TCR Signal Transduction Research Articles

Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction.

Lateral mobility and spatial organization of proteins within the plasma membrane are likely to mediate the initial events coordinating T cell activation. Lipid rafts, distinct cholesterol/sphingolipid-rich membrane microdomains, provide a mechanism for this regulation by concentrating or excluding signaling proteins. We demonstrate in peripheral blood T cell lymphoblasts that immediate early phosphotyrosine signal transduction through the TCR complex is functionally dependent on a distinct population of lipid rafts. Specifically, cholesterol extraction destabilizes the membrane microdomains containing Lck, while the rafts containing the adapter protein linker for activation of T cells remain intact. Heterogeneity in the partitioning of these proteins in resting cells was confirmed by immunoelectron microscopy. After T cell activation, both Lck and the linker for activation of T cells colocalize to 50-100 nm microdomains in the plasma membrane, indicating that sequestration of these proteins into distinct lipid rafts may function to regulate the initiation of T cell signal transduction.

Studies of T-cell activation in chronic inflammation

Chapter summaryThe strong association between specific alleles encoded within the MHC class II region and the development of rheumatoid arthritis (RA) has provided the best evidence to date that CD4+ T cells play a role in the pathogenesis of this chronic inflammatory disease. However, the unusual phenotype of synovial T cells, including their profound proliferative hyporesponsiveness to TCR ligation, has challenged the notion that T-cell effector responses are driven by cognate cartilage antigens in inflamed synovial joints. The hierarchy of T-cell dysfunction from peripheral blood to inflamed joint suggests that these defects are acquired through prolonged exposure to proinflammatory cytokines such as tumour necrosis factor (TNF)-α. Indeed, there are now compelling data to suggest that chronic cytokine activation may contribute substantially to the phenotype and effector function of synovial T cells. Studies reveal that chronic exposure of T cells to TNF uncouples TCR signal transduction pathways by impairing the assembly and stability of the TCR/CD3 complex at the cell surface. Despite this membrane-proximal effect, TNF selectively uncouples downstream signalling pathways, as is shown by the dramatic suppression of calcium signalling responses, while Ras/ERK activation is spared. On the basis of these data, it is proposed that T-cell survival and effector responses are driven by antigen-independent, cytokine-dependent mechanisms, and that therapeutic strategies that seek to restore T-cell homeostasis rather than further depress T-cell function should be explored in the future.

An approach to the analysis of gene expression in chronically activated T Lymphocytes

We have been studying the intracellular signaling pathways in chronically activated T cells involved in effector responses and promoting the inflammatory process, and have been struck by the extent to which T cells stimulated with TNF for prolonged periods in vitro resemble RA synovial T cells. For example, TNF upregulates expression of the activation antigen CD69, induces non-deletional and reversible hyporesponsiveness to TCR ligation by uncoupling proximal TCR signalling pathways, and represses CD28 gene expression. We have explored the possibility that systematic expression profiling of murine T cell hybridomas stimulated with pM concentrations of TNF under controlled conditions might provide further insight into the phenotype and function of cytokine activated T cells, as well as the mechanisms through which TNF uncouples TCR signal transduction pathways. Expression profiling has been performed using medium density spotted arrays based on the Compugen™ gene set. This comprises 9,215 oligonucleotide 50-mer elements including housekeeping genes and landing lights, and covers 7,524 known mouse genes (representing 17K RNAs and 303K ESTs). Genes whose expression are altered by TNF treatment have been identified by measuring the fluorescence ratio of Cy5- and Cy3-labeled target cDNA bound to each probe following hybridization of differentially labeled cDNA pools, prepared from TNF stimulated (Cy5) or control (Cy3) T cells. Based on this simplistic analysis, clusters of genes that appear to be differentially regulated in chronic TNF treated T cells have been identified and were found to include genes whose products may function to potentiate the inflammatory response. We report that the expression signature for chronic TNF stimulation suggests a phenotype which promotes cell survival, while enhancing Th1 differentiation, recruitment to sites of inflammation and effector responses. We are now analysing data from experiments designed to explore the possibility that this particular gene expression signature is distinct from the programme of gene transcription arising from short term TNF stimulation. We anticipate that this approach may provide further insight into the molecular mechanisms which promote chronic, as opposed to acute inflammatory responses.

CD28 as a Molecular Amplifier Extending TCR Ligation and Signaling Capabilities

Evidence has gathered that CD28 costimulation facilitates T cell activation by potentiating TCR intrinsic-signaling. However, the underlying molecular mechanism is largely unknown. Here we show that, by enhancing T cell/APC close contacts, CD28 facilitates TCR signal transduction. Moreover, the signal supplied by CD28 does not lead to increased Zap-70 and Lat phosphorylation, but amplifies PLCγ1 activation and Ca 2+ response. We provide evidence that the PTK Itk controls the latter function. Our data suggest that CD28 binding to B7 contributes to setting the level of TCR-induced phosphorylated Lat for recruiting signaling complexes, whereas the CD28 signal boosts multiple pathways by facilitating PLCγ1 activation. These results should provide a conceptual framework for understanding quantitative and qualitative aspects of CD28-mediated costimulation.

Abnormal T cell receptor signal transduction of CD4 Th cells in X-linked lymphoproliferative syndrome.

The molecular basis of X-linked lymphoproliferative (XLP) disease has been attributed to mutations in the signaling lymphocytic activation molecule-associated protein (SAP), an src homology 2 domain-containing intracellular signaling molecule known to interact with the lymphocyte-activating surface receptors signaling lymphocytic activation molecule and 2B4. To investigate the effect of SAP defects on TCR signal transduction, herpesvirus saimiri-immortalized CD4 Th cells from XLP patients and normal healthy individuals were examined for their response to TCR stimulation. CD4 T cells of XLP patients displayed elevated levels of tyrosine phosphorylation compared with CD4 T cells from healthy individuals. In addition, downstream serine/threonine kinases are constitutively active in CD4 T cells of XLP patients. In contrast, TCR-mediated activation of Akt, c-Jun-NH(2)-terminal kinases, and extracellular signal-regulated kinases in XLP CD4 T cells was transient and rapidly diminished when compared with that in control CD4 T cells. Consequently, XLP CD4 T cells exhibited severe defects in up-regulation of IL-2 and IFN-gamma cytokine production upon TCR stimulation and in MLRs. Finally, SAP specifically interacted with a 75-kDa tyrosine-phosphorylated protein upon TCR stimulation. These results demonstrate that CD4 T cells from XLP patients exhibit aberrant TCR signal transduction and that the defect in SAP function is likely responsible for this phenotype.

Functional Uncoupling of T-cell Receptor Engagement and Lck Activation in Anergic Human Thymic CD4+T Cells

Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human thymic CD1a-CD4+ T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505. Confocal microscopy demonstrated that colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.

Fine tuning of TCR signaling by CD5.

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.

Prolonged exposure of T cells to TNF down-regulates TCR zeta and expression of the TCR/CD3 complex at the cell surface.

A role for TNF-alpha in the pathogenesis of chronic inflammatory disease is now firmly established. Paradoxically, TNF also has potent immunomodulatory effects on CD4(+) T lymphocytes, because Ag-specific proliferative and cytokine responses are suppressed following prolonged exposure to TNF. We explored whether TNF attenuated T cell activation by uncoupling proximal TCR signal transduction pathways using a mouse T cell hybridoma model. Chronic TNF exposure induced profound, but reversible, T cell hyporesponsiveness, with TNF-treated T cells requiring TCR engagement with higher peptide concentrations for longer periods of time for commitment to IL-2 production. Subsequent experiments revealed that chronic TNF exposure led to a reversible loss of TCRzeta chain expression, in part through a reduction in gene transcription. Down-regulation of TCRzeta expression impaired TCR/CD3 assembly and expression at the cell surface and uncoupled membrane-proximal tyrosine phosphorylation events, including phosphorylation of the TCRzeta chain itself, CD3epsilon, ZAP-70 protein tyrosine kinase, and linker for activation of T cells (LAT). Intracellular Ca(2+) mobilization was also suppressed in TNF-treated T cells. We propose that TNF may contribute to T cell hyporesponsiveness in chronic inflammatory and infectious diseases by mechanisms that include down-regulation of TCRzeta expression. We speculate that by uncoupling proximal TCR signals TNF could also interrupt mechanisms of peripheral tolerance that are dependent upon intact TCR signal transduction pathways.

A molecular framework for two-step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events.

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.

A mathematical analysis of TCR serial triggering and down-regulation

Despite the increasing knowledge on the pathways involved in TCR signal transduction and T cell activation, the molecular mechanism of TCR triggering by ligand, MHC-peptide complexes, is still elusive and controversial. The present paper addresses the controversy on the early events of TCR engagement and triggering. Mathematical modelling techniques are applied to experimental data to infer plausible molecular mechanisms of TCR triggering and down-regulation. A similar approach has been followed by Bachmann et al. (Eur. J. Immunol. 1998, 28: 2571 - 2579), who concluded that the TCR triggering requires the formation of MHC-TCR dimers or trimers. We report here the failure to generalize this conclusion to the data reported by Valitutti et al. (Nature 1995, 375: 148 - 151). We show that there are several kinetic features in these experimental curves of TCR down-regulation that cannot be explained by the simple model proposed by Bachmann et al. unless some phenomenological extensions are considered. These extensions are: (1) a ligand independent turnover of the TCR; (2) a transient accumulation of triggered TCR; (3) a high order of TCR triggering kinetics; and (4) two pools of membrane TCR in dynamic equilibrium.

Cutting edge: B cell linker protein is dispensable for the allelic exclusion of immunoglobulin heavy chain locus but required for the persistence of CD5+ B cells.

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK(-/-) mice harboring a V(H)12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type V(H)12-expressing B cells are CD5(+), the majority of the splenic V(H)12-expressing BLNK(-/-) B cells are CD5(-). A small population of V(H)12-expressing, BLNK(-/-) CD5(+) B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.

Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction.

Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-gamma production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-zeta and the generation of inhibitory pp21zeta. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell "counterstimulator" to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21zeta-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC.

Opening a window on thymic positive selection: developmental changes in the influence of cosignaling by integrins and CD28 on selection events induced by TCR engagement.

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.

Role of CD8β Domains in CD8 Coreceptor Function: Importance for MHC I Binding, Signaling, and Positive Selection of CD8 + T Cells in the Thymus

The contribution of the CD8β subunit to CD8 coreceptor function is poorly understood. We now demonstrate that the CD8β extracellular domain increases the avidity of CD8 binding to MHC I, and that the intracellular domain of CD8β enhances association with two intracellular molecules required for TCR signal transduction, Lck and LAT. By assessing CD8 + T cell differentiation in CD8β-deficient mice reconstituted with various transgenic CD8β chimeric molecules, we also demonstrate that the intracellular and extracellular domains of CD8β can contribute independently to CD8 + T cell development, but that both CD8β domains together are most efficient. Thus, this study identifies the molecular functions of the CD8β intracellular and extracellular domains and documents their contributions to CD8 + T cell development.

Autonomous induction of proliferation, JNK and NF-alphaB activation in primary resting T cells by mobilized CD28.

Induction of proliferation in primary resting T cells requires engagement of both the antigen-specific TCR and the co-stimulatory receptor CD28. Here we report that CD28 functions as an autonomous mitogenic receptor which is mobilized by TCR signaling through cytoskeletal rearrangement. Shortcutting of TCR-dependent CD28 recruitment by stimulation with monoclonal antibodies specific for mobilized CD28 results in maximum proliferation and IL-2 secretion in primary resting T cells without activation of ZAP-70, a central component of the TCR's signal transduction machinery. Engagement of mobilized CD28 fully activates the c-Jun N-terminal kinase cascade and translocation of NF-alphaB, two key targets of signal integration in co-stimulation. We propose a two-step activation model for co-stimulation in primary resting T cells in which antigen recognition recruits co-stimulatory receptors which then autonomously transduce signals promoting T cell proliferation.

NK cytokine secretion and cytotoxicity occur independently of the SLP-76 adaptor protein.

The adapter protein SLP-76 is required for T cell development and TCR signal transduction. SLP-76 is also expressed in NK cells, yet splenic populations of NK cells develop normally in SLP-76-deficient mice. We examined the effects of SLP-76 deficiency upon cellular activation through studies of NK function in SLP-76(-/-) mice. This study presents evidence that NK populations in both spleen and liver of SLP-76(-/-) mice remain intact. Natural cytotoxic responses of SLP-76(-/-) splenocytes proceed in a manner comparable to those of wild-type control splenocytes. Similar to controls, SLP-76(-/-) splenocytes exhibit enhanced survival and augmented cytotoxic capacity after in vitro culture with IL-2. IL-2-stimulated SLP-76(-/-) splenocytes also retain normal antibody-dependent cellular cytotoxicity and the ability to secrete IFN-gamma in response to IL-12 stimulation. These results indicate that, unlike events stimulated by TCR engagement, signaling cascades engaged by IL-2 and IL-12 receptors, by Fc gammaRIIIA (which mediates antibody-dependent cellular cytotoxicity), and by natural cytotoxicity-associated receptors on murine NK cells can occur independently of SLP-76.

Membrane compartmentation and the response to antigen

Antigen-receptor engagement is a complex, highly orchestrated rearrangement of signaling molecules at, and adjacent to, the plasma membrane. Recent discoveries have shown that the plasma membrane itself is differentiated into microdomains of distinct lipid constituents and that the affiliation of lipid-modified proteins with those domains has important implications for antigen-receptor function. Disruption of lipid microdomains by a variety of methods attenuates TCR signal transduction.

T cell antigen-receptor signal transduction

During the past year, major progress has been made in understanding proximal TCR signal-transduction events. Cbl has been identified as a negative regulator of kinases from the ZAP-70/Syk family. Studies on LAT, SLP-76, Itk and Vav have revealed their role in the activation of Ras and phospholipase-Cgamma1-Ca2+ signalling pathways. TCR-induced cytoskeletal changes involve signalling through SLP-76-Vav-Nck to activate effectors of the Rho-family of GTPases. Finally, glycolipid-enriched microdomains play a crucial role in T cell activation.

Cutting Edge: The Tyrosine Phosphatase SHP-1 Regulates Thymocyte Positive Selection

The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCR-transgenic mice and support a role for SHP-1 thymocyte development.

Cutting Edge: BASH, A Novel Signaling Molecule Preferentially Expressed in B Cells of the Bursa of Fabricius

Abstract The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.