T cell regulation of p62(dok) (Dok1) association with Crk-L.

In addition to engagement of the T cell receptor-CD3 complex, T lymphocytes can be activated by a variety of cell surface molecules including the approximately 50-kDa surface receptor CD2. While the majority of biochemical signaling elements are triggered by either CD2 or TcR-CD3 receptors, a small number of proteins are engaged by only one receptor. Recently, p62(dok) (Dok1), a member of the Dok family of adapter molecules, has been reported to be activated by CD2 and not by CD3 engagement. Here we have examined the role of p62(dok) in CD2-dependent signaling in Jurkat T cells. As previously reported, we find that ligation of the CD2 molecule by mitogenic pairs of anti-CD2 mAbs led to phosphorylation of p62(dok). While CD2-induced p62(dok) tyrosine phosphorylation was independent of both the p36/38 membrane adapter protein linker of activated T cells (LAT) and the ZAP70/Syk family of kinases, it was dependent upon the Src family of kinases including Lck and Fyn. We find further that CD2 engagement induced the association of tyrosine-phosphorylated p62(dok) to Crk-L. The CD2-dependent association of p62(dok) to Crk-L was independent of expression of the ZAP70/Syk family of kinases. Of note, while T cell receptor-CD3 engagement did not induce either p62(dok) phosphorylation or Crk-L association in Jurkat T cells, it did inhibit CD2-dependent p62(dok)-Crk-L complexes; this TcR-CD3-mediated regulation was dependent upon ZAP70 kinase activity. Our data suggest that phosphorylation of p62(dok) and its interaction with other signaling proteins may depend upon integrated signals emanating from the CD2 receptor, utilizing a ZAP70/LAT-independent pathway, and the TcR-CD3 receptor, which is ZAP70/Syk-dependent.     

Evidence that Llck-mediated phosphorylation of p56dok and p62dok may play a role in CD2 signaling

The Lck tyrosine kinase is involved in signaling by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other downstream protein-tyrosine kinases are activated upon stimulation of these receptors, it is difficult to assign which tyrosine-phosphorylated proteins represent bona fide Lck substrates and which are phosphorylated by other tyrosine kinases. We have developed a system in which Lck can be activated independently of TCR/CD3. We have shown that activation of an epidermal growth factor receptor/Lck chimera leads to the specific phosphorylation of Ras GTPase-activating protein (RasGAP) and two RasGAP-associated proteins, p56(dok) and p62(dok). Activation of the chimeric protein correlates with an increase in cellular Ca(2+) in the absence of ZAP-70 and phospholipase Cgamma1 phosphorylation. Furthermore, we have found that p62(dok) co-immunoprecipitates with the activated epidermal growth factor receptor/LckF505 and that phosphorylated Dok proteins bind to the Src homology 2 domain of Lck in vitro. In addition, we have shown that activation via the CD2 but not the TCR/CD3 receptor leads to the phosphorylation of p56(dok) and p62(dok). Using JCaM1.6 cells, we have demonstrated that Lck is required for CD2-mediated phosphorylation of Dok proteins. We propose that phosphorylation and Src homology 2-mediated association of p56(dok) and p62(dok) with Lck play a selective function in accessory receptor signal transduction mechanisms.     

CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases

The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4(+) T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR zeta-chain phosphorylation and subsequent zeta-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56(lck) with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56(lck) in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.     

Interaction of SAP-1, a transmembrane-type protein-tyrosine phosphatase,with the tyrosine kinase Lck. Roles in regulation of T cell function

SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function.     

Critical role of TNF receptor type-2 (p75) as a costimulator for IL-2 induction and T cell survival: a functional link to CD28

CD28 provides important signals that lower the threshold of T cell activation, augment the production of IL-2, and promote T cell survival. The recent identification of a second family of costimulatory molecules within the TNFR family has reshaped the "two-signal" model of T cell activation. In this study the role of p75 as a T cell costimulatory molecule in controlling cell fate during TCR/CD28-mediated stimulation was examined. We found that p75-deficient T cells possess a profound defect in IL-2 production in response to TCR/CD28-mediated stimulation. Examination of key signaling intermediates revealed that TCR proximal events such as global tyrosine phosphorylation and ZAP70 phosphorylation, as well as downstream MAPK cascades are unperturbed in p75-deficient T cells. In contrast, p75 is nonredundantly coupled to sustained AKT activity and NF-kappaB activation in response to TCR/CD28-mediated stimulation. Moreover, p75-deficient T cells possess a defect in survival during the early phase of T cell activation that is correlated with a striking defect in Bcl-x(L) expression. These data indicate discrete effects of p75 on the intracellular signaling milieu during T cell activation, and reveal the synergistic requirement of TCR, CD28, and p75 toward optimal IL-2 induction and T cell survival. We propose that p75 acts as one of the earliest of the identified costimulatory members of the TNFR family, and is functionally linked to CD28 for initiating and determining T cell fate during activation.     

Ligation of the T cell receptor complex results in phosphorylation of Smad2 in T lymphocytes

TGF-beta modulates immune responses by regulating T cell function. The Smad family of proteins has been recently shown to transduce signals for the TGF-beta superfamily and Smad2 mediates TGF-beta signaling. Here, we showed that TGF-beta phosphorylated Smad2 and induced interaction between Smad2 and Smad4 in primary T cells and the Jurkat T cell line. Interestingly, ligation of the T cell receptor (TCR)/CD3 complex with anti-CD3 mAb also phosphorylated Smad2, but failed to induce interaction between Smad2 and Smad4 in the Jurkat T cell line. Phosphorylation of Smad2 via the TCR/CD3 complex was not abrogated by treatment with neutralizing antibody against TGF-beta. Furthermore, PD98059, a MEK inhibitor, suppressed Smad2 phosphorylation by stimulation with anti-CD3 mAb in Jurkat T cell line. These findings indicated that not only TGF-beta but also stimulation via the TCR/CD3 complex phosphorylated Smad2 through mitogen-activated protein (MAP) kinase cascades, suggesting that Smad2 may function in both TGF-beta- and TCR/CD3 complex-mediated signaling pathways in T cells.     

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.     

CD28 is not required for c-Jun N-terminal kinase activation in T cells.

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.     

Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells

The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca²⁺ imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca²⁺ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca²⁺ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses.     

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.     

Negative regulation of interleukin-2 and p38 mitogen-activated protein kinase during T-cell activation by the adaptor ALX

Activation of na?ve T cells requires synergistic signals produced by the T-cell receptor (TCR) and by CD28. We previously identified the novel adaptor ALX, which, upon overexpression in Jurkat T cells, inhibited activation of the interleukin-2 (IL-2) promoter by TCR/CD28, suggesting that it is a negative regulator of T-cell activation. To further understand the physiological role of ALX, ALX-deficient mice were generated. Purified T cells from ALX-deficient mice demonstrated increased IL-2 production, CD25 expression, and proliferation in response to TCR/CD28 stimulation. Enhanced IL-2 production and proliferation were also observed when ALX-deficient mice were primed in vivo with ovalbumin-complete Freund's adjuvant and then restimulated ex vivo. Consistent with our initial overexpression studies, these data demonstrate that ALX is a negative regulator of T-cell activation. While TCR/CD28-mediated activations of phosphotyrosine induction, extracellular signal-regulated kinase 1/2, Jun N-terminal protein kinase, IkappaB kinase alpha/beta, and Akt were unaltered, constitutive activation of p38 mitogen-activated protein kinase and its upstream regulators MKK3/6 were observed for ALX-deficient splenocytes. The phenotype of ALX-deficient mice resembled the phenotype of those deficient in the transmembrane adaptor LAX, and an association between ALX and LAX proteins was demonstrated. These results suggest that ALX, in association with LAX, negatively regulates T-cell activation through inhibition of p38.     

The CD3-gamma delta epsilon transducing module mediates CD38-induced protein-tyrosine kinase and mitogen-activated protein kinase activation in Jurkat T cells.

We have examined the ability of the CD3-gamma delta epsilon and CD3-zeta signaling modules of the T cell receptor (TCR) to couple CD38 to intracellular signaling pathways. The results demonstrated that in TCR+ T cells that express the whole set of CD3 subunits CD38 ligation led to complete tyrosine phosphorylation of both CD3-zeta and CD3-epsilon polypeptide chains. In contrast, in TCR+ cells with a defective CD3-zeta association CD38 engagement caused tyrosine phosphorylation of CD3-epsilon but not of CD3-zeta. Despite these differences, in both cell types CD38 ligation resulted in protein-tyrosine kinase and mitogen-activated protein kinase activation. However, in cells expressing chimerical CD25-zeta or CD25-epsilon receptors or in a TCR-beta- Jurkat T cell line, CD38 ligation did not result in tyrosine phosphorylation of the chimeric receptors, or CD3 subunits, or protein-tyrosine kinase or mitogen-activated protein kinase activation. In summary, these results support a model in which CD38 transduces activating signals inside the cell by means of CD3-epsilon and CD3-zeta tyrosine phosphorylation. Moreover, these data identify the CD3-gamma delta epsilon signaling module as a necessary and sufficient component of the TCR/CD3 complex involved in T cell activation through CD38.     

Selective regulation of TCR signaling pathways by the CD45 protein tyrosine phosphatase during thymocyte development

In CD45-deficient animals, there is a severe defect in thymocyte-positive selection, resulting in an absence of mature T cells and the accumulation of thymocytes at the DP stage of development. However, the signaling defect(s) responsible for the block in development of mature single-positive T cells is not well characterized. Previous studies have found that early signal transduction events in CD45-deficient cell lines and thymocytes are markedly diminished following stimulation with anti-CD3. Nevertheless, there are also situations in which T cell activation and TCR signaling events can be induced in the absence of CD45. For example, CD45-independent TCR signaling can be recovered upon simultaneous Ab cross-linking of CD3 and CD4 compared with cross-linking of CD3 alone. These data suggest that CD45 may differentially regulate TCR signaling events depending on the nature of the signal and/or on the differentiation state of the cell. In the current study, we have assessed the role of CD45 in regulating primary thymocyte activation following physiologic stimulation with peptide. Unlike CD3-mediated stimulation, peptide stimulation of CD45-deficient thymocytes induces diminished, but readily detectable TCR-mediated signaling events, such as phosphorylation of TCR-associated zeta, ZAP70, linker for activation of T cells, and Akt, and increased intracellular calcium concentration. In contrast, phosphorylation of ERK, which is essential for positive selection, is more severely affected in the absence of CD45. These data suggest that CD45 has a selective role in regulating different aspects of T cell activation.