Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alone does not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMP was generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation, β-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) was recruited to lipid rafts which down-regulated cAMP levels. Whereas inhibition of protein kinase A increased TCR-induced immune responses, inhibition of PDE4 blunted T cell cytokine production. Conversely, overexpression of either PDE4 or β-arrestin augmented TCR/CD28-stimulated cytokine production. We show here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipid rafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus.

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced ζ-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.

BackgroundTCR and CXCR4-mediated signaling appears to be reciprocally regulated pathways. TCR activation dampens the chemotactic response towards the CXCR4 ligand CXCL12, while T cells exposed to CXCL12 are less prone to subsequent TCR-activation. The heterotrimeric G proteins Gαq and Gαi2 have been implicated in CXCR4-signaling and we have recently also reported the possible involvement of Gαq in TCR-dependent activation of Lck (Ngai et al., Eur. J. Immunol., 2008, 38: 32083218). Here we examined the role of Gαq in migration and TCR activation.ResultsPre-treatment of T cells with CXCL12 led to significantly reduced Lck Y394 phosphorylation upon TCR triggering indicating heterologous desensitization. We show that knockdown of Gαq significantly enhanced basal migration in T cells and reduced CXCL12-induced SHP-1 phosphorylation whereas Gαi2 knockdown inhibited CXCL12-induced migration.ConclusionOur data suggest that Gαi2 confers migration signals in the presence of CXCL12 whereas Gαq exerts a tonic inhibition on both basal and stimulated migrational responses. This is compatible with the notion that the level of Gαq activation contributes to determining the commitment of the T cell either to migration or activation through the TCR.

Here, we examined the functional involvement of heterotrimeric G-proteins in TCR-induced immune responses. TCR/CD3 crosslinking resulted in activation of both Gaq and Gas, but not Gai-2. Targeting of Gas, Gai-2 and Gaq using siRNA demonstrated a specific role of Gaq in TCR signaling. Jurkat TAg T cells with Gaq knockdown displayed reduced activation of Lck and LAT phosphorylation, but paradoxically showed sustained ERK1/2 phosphorylation and increased NFAT-AP-1-reporter activity implicating Gaq in the negative control of downstream signaling and IL-2-promoter activity. Primary T cells isolated from Gaq-deficient mice had a similar TCR signaling response with reduced proximal LAT phosphorylation, sustained ERK1/2 phosphorylation and augmented immune responses including increased secretion of IL-2, IL-5, IL-12 and TNF-a. The effects on NFAT-AP-1-reporter activity were sensitive to the Src family kinase inhibitor PP2 and were reversed by transient expression of constitutively active Lck. Furthermore, expression of constitutively active Gaq Q209L elevated Lck activity and Zap-70 phosphorylation. Together these data argue for a role of Gaq in the fine-tuning of proximal TCR signals at the level of Lck and a negative regulatory role of Gaq in transcriptional activation of cytokine responses. Key words: Signal transduction . T cells . TCR . Transgenic/knockout mice IntroductionThe TCR/CD3 complex is composed of a ligand-binding TCR-ab heterodimer and signal transducing dimers of CD3eg, CD3ed and zz that upon interaction with its MHC-peptide ligand initiate an activation process that, although not fully understood, probably involves both receptor clustering and conformational changes of the cytoplasmic part of the CD3 complex [1,2]. Considering the physiological significance of correct T-cell activation, this signal interpretation probably involves complex molecular integration proximal to the TCR. A key early event is the mobilization and activation of the protein tyrosine kinase Lck, which in turn phosphorylates ITAM within the CD3 component that can recruit ZAP-70 through its two SH2-domains [3]. ZAP-70 is then phosphorylated by Lck on Y493 leading to full catalytic activation [4]. One important substrate of activated ZAP-70 is the transmembrane adaptor LAT, which acts as a scaffold to assemble signaling complexes essential for T-cell activation [5].The 3208TCR necessary for the development and activation of T cells. In addition, several lines of evidence suggest that initiation of modulating pathways is controlled by Lck. Both TCR desensitization by internalization and induction of apoptosis through the mitochondrial death pathway are regulated by Lck [7,8]. Furthermore, the activation of Lck by partial antagonist and the ability of Lck to negatively regulate superantigen-induced T-cell activation support a dual role of Lck [9,10]. Interestingly, acute elimination of Lck in either Jurkat or primary T cells using RNA interference results in sustained and elevated ERK1/2 phosphorylation and augmented NFAT-reporter ac...

Despite the loss of proximal TCR‐dependent signaling events, downstream T cell responses are paradoxically augmented in T cells with siRNA‐mediated Lck knockdown (Methi et al., J. Immunol. 2005. 175: 7398–7406). This indicates that alternative Lck‐independent pathways of T cell activation exist or that low levels of Lck elicit other signals than normal T cell activation. Here we report the recruitment of Grb2‐SOS1 to CD3ζ of the TCR complex after prolonged anti‐CD3 (OKT3) stimulation in T cells with Lck knockdown. Grb2 bound to incompletely phosphorylated ITAM1 with the pY‐Y configuration in a solid‐phase assay, but was excluded by ZAP‐70 in the doubly phosphorylated pY‐pY conformation. Ras and ERK1/2 activation was augmented after prolonged stimulation in T cells with Lck knockdown compared to control, leading to increased activation of the proximal IL‐2 promoter (NFAT‐AP‐1). Finally, the phosphorylation of Ras‐GAP was strongly suppressed in Lck knockdown cells, indicating that a Ras negative feedback mechanism is dependent on Lck.