Essential role of Rap signal in pre-TCR–mediated -selection checkpoint in 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 T-cell development at the CD4/CD8 doublenegative (DN) stage due to enhanced cell death without affecting 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 dominantnegative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2 / fetal thymocytes in vitro in the presence of anti-CD3 antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3 chimera), which substituted autonomous pre–T-cell receptor (TCR) signal, inducing CD69 expression and CD25 downregulation. 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-CD3 antibody, thus bypassing pre-TCR. Defective 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 preT cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation. (Blood. 2008;112:4565-4573)