Requirement for CD28 co-stimulation is lower in SHP-1-deficient T cells.

This study provides biochemical and functional evidence pertaining to the role of the intracellular protein tyrosine phosphatase, SHP-1, in influencing thresholds for TCR activation. Although the loss of SHP-1 in thymocytes from motheaten mice had minimal effects on the initial rise of cytosolic Ca(2+) concentration following TCR triggering, the post-stimulation equilibrium levels of Ca(2+) were consistently elevated. In keeping with a SHP-1 effect on PLCgamma function, IP3 generation was increased in SHP-1 deficient thymocytes. Importantly, we demonstrate that loss of SHP-1 results in a relaxation of the normally stringent co-stimulatory requirements for IL-2 production. SHP-1 deficient single-positive CD4(+) thymocytes revealed a significantly enhanced capacity to produce IL-2 in response to anti-CD3 stimulation alone. In contrast, the simultaneous triggering of CD3 and CD28 was required for equivalent IL-2 production in control single-positive CD4(+) thymocytes. Furthermore, SHP-1 deficient thymocytes generated an increased and prolonged proliferative response to anti-CD3 stimulation alone. In addition, the simultaneous triggering of CD28 and CD3 resulted in equivalent proliferative responses in SHP-1-deficient and control thymocytes, suggesting that a strong co-stimulatory signal is able to override the effect of SHP-1 loss on TCR hyperresponsiveness. Collectively, these results suggest that SHP-1, rather than acting directly on TCR signaling, may indirectly raise thresholds for TCR triggering by modulating co-stimulatory signals.

Constitutive association of SHP-1 with leukocyte-associated Ig-like receptor-1 in human T cells.

The intracellular Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP-1) is a negative regulator of cell signaling and contributes to the establishment of TCR signaling thresholds in both developing and mature T lymphocytes. Although there is much functional data implicating SHP-1 as a regulator of TCR signaling, the molecular basis for SHP-1 activation in T lymphocytes is poorly defined. A modification of the yeast two-hybrid system was employed to identify in T cells phosphotyrosine-containing proteins capable of binding the SH2 domains of SHP-1. From this yeast tri-hybrid screen, the p85beta subunit of phosphatidylinositol 3-kinase and the immunoreceptor tyrosine-based inhibitory motif-containing receptors, leukocyte-associated Ig-like receptor-1 (LAIR-1) and programmed death-1 (PD-1), were identified. Coimmunoprecipitation studies demonstrated that the exclusive phosphotyrosine-containing protein associated with SHP-1 in Jurkat T cells under physiological conditions is LAIR-1. Significantly, this interaction is constitutive and was detected only in the membrane-enriched fraction of cell lysates. Ligand engagement of the SH2 domains of SHP-1 is a prerequisite to activation of the enzyme, and, consistent with an association with LAIR-1, SHP-1 was found to be constitutively active in unstimulated Jurkat T cells. Importantly, a constitutive interaction between LAIR-1 and SHP-1 was also detected in human primary T cells. These results illustrate the sustained recruitment and activation of SHP-1 at the plasma membrane of resting human T cells by an inhibitory receptor. We propose that this mechanism may exert a constitutive negative regulatory role upon T cell signaling.

TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1.

An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4-CD8- double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.