RasGRP1 Transduces Low-Grade TCR Signals which Are Critical for T Cell Development, Homeostasis, and Differentiation

Abstract

Two important Ras-guanyl nucleotide exchange factors, Sos and RasGRP1, control Ras activation in thymocytes. However, the relative contribution of these two exchange factors to Ras/ERK activation and their resulting impact on positive and negative selection is unclear. We have produced two lines of RasGRP1(-/-) TCR transgenic mice to determine the effect of RasGRP1 in T cell development under conditions of defined TCR signaling. Our results demonstrate that RasGRP1 is crucial for thymocytes expressing weakly selecting TCRs whereas those that express stronger selecting TCRs are more effective at utilizing RasGRP1-independent mechanisms for ERK activation and positive selection. Analysis of RasGRP1(-/-) peripheral T cells also revealed hitherto unidentified functions of RasGRP1 in regulating T cell homeostasis and sustaining antigen-induced developmental programming.

Calcium signaling in lymphocytes

“…The RasGEF activity of RasGRP1 in the Golgi depends both on DAG and on increased intracellular Ca 2+ concentration [22]. RasGRP1 is barely expressed in thymocytes at the double-negative (CD4 − CD8 − , DN) to double-positive (CD4 + CD8 + , DP) stages [24]; instead, the calcineurin/ NFAT pathway was shown to modulate Ras activation in CD4 + CD8 + CD69 − DP thymocytes that have not yet undergone positive selection, altering the threshold for ERK activity and allowing weak TCR signals to induce positive selection [25]. B-Raf, MEK and ERK activation are all impaired in DP thymocytes from mice lacking the calcineurin regulatory subunit CnB1, suggesting that calcineurin/ NFAT-dependent transcription regulates the Raf/ Ras/ MAP kinase pathway at several levels, both upstream and downstream of Raf.…”

Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors

“…The lymphoma cells are a combination of DP and CD8 SP, as might be expected if they arose from exaggerated positive selection of the H-Y TCR which directs survival of DP thymocytes and their differentiation into the CD8 lineage. However, none of the CD8 SP cells of this mouse expressed CD69, which is induced in response to RasGRP1-dependent positive selection of the H-Y TCR (Priatel et al, 2002). The lymphoma CD8 SP cells also differed from the positively selected CD8 SP thymocytes of H-Y TCR transgenic mice by being predominantly TCR low and CD24 high .…”

“…In female mice transgenic for the MHC class I-specific H-Y TCR, a high proportion of DP thymocytes are positively selected on H-2D b and differentiate into mature CD8 SP thymocytes (Kisielow et al, 1988). The efficiency of positive selection of DP thymocytes expressing the H-Y TCR on H-2D b is attenuated in RasGRP1-deficient mice (Priatel et al, 2002) and is enhanced in AM1268 RasGRP1 transgenic mice (data not shown). Expression of the H-Y TCR markedly increased and accelerated the incidence of thymic lymphomas in female mice of the AM1268 RasGRP1 transgenic line (Figure 4, in comparison to Figure 1).…”

“…RasGRP1 is activated in response to TCR ligation Bivona et al, 2003), and transduces signals that enable TCR-mediated positive selection at the DP stage (Dower et al, 2000;Priatel et al, 2002;Layer et al, 2003) as well as enhanced survival and TCR-induced proliferation at the SP stage (Norment et al, 2003). This raised the question of whether the lymphomas in RasGRP1 transgenic mice were caused by amplified signaling downstream of TCR, leading to an exaggeration of the survival and proliferative responses which confer positive selection.…”

“…Although RasGRP1 is not required for pre-TCR-directed b selection in normal mice (Dower et al, 2000), constitutive transcription of RasGRP1 from a transgene confers partial developmental progression up to the DP stage in the absence of pre-TCR (Norment et al, 2003). The efficiency of TCR-directed positive selection of DP thymocytes is severely compromised in RasGRP1-deficient mice (Dower et al, 2000;Priatel et al, 2002;Layer et al, 2003). Conversely, RasGRP1 transgenic mice have increased numbers of CD8 SP thymocytes that have enhanced proliferative and survival responses to TCR ligation (Norment et al, 2003).…”