Intracellular Signals Controlling Lymphocyte Development

细胞内TCR信号的调控一直是一个有趣的课题，因为它在T细胞的发育和功能中起着重要的作用。有证据表明，细胞内信号转导的失调也是许多疾病的原因，包括自身免疫和免疫缺陷。我们以前证明，衔接分子Cbl和Cbl-b参与组织TCR信号在胸腺细胞和外周T细胞，这两个分子的缺陷导致增强的CD 4+胸腺细胞的发展或CD 28独立的高反应性的成熟T细胞，分别在体内。我们现在表明，在T谱系细胞中Cbl和Cbl-b的破坏导致更高的死亡率，主要是由于突变小鼠中自身免疫性动脉炎的自发发展。我们发现Cbl/Cbl-b双突变（dKO）T细胞对抗原刺激具有高应答性。然而，生化分析表明，主要的TCR信号通路，如酪氨酸磷酸化，Ca++动员，MAP激酶和Vav激活没有显着增强，尽管在细胞核中观察到转录因子NF κ B，AP-1，NFAT的水平显着增加通过TCR刺激后。进一步的分析表明，突变T细胞在抗原刺激后未能下调其TCR，导致活化细胞中持续的信号传导。在不存在通过TCR的刺激的情况下，TCR内吞作用在dKO细胞中是正常的。然而，在TCR刺激后，内化的TCR未能被转运到溶酶体，表明内化的TCR被阻断蛋白分选进入溶酶体区室。这些结果表明，Cbl和Cbl-b可能通过调节胞吞过程中的细胞内膜分选来控制TCR内化，从而控制TCR信号传导。此外，dKO小鼠中动脉炎的发展表明抗原刺激后TCR下调的生理重要性，并且未能这样做将导致自身免疫性疾病的发展。Grb 2是一种衔接分子，被认为连接受体酪氨酸激酶信号传导和MAP激酶途径。我们决定利用基因打靶技术研究它在TCR信号传导和细胞发育中的功能。我们已经产生了Grb 2条件ko小鼠品系，其中Grb 2在T谱系细胞中特异性失活。我们发现Grb 2缺乏阻断了胸腺的阳性选择和阴性选择。该缺陷减弱TCR诱导的酪氨酸激酶级联。在Grb 2 ko T细胞中，p38 MAP激酶减弱，而MAP激酶Erk 1/2和JNK不受影响。有趣的是，Grb 2突变显著减弱了抗CD 3或抗CD 3和CD 4刺激的Lck酪氨酸激酶活化，首次表明Grb 2是Lck的正调节剂。Grb 2与胸腺细胞中的Lck组成性相关，这表明这种关联可能指导Lck细胞内定位或在T细胞发育过程中与其他调节因子的关联。

Intracellular regulation of TCR signaling has been a fascinating subject because it palys an important role for T cell development and function. Evidences indicated that dysregulation of intracellular signaling is also responsible for many diseases including autoimmunity and immune deficiency. We previously demonstrated that adaptor molecule Cbl and Cbl-b are involved in organizing TCR signals in thymocytes and peripheral T cells, and deficiency of these two molecules leads to an enhanced CD4+ thymocyte development or CD28 independent-hyperresponsiveness of mature T cells, respectively, in vivo. We now show that disruption of both Cbl and Cbl-b in T lineage cells lead to higher mortality primarily due to the spontaneous development of autoimmune arteritis in the mutant mice. We found that the Cbl/Cbl-b double mutant (dKO) T cells were hyperresponsive to antigen stimulation. However, the biochemistry analysis indicated that major TCR signaling pathways, such as tyrosine-phosphorylation, Ca++ mobilization, MAP kinase and Vav activation, were not significantly enhanced, despite that the dramatically increased levels of transcription factors NFkB, AP-1, NFAT were observed in the cell nucleus after stimulation through the TCR. Further analysis indicated that the mutant T cells failed to downmodulate their TCR after antigen stimulation, resulting in a sustained signaling in the activated cells. TCR endocytosis is normal in the dKO cells in the absence of stimulation through TCR. However, internalized TCR failed to be transported to the lysosomes after TCR stimulation, indicating a blocked protein sorting of internalized TCR into the lysosome compartment. These results demonstrate a novel mechanis that Cbl and Cbl-b may control TCR internalization, thus TCR signaling through regulating the intracellular membrane sorting during endocytosis. Furthermore, development of arteritis in the dKO mice indicate the physiological importance of TCR downmodulation after antigen stimulation, and failed to do so will lead to the development of autoimmune diseases. Grb2 is an adaptor molecule that has been thought to link receptor tyrosine kinase signaling and MAP kinase pathways. We decided to study it function in TCR signaling and cell development using gene targeting technology. We have generated an Grb2 conditional ko mouse strain in which the Grb2 was inactivated specifically in T lineage cells. We found that Grb2 deficiency blocked both thymic positive and negative selection. The deficiency attenuated TCR induced tyrosine kinase cascades. p38 MAP kinase was weakened whereas MAP kinases Erk1/2 and JNK were not affected in Grb2 ko T cells. Interestingly, the Grb2 mutation significantly attenuated Lck tyrosine kinase activation up anti-CD3 or anti-CD3 and CD4 stimulation, indicating for the first time that Grb2 is a positive regulator for Lck. Grb2 is constitutively associated with Lck in thymocyte, suggesting that this association may guide Lck intracellular locolization or association with other regulators during T cell development.