Vav1 transduces T cell receptor signals to the activation of the Ras/ERK pathway via LAT, Sos, and RasGRP1

Vav1 is a signaling protein required for both positive and negative selection of CD4(+)CD8(+) double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase Cgamma1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase Cgamma1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells.     

The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.     

CD8+CD45RA+CCR7+FOXP3+ T Cells with Immunosuppressive Properties: A Novel Subset of Inducible Human Regulatory T Cells

CD8 T cells stimulated with a suboptimal dose of anti-CD3 Abs (100 pg/ml) in the presence of IL-15 retain a naive phenotype with expression of CD45RA, CD28, CD27, and CCR7 but acquire new functions and differentiate into immunosuppressive T cells. CD8(+)CCR7(+) regulatory T cells (Tregs) express FOXP3 and prevent CD4 T cells from responding to TCR stimulation and entering the cell cycle. Naive CD4 T cells are more susceptible to inhibition than memory cells. The suppressive activity of CD8(+)CCR7(+) Tregs is not mediated by IL-10, TGF-β, CTLA-4, CCL4, or adenosine and relies on interference with very early steps of the TCR signaling cascade. Specifically, CD8(+)CCR7(+) Tregs prevent TCR-induced phosphorylation of ZAP70 and dampen the rise of intracellular calcium in CD4 T cells. The inducibility of CD8(+)CCR7(+) Tregs is correlated with the age of the individual with PBLs of donors older than 60 y yielding low numbers of FOXP3(low) CD8 Tregs. Loss of CD8(+)CCR7(+) Tregs in the elderly host may be of relevance in the aging immune system as immunosenescence is associated with a state of chronic smoldering inflammation.     

Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4(+)CD8(+) thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4(+)CD8(+) thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.     

Distinct signaling pathways regulate TLR2 co-stimulatory function in human T cells

Toll-like receptor 2 (TLR2) serves as a co-stimulatory receptor for human T cells by enhancing T cell receptor (TCR)-induced cytokine production and proliferation. However, it is unknown where signals from the TCR and TLR2 converge to enhance T cell activation. To address this gap, we examined changes in TCR-induced signaling following concurrent TLR2 activation in human T cells. Both proximal TCR-mediated signaling and early NFκB activation were not enhanced by TCR andTLR2 co-activation, potentially due to the association of TLR2 with TLR10. Instead, TLR2 co-induction did augment Akt and Erk1/Erk2 activation in human T cells. These findings demonstrate that TLR2 activates distinct signaling pathways in human T cells and suggest that alterations in expression of TLR2 co-receptors may contribute to aberrant T cell responses.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

Expression of CD45 lacking the catalytic protein tyrosine phosphatase domain modulates Lck phosphorylation and T cell activation

The function of the second protein tyrosine phosphatase domain (D2) in two-domain protein tyrosine phosphatases (PTP) is not well understood. In CD45, D2 can interact with the catalytic domain (D1) and stabilize its activity. Although D2 itself has no detectable catalytic activity, it can bind substrate and may influence the substrate specificity of CD45. To further explore the function of D2 in T cells, a full-length construct of CD45 lacking the D1 catalytic domain (CD45RABC-D2) was expressed in CD45+ and CD45- Jurkat T cells. In CD45- Jurkat T cells, CD45RABC-D2 associated with Lck but, unlike its active counterpart CD45RABC, did not restore the induction of tyrosine phosphorylation or CD69 expression upon T cell receptor (TCR) stimulation. Expression of CD45RABC-D2 in CD45+ Jurkat T cells resulted in its association with Lck, increased the phosphorylation state of Lck, and reduced T cell activation. TCR-induced tyrosine phosphorylation was delayed, and although MAPK phosphorylation and CD69 expression were not significantly affected, the calcium signal and IL2 production were severely reduced. This indicates that the non-catalytic domains of CD45 can interact with Lck in T cells. CD45RABC-D2 acts as a dominant negative resulting in an increase in Lck phosphorylation and a preferential loss of the calcium signaling pathway, but not the MAPK pathway, upon TCR signaling. This finding suggests that, in addition to their established roles in the initiation of TCR signaling, CD45 and Lck may also influence the type of TCR signal generated.     

CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.     

The cGMP/protein kinase G pathway contributes to dihydropyridine-sensitive calcium response and cytokine production in TH2 lymphocytes

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.     

Differential regulation of primary and memory CD8 T cell immune responses by diacylglycerol kinases

The manipulation of signals downstream of the TCR can have profound consequences for T cell development, function, and homeostasis. Diacylglycerol (DAG) produced after TCR stimulation functions as a secondary messenger and mediates the signaling to Ras-MEK-Erk and NF-κB pathways in T cells. DAG kinases (DGKs) convert DAG into phosphatidic acid, resulting in termination of DAG signaling. In this study, we demonstrate that DAG metabolism by DGKs can serve a crucial function in viral clearance upon lymphocytic choriomeningitis virus infection. Ag-specific CD8(+) T cells from DGKα(-/-) and DGKζ(-/-) mice show enhanced expansion and increased cytokine production after lymphocytic choriomeningitis virus infection, yet DGK-deficient memory CD8(+) T cells exhibit impaired expansion after rechallenge. Thus, DGK activity plays opposing roles in the expansion of CD8(+) T cells during the primary and memory phases of the immune response, whereas consistently inhibiting antiviral cytokine production.