The molecular mechanism of TCR repertoire generation and coreceptor expression

为了研究正选择(PS)过程产生TCR谱系的分子机制，我们分析了调节TCR和CD4、CD8共受体表达的相关分子。在基因操作小鼠的实验中，我们证明：(1)在没有PS的情况下，在DP期随机增加的TCR的表达在没有PS的情况下被下调，但在没有合适的TCR介导的信号的情况下，TCR的密度保持不变。这一结论与以前的假设不一致，即DP细胞上TCR的表达本来就很低，但当DP细胞收到TCR介导的PS信号时，TCR的表达就会上调，而在没有TCR信号的情况下，TCR的表达没有增加。然而，我们的发现有助于解决先前提出的假设中提出的问题，即低TCR可以识别低亲和力的MHC分子，这可能会积极地选择DP细胞，导致DP细胞激活以获得高TCR表达。(2)DP细胞表面CD45与TCR表达下调密切相关，并被TCR介导的信号所阻断。(3)在体内条件下检测胸腺细胞胞内底物的酪氨酸磷酸化，证明ZAP-70在PS过程中对CD8SP的产生是高度磷酸化的，而对CD4SP细胞系不是。这一结果与最近报道的一例ZAP-70异常患者的观察结果一致。在该患者中，CD8SP细胞没有发育，但发现了CD4SP细胞，但数量很少。这些发现提示DP胸腺细胞中CD4和CD8的下调是一种不对称的机制。(4)CD4成熟T细胞通过非MHC相关调节功能进一步发育为Th1和Th2细胞。

To study molecular mechanism of positive selection (PS) process for generating TCR repertoire, we analyzed related molecules for regulating expression of TCR and CD4 and_CD8 co-receptor. In use of gene manipulating mice, we proved followings.(1) TCR expression which increased randomly at DP stage was down-regulated without PS by TCR-MHC interaction but maintained its density without an appropriate TCR mediated signal. This conclusion is not consistent with the previous hypothesis that TCR expression on DP cells is originally low but it is induced to raise up when DP cells receive TCR mediated PS signal while TCR expression is not increased without TCR signal. However, our findings is helpful to resolve the question raised in the previous hypothesis how low TCR can recognize MHC molecules with low avidity which may positively select DP cells, resulting in DP cells activation for high TCR expression. (2) CD45 on DP cells was closely involved in down-regulation of TCR expression, which was overcome by TCR mediated signal. (3) We explored to detect tyrosine phosphorylation of intracellular substrates of thymocytes in the in vivo condition, and demonstrated that ZAP-70 is highly phosphorylated in PS process for CD8SP generation but not for CD4SP cell lineage. This result is consistent with the recent observation reported in a human patient possessing with abnormal ZAP-70, in which CD8SP cells did not develop but CD4SP cells were found though their number was small. These findings suggest an asymmetric mechanism for down regulation of CD4 and CD8 in DP thymocytes. (4) CD4 mature T cells are induced further functional development into Th1 and Th2 cells by non-MHC related regulation.