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Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP-CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4(+) and CD8(+) T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4(+) and CD8(+) primary human T cells upon T cell activation. This was also true in mouse CD4(+) T cells, but less striking in mouse CD8(+) T cells. PTP-PEST reintroduced into Jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-kappaB elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells.

A SNP in the gene PTPN22 is associated with type 1 diabetes, rheumatoid arthritis, lupus, Graves thyroiditis, Addison disease and other autoimmune disorders. T cells from carriers of the predisposing allele produce less interleukin-2 upon TCR stimulation, and the encoded phosphatase has higher catalytic activity and is a more potent negative regulator of T lymphocyte activation. We conclude that the autoimmune-predisposing allele is a gain-of-function mutant.

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 zeta-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.

The Csk tyrosine kinase negatively regulates the Src family kinases Lck and Fyn in T cells. Engagement of the T-cell antigen receptor results in a removal of Csk from the lipid raft-associated transmembrane protein PAG/Cbp. Instead, Csk becomes associated with an approximately 72-kDa tyrosine-phosphorylated protein, which we identify here as G3BP, a phosphoprotein reported to bind the SH3 domain of Ras GTPase-activating protein. G3BP reduced the ability of Csk to phosphorylate Lck at Y505 by decreasing the amount of Csk in lipid rafts. As a consequence, G3BP augmented T-cell activation as measured by interleukin-2 gene activation. Conversely, elimination of endogenous G3BP by RNA interference increased Lck Y505 phosphorylation and reduced TCR signaling. In antigen-specific T cells, endogenous G3BP moved into a intracellular location adjacent to the immune synapse, but deeper inside the cell, upon antigen recognition. Csk colocalization with G3BP occurred in this "parasynaptic" location. We conclude that G3BP is a new player in T-cell-antigen receptor signaling and acts to reduce the amount of Csk in the immune synapse.

C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h. No alterations in surface expression of CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4-5-fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2-3-fold higher Lck activity. In Jurkat cells, basal levels of zeta chain phosphorylation were elevated, and spontaneous NFAT-AP-1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and zeta chains compared to control. TCR-induced activation of NFAT-AP-1 and TCR/CD28-stimulated IL-2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR-stimulated responses.

Background: Lipopolysaccharide (LPS) is the main initiator of the early signaling events leading to sepsis caused by Gram-negative bacteria. Late stages of sepsis are associated with impairments of T lymphocyte function, a condition associated with nosocomial infection and poor outcome. The molecular basis for septic immunosuppression is not fully understood., Material/methods: Human whole blood was incubated ex vivo with purified LPS. Cytokine responses and T cell proliferation were assessed, and the role of cyclic 3',5'-adenosine monophosphate (cAMP) in T cell suppression by LPS was studied using a cAMP-antagonist (Rp-8-Br-cAMPS)., Results: Adding LPS (0.01 to 10 microg/ml) to human blood ex vivo caused a release of prostaglandin E2 (PGE2) in a concentration- and time-dependent manner, with maximal levels of PGE2 obtained with 10 microg LPS per ml blood after 10 hours of incubation. Adding PGE2-concentrations ranging from 0.03 to 10 microM to purified T cells completely abrogated T cell activation and proliferative response, which was largely reversed by adding Rp-8-Br-cAMPS. Peripheral blood mononuclear cells (PBMC) and T cells harvested from whole blood cultured in the presence of LPS ex vivo showed attenuated proliferative response (30-70%) (purified T cells and PBMC) and reduced IL-2 production (85%) upon T cell receptor (TCR)/CD3 activation with anti-CD3. The proliferation in T cells and PBMC was in part restored by Rp-8-Br-cAMPS., Conclusions: The human whole blood model of LPS-mediated T lymphocyte suppression described in this paper is time and cost efficient, as well as easy to use.

Protein kinase A (PKA) is a serine/threonine kinase that regulates a number of cellular processes important for immune activation and control. Modulation of signal transduction by PKA is a complex and diverse process, and differential isozyme expression, holoenzyme composition and subcellular localization contribute specificity to the PKA signalling pathway. In lymphocytes, phosphorylation by PKA has been demonstrated to regulate antigen receptor-induced signalling both by altering protein-protein interactions and by changing the enzymatic activity of target proteins. PKA substrates involved in immune activation include transcription factors, members of the MAP kinase pathway and phospholipases. The ability of PKA type I to regulate activation of signalling components important for formation of the immunological synapse, demonstrates that the cAMP signalling pathway can directly modulate proximal events in lymphocyte activation. Furthermore, the recent discovery that PKA regulates Src kinases through modulation of Csk, supports the notion that PKA is involved in the fine-tuning of immune receptor signalling in lipid rafts.