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.
