Kometani Kohei, Moriyama Masaki, Nakashima Yasuhiro, Katayama Yoshinori, Wang Shu-Fang, Yamasaki Sho, Saito Takashi, Hattori Masakazu, Minato Nagahiro
Department of Immunology and Cell Biology, Kyoto University, Kyoto, Japan.
Blood. 2008 Dec 1;112(12):4565-73. doi: 10.1182/blood-2008-06-164517. Epub 2008 Sep 18.
We demonstrate that lck promoter-driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of alphabeta T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting gammadelta 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-CD3epsilon antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3epsilon 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-CD3epsilon antibody, thus bypassing pre-TCR. Defective alphabeta 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.
我们证明，由lck启动子驱动的转基因SPA-1（一种Rap GTP酶激活蛋白）的条件性表达，会导致αβ T细胞在CD4/CD8双阴性（DN）阶段的发育出现严重缺陷，这是由于细胞死亡增加所致，而γδ T细胞的发育不受影响。这种效应在DN阶段具有特异性，因为由CD4启动子驱动的SPA-1表达几乎不影响T细胞发育。Rap1A17是一种显性负性Rap突变体，在存在抗CD3ε抗体和Notch配体的情况下，它在体外干扰了Rag2(-/-)胎儿胸腺细胞中双阳性（DP）细胞的产生。通过表达自寡聚化的CD3（CD8:CD3ε嵌合体）激活了DN细胞系中的Rap GTP酶，该嵌合体替代了自主前T细胞受体（TCR）信号，诱导了CD69表达和CD25下调。相反，在正常和Rag2(-/-) DN细胞中表达Rap鸟嘌呤核苷酸交换因子C3G，在没有额外抗CD3ε抗体的情况下，显著增强了Notch依赖性DP细胞的产生和扩增，从而绕过了前TCR。通过引入p53(-/-)突变，条件性SPA-1转基因小鼠中缺陷的αβ T细胞发育得以完全恢复。这些结果表明，前TCR下游的内源性Rap GTP酶在将前T细胞从p53介导的检查点反应中拯救出来方面起着至关重要的作用，从而允许Notch介导的扩增和分化。
