Essential role of Rap signal in pre-TCR-mediated Β-selection checkpoint in άΒ T-cell development

We demonstrate that lck promoter-driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of αβ T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting γδ T-cell development. The effect was specific to the DN stage, because CD4 promoter-driven SPA-1 expression hardly affected T-cell development. Rap1A17, a dominant-negative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2^-/- fetal thymocytes in vitro in the presence of anti-CD3ε antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3ε chimera), which substituted autonomous pre-T-cell receptor (TCR) signal, inducing CD69 expression and CD25 down-regulation. Reciprocally, expression of C3G, a Rap guanine nucleotide exchange factor, in both normal and Rag2^-/- DN cells markedly enhanced Notch-dependent generation and expansion of DP cells without additional anti-CD3ε antibody, thus bypassing pre-TCR. Defective aβ T-cell development in the conditional SPA-1-transgenic mice was restored completely by introducing a p53^-/- mutation. These results suggest that endogenous Rap GTPases downstream of pre-TCR play an essential role in rescuing pre-T cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation.