The Effects Of RACK1 Conditional Knock Out On Mice T Cells

Objective:To investigate the effects of RACK1 on T cell development, survival, activation, and proliferation in mice.Method:Using the Cre-LoxP system to get the mice with T cell specific RACK1 depletion, using FACS to detect some cell surface markers, e. g. CD4, CD8, CD62L, CD44 and nuclear protein Foxp3 in peripheral lymphoid organs and thymus, using BrdU and FACS to detect the proliferation of cells, using Annexin V and FACS to detect the apoptosis of cells, using the chimeric mice and FACS to detect the percentages of CD45.1+CD45.2+ WT mice-derived T cells and CD45.1-CD45.2+ KO mice-derived T cells in CD45.1+CD45.2-myeloablative mice, using western blot to detect the level of intra-cellular autophagy marker protein LC3BⅡ and pro-apoptosis protein Bim.Results:l.The peripheral CD4 and CD8 T cells in KO mice were decreased to different extent as compared to their counterparts in WT mice. Data from chimeric mice indicate that reduced peripheral T cells upon RACKl deficiency came from cell-intrinsic defects. However, RACK1 deficiency didn’t affect T cell development in the thymus. Moreover, the generation of Tregs in the thymus was not affected upon RACK1 deficiency.2. RACK1-deficient peripheral T cells didn’t show aberrantly enhanced basal activation, as compared to their RACK1-sufficient counterparts. However, RACK1-deficient peripheral T cells exhibited distinct expression pattern of CD44 and CD62L, as compared to their RACK1-sufficient counterparts,8 days after listeria monocytogenes infection. These data suggest that RACK1 affect the T cell activation in some circumstances.3. RACK1-deficient CD4 T cells in chimeric mice exhibited impaired proliferation as compared to their RACK1-sufficient counterparts. However, CD8 T cells didn’t show the same phenomenon.4. Less mitochondrion clearance combined with less LC3B II protein and more pro-apoptosis Bim protein were observed in peripheral T cells from KO mice, indicating an impaired autophagy. Consistently, more severe apoptosis was observed in RACK1-deficient T cells.Conclusion:RACK1 does not affect the development of thymocytes. RACK1 affects T cell-activation and -proliferation under certain circumstances, although its clear roles remain to be explored. RACK1 depletion in T cells leads to impaired autophagy and autophagy-dependent mitochondrion clearance. Consequently, RACK1-deficient peripheral T cells undergo enhanced apoptosis and thereby show reduced numbers.