Cutting Edge: Codeletion of the Ras GTPase-Activating Proteins (RasGAPs) Neurofibromin 1 and p120 RasGAP in T Cells Results in the Development of T Cell Acute Lymphoblastic Leukemia.

Ras GTPase-activating proteins (RasGAPs) inhibit signal transduction initiated through the Ras small GTP-binding protein. However, which members of the RasGAP family act as negative regulators of T cell responses is not completely understood. In this study, we investigated potential roles for the RasGAPs RASA1 and neurofibromin 1 (NF1) in T cells through the generation and analysis of T cell-specific RASA1 and NF1 double-deficient mice. In contrast to mice lacking either RasGAP alone in T cells, double-deficient mice developed T cell acute lymphoblastic leukemia/lymphoma, which originated at an early point in T cell development and was dependent on activating mutations in the Notch1 gene. These findings highlight RASA1 and NF1 as cotumor suppressors in the T cell lineage.

Blood vascular abnormalities in Rasa1(R780Q) knockin mice: implications for the pathogenesis of capillary malformation-arteriovenous malformation.

Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant blood vascular (BV) disorder characterized by CM and fast flow BV lesions. Inactivating mutations of the RASA1 gene are the cause of CM-AVM in most cases. RASA1 is a GTPase-activating protein that acts as a negative regulator of the Ras small GTP-binding protein. In addition, RASA1 performs Ras-independent functions in intracellular signal transduction. Whether CM-AVM results from loss of an ability of RASA1 to regulate Ras or loss of a Ras-independent function of RASA1 is unknown. To address this, we generated Rasa1 knockin mice with an R780Q point mutation that abrogates RASA1 catalytic activity specifically. Homozygous Rasa1(R780Q/R780Q) mice showed the same severe BV abnormalities as Rasa1-null mice and died midgestation. This finding indicates that BV abnormalities in CM-AVM develop as a result of loss of an ability of RASA1 to control Ras activation and not loss of a Ras-independent function of this molecule. More important, findings indicate that inhibition of Ras signaling is likely to represent an effective means of therapy for this disease.

The Ras GTPase-activating protein neurofibromin 1 promotes the positive selection of thymocytes.

TCR-mediated activation of the Ras signaling pathway is critical for T cell development in the thymus and function in the periphery. However, which members of a family of Ras GTPase-activating proteins (RasGAPs) negatively regulate Ras activation in T cells is unknown. In this study we examined a potential function for the neurofibromin 1 (NF1) RasGAP in the T cell lineage with the use of T cell-specific NF1-deficient mice. Surprisingly, on an MHC class I-restricted TCR transgenic background, NF1 was found to promote thymocyte positive selection. By contrast, NF1 neither promoted nor inhibited the negative selection of thymocytes. In the periphery, NF1 was found to be necessary for the maintenance of normal numbers of naïve CD4⁺ and CD8⁺ T cells but was dispensable as a regulator of TCR-induced Ras activation, cytokine synthesis, proliferation and differentiation and death. These findings point to a novel unexpected role for NF1 in T cell development as well as a regulator of T cell homeostasis.

IL-4 acts as a potent stimulator of IFN-γ expression in CD8+ T cells through STAT6-dependent and independent induction of Eomesodermin and T-bet.

CD8+ T cell synthesis of IFN-γ is an important component of the CD8+ T cell immune response. In short-term cultures of murine pan-T cells, we found that IL-4 was the principal cytokine responsible for driving IFN-γ synthesis by CD3/CD28-activated CD8+ T cells. IL-4 was able to induce low levels of IFN-γ mRNA in CD8+ T cells even in the absence of CD3/CD28 engagement, although concomitant CD3/CD28 stimulation was necessary for IFN-γ secretion. IL-4 induction of IFN-γ was explained by its ability to induce Eomesodermin and T-bet transcription factors whose expression was further increased by CD3/CD28. Expression of Eomesodermin, T-bet and IFN-γ induced by IL-4 was partially dependent upon activation of MAPK and PI3K but independent of the canonical IL-4-activated transcription factor, STAT6. In contrast, expression of IFN-γ induced by IL-4/CD3/CD28 stimulation showed additional dependency upon STAT6 which functions to increase expression of Eomesodermin specifically. These novel findings point to a function for IL-4 as a direct regulator of IFN-γ expression in CD8+ T cells and reveal the molecular mechanisms involved.

The T-cell-specific adapter protein family: TSAd, ALX, and SH2D4A/SH2D4B.

Adapter proteins play key roles in intracellular signal transduction through complex formation with catalytically active signaling molecules. In T lymphocytes, the role of several different types of adapter proteins in T-cell antigen receptor signal transduction is well established. An exception to this is the family of T-cell-specific adapter (TSAd) proteins comprising of TSAd, adapter protein of unknown function (ALX), SH2D4A, and SH2D4B. Only recently has the function of these adapters in T-cell signal transduction been explored. Here, we discuss advances in our understanding of the role of this family of adapter proteins in T cells. Their function as regulators of signal transduction in other cell types is also discussed.

Genetic analysis of SH2D4A, a novel adapter protein related to T cell-specific adapter and adapter protein in lymphocytes of unknown function, reveals a redundant function in T cells.

T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.