Sin: good or bad? A T lymphocyte perspective

Summary: Stimulation of T cells through their antigen receptor induces a multitude of signaling networks that regulate T cell activation in the form of cytokine production and T cell proliferation. Multiple signal integration sites exist along these pathways in the form of multiprotein signaling complexes, the formation of which is facilitated by adapter and scaffold molecules. In recent years a number of adapter and scaffold molecules have been described in T cells and shown to play an integral part in T cell function. Among these molecules are proteins that function as positive or negative regulators of T cell activation downstream of the activated T cell receptor (TCR). Here, we discuss the role of a small family of multiadapter proteins on T cell activation, the p130Cas family, with emphasis on one of its members, Sin (Src‐interacting protein). Our results suggest that Sin inhibits thymocyte development and T cell activation and is a novel negative regulator of T lymphocyte function.     

Known and potential functions for the SLP-76 adapter protein in regulating T-cell activation and development

The hematopoietic adapter protein SLP‐76 is a critical component of multiple biochemical signaling ‘circuits’ in T cells that integrate proximal signaling events initiated by ligation of the T‐cell receptor (TCR) into more distal pathways. Given the important role ascribed to TCR signaling in directing the outcome of thymocyte selection, it seems likely that SLP‐76 may also function in signaling pathways that ultimately impact the establishment of the peripheral T‐cell repertoire. It is generally accepted that the peripheral T‐cell repertoire is selected in large part during T‐cell development in the thymus. Molecular interactions between the TCR and self‐peptide/major histocompatibility complexes expressed on thymic stromal elements dictate the fate of developing thymocytes. Thymocyte survival and further maturation (positive selection) require an active signal delivered to the cell as a consequence of TCR ligation. This raises the intriguing question of how a thymocyte can, for a narrow window of developmental time, obtain responsiveness to self while maintaining tolerance to these same determinants upon export to the periphery. This article reviews the current literature describing SLP‐76‐dependent signaling pathways in mature T cells and developing thymocytes. A potential role for this critical signaling intermediate in integrating signals leading to positive and negative selection of the peripheral T‐cell repertoire is also discussed.     

Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors

Summary: Antigen recognition by the T cell receptor (TCR) complex induces the formation of a TCR signalosome by recruiting various signaling molecules, generating the recognition signals for T cell activation. The activation status and functional outcome are positively and negatively regulated by dynamic organization of the signalosome and by costimulation signals. We have studied the negative regulation of T cell activation, particularly through inhibitory adapters and costimulation receptors that are little expressed in resting cells but are induced upon T cell activation. We described Grb‐associated binder 2 (Gab2) and cytotoxic T lymphocyte antigen‐4 (CTLA‐4) as a representative inhibitory adapter and a negative costimulation receptor, respectively, both of which exhibit negative feedback. Gab2 functions as a signal branch for activation vs. inhibition, as phosphorylation of either Src homology 2 (SH2) domain‐containing leukocyte phosphoprotein of 76 kDa (SLP‐76) or Gab2 by zeta‐associated protein of 70 kDa (ZAP‐70) determines the fate of the response. As a professional inhibitory receptor, CTLA‐4 inhibits T cell response by competition of ligand binding with positive costimulator receptor CD28, and also induces inhibitory signaling. The trafficking and the cell surface expression of CTLA‐4 are dynamically regulated and induced. CTLA‐4 is accumulated in lysosomes and secreted to the T cell–APC contact site upon TCR stimulation. As T cell activation proceeds, these inhibitory adapters and costimulation receptors are induced and suppress/regulate the responses as negative feedback.     

Obligatory cross-talk with the tyrosine kinases assembled with the TCR/CD3 complex in CD4 signal transduction.

Dissection of the CD4 signal transduction pathway has revealed striking similarities with the TCR/CD3 pathway. Furthermore, downstream signaling by CD4 is impaired in cells lacking surface TCR, suggesting a role for the TCR/CD3 complex in CD4 signal transduction. We have investigated the molecular basis for the dependence of CD4 signaling on TCR/CD3 expression. Using the phosphotyrosine binding domains of the Shc adaptor and the Fyn kinase, which both participate in CD4 signaling, as baits, we show that CD4 induces tyrosine phosphorylation of a subset of the proteins phosphorylated in response to TCR/CD3 engagement. The phosphoprotein patterns were dramatically altered in cells defective for TCR/CD3 expression, and were recoverable by reconstitution of correctly assembled TCR, suggesting that CD4 uses TCR/CD3-associated tyrosine kinases to signal. Among the tyrosine kinases associated with the resting TCR/CD3 complex, only Fyn is activated following CD4 engagement. The failure of Fyn to become phosphorylated in cells defective for TCR expression underlines the unique role of TCR/CD3 associated Fyn in CD4 signal transduction. While no calcium mobilization was measurable in cells defective for TCR/CD3 expression in response to CD4 engagement, the Ras/MAP kinase pathway could be partially activated. Thus, CD4 activates at least two signaling pathways, and tyrosine kinases associated with the TCR/CD3 complex are key components of one of these pathways.     

The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor ζ subunits and the CD3 γ,δ and ϵ chains

Immunoglobulin family tyrosine-based activation motifs (ITAM), which define the conserved signaling sequence EX₂YX₂L/IX₇YX₂L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3γ, δ and ? that each contain a single ITAM and the TCR ζ chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 γ, δ, ? ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP-70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb-2 and the p85 regulatory subunit of phosphoinositol 3′ kinase. The CD3 and ζ ITAM display a hierarchy of ZAP-70 binding: ζ1 = γ = δ > ζ3 > ζ2 = ?. Shc, Grb-2 and p85 could bind the ζ ITAM and the CD3 γ and δ ITAM, but not the CD3 ? ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 γ,δ and ζ ITAM that show that the ζ, CD3 γ, δ and ? ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.     

A nonredundant role for the adapter protein Shc in thymic T cell development

Signaling via the pre-T cell receptor (pre-TCR) regulates survival, proliferation, allelic exclusion and differentiation of thymocytes. The role played by the adapter protein Shc in T cells has remained controversial, and its role in pre-TCR signaling has not been addressed. We examined Shc function in thymic T cell development using two genetic approaches. Cre-loxP-mediated inducible expression in transgenic mice of a phosphorylation-defective mutant of Shc impaired signaling through the pre-TCR as well as subsequent proliferation and differentiation. Conditional deletion of the Shc locus in thymocytes also affected thymic maturation at the same pre-TCR developmental stage. Thus, both Shc expression and its tyrosine phosphorylation play an essential and nonredundant role in thymic T cell development.     

Roles of Lck,Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk -family and scr -family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.     

Regulation of ZAP-70 activation and TCR signaling by two related proteins, Sts-1 and Sts-2

T cells play a central role in the recognition and elimination of foreign pathogens. Signals through the T cell receptor (TCR) control the extent and duration of the T cell response. To ensure that T cells are not inappropriately activated, signaling pathways downstream of the TCR are subject to multiple levels of positive and negative regulation. Herein, we describe two related proteins, Sts-1 and Sts-2, that negatively regulate TCR signaling. T cells from mice lacking Sts-1 and Sts-2 are hyperresponsive to TCR stimulation. The phenotype is accompanied by increased Zap-70 phosphorylation and activation, including its ubiquitinylated forms. Additionally, hyperactivation of signaling proteins downstream of the TCR, a marked increase in cytokine production by Sts1/2(-/-) T cells, and increased susceptibility to autoimmunity in a mouse model of multiple sclerosis is observed. Therefore, Sts-1 and Sts-2 are critical regulators of the signaling pathways that regulate T cell activation.     

The activation of Csk by CD4 interferes with TCR-mediated activatory signaling

CD4-Lck recruitment to TCR/CD3, as well as Lck activation is essential for T cell activation. Indeed, the blockage of CD4-Lck recruitment to TCR during antigen recognition exerts a drastic inhibitory effect on T cell activation by interfering with both early and late phases of T cell signaling. In the present work, we report a novel inhibitory mechanism by which CD4 can shut down proximal T cell-activating signals. Indeed, we show that upon ligation of CD4 by antibodies the inhibitory kinase, p50(csk), is strongly induced and prolonged during the time. In contrast, p50(csk) was not activated when TCR and CD4 were properly engaged by their ligands. We also demonstrate that anti-CD4 treatment stimulated Csk kinase associated to the membrane adapter, PAG/Cbp, without affecting the total amount of Csk bound to PAG/Cbp. As a consequence, early tyrosine phosphorylation events as well as downstream signaling pathways leading to IL-2 gene expression induced by TCR were inhibited in anti-CD4 pretreated cells. We suggest a new model to explain the activation of negative signals by CD4 molecule.     

Rapid CD4+ T‐cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

Toll‐like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as‐MyD88 or (TRIF TIR‐domain‐containing adapter‐inducing interferon‐β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin‐specific T‐cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR‐signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin‐specific CD4⁺ T‐cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin‐specific T‐cell responses.     

N‐Ethyl‐N‐nitrosourea mutagenesis in the mouse provides strong genetic and in vivo evidence for the role of the Caspase Recruitment Domain (CARD) of CARD‐MAGUK1 in T regulatory cell development

Natural regulatory T (nTreg) cells generated in the thymus are essential throughout life for the maintenance of T‐cell homeostasis and the prevention of autoimmunity. T‐cell receptor (TCR)/CD28‐mediated activation of nuclear factor‐κB and (J)un (N)‐terminal kinase pathways is known to play a key role in nTreg cell development but many of the predicted molecular interactions are based on extrapolations from non‐Treg cell TCR stimulation with non‐physiological ligands. For the first time, we provide strong genetic evidence of a scaffold function for the Caspase Recruitment Domain (CARD) of the TCR signalling protein CARD‐MAGUK1 (CARMA1) in nTreg cell development in vivo. We report two, new, N‐ethyl‐N‐nitrosourea‐derived mutant mice, Vulpo and Zerda, with a profound block in the development of nTreg cells in the thymus as well as impaired inducible Treg cell differentiation in the periphery. Despite independent heritage, both mutants harbour different point mutations in the CARD of the CARMA1 protein. Mutations in vulpo and zerda do not affect expression levels of CARMA1 but still impair signalling through the TCR due to defective downstream Bcl‐10 recruitment by the mutated CARD of CARMA1. Phenotypic differences observed between Vulpo and Zerda mutants suggest a role for the CARD of CARMA1 independent of Bcl‐10 activation of downstream pathways. We conclude that our forward genetic approach demonstrates a critical role for the CARD function of CARMA1 in Treg cell development in vivo.     

Changes in actin dynamics at the T‐cell/APC interface: implications for T‐cell anergy?

Summary: Over the past 20 years the role of the actin cytoskeleton in the formation of the immunological synapse and in T‐cell activation has been the subject of intense scrutiny. T‐cell receptor (TCR) signaling leads to tyrosine phosphorylation of numerous adapter proteins whose function is to relay signals to downstream components of the TCR signaling pathway and, in particular, to molecules implicated in remodeling the actin cytoskeleton. Here, we discuss how signals from the TCR converge on two key regulators of the actin cytoskeleton, Ena/vasodilator‐stimulated phosphoproteins (VASPs) and the actin‐related protein (ARP2/3) complex. We also discuss the implications of TCR signaling in the process of T‐cell anergy with particular emphasis on the actin remodeling and molecules involved in the control of T‐cell proliferation.     

The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation

The src‐family kinases p56^lck (Lck) and p59^fyn (Fyn) are expressed in T cells and are among the first signaling molecules to be activated downstream of the T cell receptor (TCR). Evidence is emerging that although closely related, these signaling molecules have discrete functions during development, maintenance and activation of peripheral T cells. For example, during thymopoiesis Lck is uniquely able to provide all the signals required for pre‐TCRβ selection, although Fyn can substitute for a subset of these. Positive selection of CD4 single‐positive (SP) cells is also critically dependent on the expression of Lck but not Fyn, while differentiation of CD8 SP cells proceeds relatively efficiently in the absence of Lck. In naïve peripheral T cells either Lck or Fyn can transmit TCR‐mediated survival signals, and yet only Lck is able to trigger TCR‐mediated expansion signals under conditions of lymphopenia. Stimulation of naïve T cells by antigenic stimuli is also severely compromised in the absence of Lck, but more subtly impaired by the absence of Fyn. We discuss recent experiments addressing how these two src‐kinase family members interface with downstream signaling pathways to regulate these diverse aspects of T cell behavior.     

Activation of T cells by superantigen: cytokine production but not apoptosis depends on MEK-1 activity

Engagement of the TCR may result in proliferation and cytokine release or programmed cell death. These two outcomes may be the consequence of distinct T cell receptor-coupled signal transduction pathways or may reflect quantitative differences in signaling strength via a single pathway. Here we show that genetic inhibition of MAP kinase kinase (MEK) by a dominant negative mutant or through chemical inhibition by PD98059 inhibits IL-2 secretion but not programmed cell death after TCR ligation by superantigen. This supports the hypothesis that T cell cytokine release and apoptosis result from signaling through distinct pathways and implies that the molecular signaling mechanisms regulating apoptosis of mature T cells and negative selection of thymocytes may be similar.     

SWAP-70-like adapter of T cells,an adapter protein that regulates early TCR-initiated signaling in Th2 lineage cells

We describe the isolation of a protein, SWAP-70-like adapter of T cells (SLAT), which is expressed at high levels in thymocytes and differentiated Th2 cells. SLAT expression was upregulated in differentiating Th2 cells and downregulated in Th1 cells. Ectopic SLAT expression exerted positive or negative effects on IL-4 versus IFNgamma induction, respectively. TCR signaling induced translocation of SLAT to the immunological synapse and its association with ZAP-70 kinase. SLAT reduced the association of ZAP-70 with TCR-zeta and interfered with ZAP-70 but not Lck signaling. Consistent with these results, pharmacological inhibition of ZAP-70 also induced Th2 skewing. Thus, SLAT is a protein which plays a role in Th2 development and/or activation, perhaps by interfering with ZAP-70 signaling.     

Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses

The adaptor protein Src homology 2 domain‐containing leukocyte‐specific protein of 76 kDa (SLP‐76) is central to the organization of intracellular signaling downstream of the T‐cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP‐76 protein in thymocytes. Here, we show that following tamoxifen‐regulated conditional deletion of SLP‐76, mature, antigen‐inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR‐generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP‐76 are resistant to induction of the CD4⁺ T‐cell‐mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP‐76‐mediated signaling in initiating T‐cell‐directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.     

Antigen specific T lymphocyte activation

Following recognition of foreign antigens, the T cell antigen receptor (TCR) transduces signals leading to clonal expansion and differentiation into effector cells. Engagement of the TCR results first in the activation of cytosolic protein tyrosine kinases, followed by initiation of multiple signaling cascades. Recently, it has been shown that regulation and integration of these signaling pathways requires formation of multimolecular complexes and the recruitment of these complexes to specific regions within the cell. This review focuses on the events leading to T cell activation including the signaling cascades and the role of adapter molecules in coordination of these pathways. In addition, the mechanism by which TCR ligation can lead to T cell removal following elimination of an antigenic challenge is discussed.     

Phospholipase Cγ1 is required for pre‐TCR signal transduction and pre‐T cell development

Pre‐T cell receptor (TCR) signaling is required for pre‐T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre‐TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5‐trisphosphate, that are important to mediate PKC activation and intracellular Ca²⁺ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR‐mediated signaling, development and T‐cell activation, but the role of PLCγ1 in pre‐TCR signal transduction and pre‐T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre‐T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre‐TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre‐T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre‐TCR mediated Ca²⁺ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre‐TCR mediated signal transduction and pre‐T cell development.     

Linker for activation of T cells is displaced from lipid rafts and decreases in lupus T cells after activation via the TCR/CD3 pathway

Systemic lupus erythematosus (SLE) is characterized by abnormal signal transduction mechanisms in T lymphocytes. Linker for activation of T cells (LAT) couples TCR/CD3 activation with downstream signaling pathways. We reported diminished ERK 1/2 kinase activity in TCR/CD3 stimulated lupus T cells. In this study we evaluated the expression, phosphorylation, lipid raft and immunological synapse (IS) localization and colocalization of LAT with key signalosome molecules. We observed a diminished expression and an abnormal localization of LAT in lipid rafts and at the IS in activated lupus T cells. LAT phosphorylation, capture by GST-Grb2 fusion protein, and coupling to Grb2 and PLCγ1, was similar in healthy control and lupus T cells. Our results suggest that an abnormal localization of LAT within lipid rafts and its accelerated degradation after TCR/CD3 activation may compromise the assembly of the LAT signalosome and downstream signaling pathways required for full MAPK activation in lupus T cells.