Maillard Ivan, Tu LiLi, Sambandam Arivazhagan, Yashiro-Ohtani Yumi, Millholland John, Keeshan Karen, Shestova Olga, Xu Lanwei, Bhandoola Avinash, Pear Warren S
Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA 19104, USA.
J Exp Med. 2006 Oct 2;203(10):2239-45. doi: 10.1084/jem.20061020. Epub 2006 Sep 11.
Genetic inactivation of Notch signaling in CD4(-)CD8(-) double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4(+)CD8(+) double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein-tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRbeta expression. DNMAML attenuated the pre-TCR-associated increase in cell size and CD27 expression. TCRbeta or TCRalphabeta transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML(-) or DNMAML(+) DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML(+) DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the beta-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J-MAML complex.
先前的研究表明，CD4(-)CD8(-)双阴性（DN）胸腺细胞中Notch信号的基因失活会损害T细胞受体（TCR）基因重排，并导致小鼠CD4(+)CD8(+)双阳性（DP）胸腺细胞发育出现部分阻滞。相比之下，体外培养表明，Notch对于DP胸腺细胞的产生是绝对必需的，且独立于前TCR的表达和活性。为了明确Notch和前TCR各自的作用，我们在体内用绿色荧光蛋白标记的显性负性Mastermind样蛋白1（DNMAML）抑制Notch介导的转录激活，这使我们能够追踪无法进行Notch信号传导的单细胞。DN细胞中DNMAML的表达导致DP胸腺细胞的产生减少，但细胞内TCRβ表达仅适度降低。DNMAML减弱了前TCR相关的细胞大小增加和CD27表达。TCRβ或TCRαβ转基因未能挽救与DNMAML相关的缺陷。胸腺内注射DNMAML(-)或DNMAML(+) DN胸腺细胞显示出完全的DN/DP转变阻滞，只有经历晚期Notch失活的细胞才能产生DNMAML(+) DP胸腺细胞。这些发现表明，体内β选择检查点期间对Notch的需求是绝对的，且独立于前TCR，并且它取决于Notch通过CSL/RBP-J-MAML复合物进行的转录激活。
