signals in vivo, and identify schnurri-2 (Shn2; also known as Hivep2) as a crucial death dampener. Our results indicate that Shn2(-/-) double-positive thymocytes inappropriately undergo negative selection in response to positive selecting signals, thus leading to disrupted T-cell development. Shn2(-/-) double-positive thymocytes are more sensitive to TCR-induced death in vitro and die in response to positive selection interactions in vivo. However, Shn2-deficient thymocytes can be positively selected when TCR-induced death is genetically ablated. Shn2 levels increase after TCR stimulation, indicating that integration of multiple TCR-MHC-peptide interactions may fine-tune the death threshold. Mechanistically, Shn2 functions downstream of TCR proximal signalling compenents to dampen Bax activation and the mitochondrial death pathway. Our findings uncover a critical regulator of T-cell development that controls the balance between death and differentiation.     

The B-cell surface protein CD72/Lyb-2 is the ligand for CD5.

The glycoprotein CD5 is expressed on the surface membrane of all mature T cells and a small proportion of B lymphocytes. Its exact role in immune interactions is still unknown. Studies indicate that CD5 functions both in mice and humans as a receptor, delivering co-stimulatory signals to T cells in a manner similar to CD2 (ref. 11) and CD28 (ref. 12). Anti-CD5 antibodies stimulate both T-cell proliferation mediated by CD3 in association with the T-cell receptor and secretion of interleukin-2 and expression of its receptor, as well as inducing an increase in intracellular Ca2+ concentration (refs 5-10). To identify the ligand for CD5 we purified the human CD5 protein, labelled it with biotin and used it as a probe. Here we report that CD5 specifically interacts with the cell-surface protein CD72 exclusive to B cells. This interaction is blocked by anti-CD72 antibodies, but not by any other anti-B-cell antibodies. Moreover, non-B cells (mouse L-cell fibroblasts and human Jurkat T cells) expressing a transfected human CD72 complementary DNA could bind to the CD5-biotin conjugate. The results demonstrate that the B-cell surface protein CD72 (Lyb-2 in mice) is the ligand for CD5.     

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.     

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.     

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.     

Regulatory properties of LFA-1 alpha and beta chains in human T-lymphocyte activation

Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimer composed of an alpha and beta chain that is expressed on the surface of most leukocytes and is an essential molecule for adhesion reactions between cells participating in the immune response. A putative ligand for LFA-1 is the intercellular adhesion molecule ICAM-1 (refs 3-5). Leukocyte adhesion abnormality is found in patients with LFA-1 deficiency. It is not clear whether binding of ligand to the LFA-1 molecule merely spatially orientates cells towards each other or can also induce signals that regulate cell activation and differentiation. We have recently developed a T-cell proliferation assay which uses immobilized anti-CD3 monoclonal antibodies as stimulant and is independent of LFA-1-mediated cellular adhesion. As there is no interference by anti-LFA-1 monoclonal antibodies with the adhesion-dependent activation steps, this T-cell activation system allows us to investigate whether transmembrane signals are induced by binding of ligand to LFA-1 on T cells. Our data indicate that binding of ligand to LFA-1 results in the transduction of regulatory signal across the plasma membrane, rather like other molecules (CD2, CD4, CD8) (refs 8-11) with signal-modifying properties involved in the adhesion of T cells to target/stimulator cells. Indeed, adhesion molecules might generally be important in signal transduction, even in cells not belonging to the immune system.     

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.     

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.     

Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation

T-cell activation requires clustering of a threshold number of T-cell receptors (TCRs) at the site of antigen presentation, a number that is reduced by CD28 co-receptor recruitment of signalling proteins to TCRs. Here we demonstrate that a deficiency in beta1,6 N-acetylglucosaminyltransferase V (Mgat5), an enzyme in the N-glycosylation pathway, lowers T-cell activation thresholds by directly enhancing TCR clustering. Mgat5-deficient mice showed kidney autoimmune disease, enhanced delayed-type hypersensitivity, and increased susceptibility to experimental autoimmune encephalomyelitis. Recruitment of TCRs to agonist-coated beads, TCR signalling, actin microfilament re-organization, and agonist-induced proliferation were all enhanced in Mgat5-/- T cells. Mgat5 initiates GlcNAc beta1,6 branching on N-glycans, thereby increasing N-acetyllactosamine, the ligand for galectins, which are proteins known to modulate T-cell proliferation and apoptosis. Indeed, galectin-3 was associated with the TCR complex at the cell surface, an interaction dependent on Mgat5. Pre-treatment of wild-type T cells with lactose to compete for galectin binding produced a phenocopy of Mgat5-/- TCR clustering. These data indicate that a galectin-glycoprotein lattice strengthened by Mgat5-modified glycans restricts TCR recruitment to the site of antigen presentation. Dysregulation of Mgat5 in humans may increase susceptibility to autoimmune diseases, such as multiple sclerosis.     

Cytotoxic T-lymphocyte activation involves a cascade of signalling and adhesion events.

In addition to the antigen-specific T-cell receptor (TCR), T cells bear an array of 'accessory' molecules that can contribute to stable adhesion to the antigen-bearing cell and provide costimulatory signals. For several of these, T-cell adhesion to the ligand can be activated by TCR-dependent signalling (a signal from the TCR primes the coreceptor to bind to its ligand). It is unclear whether the individual coreceptors share common mechanisms of priming and cosignalling, and perhaps act in a redundant manner, or whether they act in a distinct way and contribute uniquely to the activation process. We report here the use of isolated alloantigen, class I proteins and fibronectin ligands to show that coreceptors on cytotoxic T lymphocytes are activated sequentially and deliver distinct biochemical signals on binding to their ligands. TCR engagement activates CD8 by a protein tyrosine kinase-dependent pathway, and CD8 then acts as a signal for initiation of polyphosphoinositide hydrolysis on binding to class I. In contrast, activated adhesion to fibronectin does not initiate polyphosphoinositide hydrolysis, but amplifies hydrolysis once it has been initiated. Thus, cytotoxic T-lymphocyte activation involves a TCR-initiated cascade of adhesion and signalling events leading to response.     

Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

Lymphocyte-specific tyrosine protein kinase p56lck is physically associated with CD4 and CD8 T-cell surface molecules, suggesting that it may transduce CD4/CD8-triggered tyrosine phosphorylation signals during antigen stimulation. Indeed, antibody-mediated aggregation of CD4 (to mimic interaction with its ligand, major histocompatibility complex (MHC) class II molecules), rapidly elevates the kinase activity of p56lck and is associated with marked changes in tyrosine protein phosphorylation. Genetic analyses suggest that the interaction of CD4/CD8 with p56lck results in a positive signal during antigen-induced T-cell activation. To evaluate directly the role of p56lck in T-cell activation, we introduced a constitutively activated form of Lck protein (tyrosine 505 to phenylalanine 505 mutant); in a CD4-negative, MHC-class II restricted mouse T-cell hybridoma. We report here that, as for transfection of CD4, expression of the Lck mutant enhanced T-lymphocyte responsiveness. This finding provides direct evidence that p56lck can positively regulate T-cell functions and that it mediates at least some of the effects of CD4 and CD8 on T-cell activation.