The Effects Of T-cell Specific Rack1 Knockout On Gamma Delta T Cells And Nkt Cells

Objective: To investigate the development of γδT and NKT cells and the percentages and numbers of peripheral γδT and NKT cells in mice with Tcell specific RACK1 knockout.In addition,we explore how the above effects might switch the responses of the mice to infection and inflammatory injury.Method: With Cre-Lox P system,we obtained mice in which RACK1 gene was specifically deleted in T cells(KO mice,control littermates were called WT mice).We monitored the mortality and the bacterial burden in the liver and spleen of mice challenged with Listeria monocytogenes.Some surface markers were detected using flow cytometry,Such as CD3,CD4,CD8,γδTCR,Vγ1,Vγ2,etc.We also detected changes in the expression level of intracellular cytokine IL-17 in WT and KO mice after infection.Using the chimeric mice and flow cytometry analysis,we detected the percentages of CD45.1+CD45.2+ WT mice-derived γδT cells and CD45.1-CD45.2+ KO mice-derived γδT cells in CD45.1+CD45.2-myeloablative mice.The acute liver injury model of mice was induced by Con A,and the mortality,liver H&E staining and serum transaminase expression levels were measured in WT and KO mice.Using flow cytometry to detect certain surface markers(Such as CD3,NK1.1,CD1dtetramer)as well as intracellular cytokines TNF-α and IFN-γ,the differences in these aspects in WT and KO mice were analyzed.Results: 1.After infection with Listeria monocytogenes,KO mice showed decreased mortality and bacterial burden in the liver and spleen,as compared with WT mice.The percentages and absolute numbers of peripheral γδT cells in KO mice increased significantly compared to WT mice.γδT cell subsets Vγ1,Vγ2 and secreted IL-17 all significantly increased.2.Data from chimeric mice demonstrated that there was no significant increase in KO micederived γδT cells.3.CD3+CD1d-tetramer+ i NKT development was impaired in KO mice.In Con A-induced acute liver injury model,the mortality rate of KO mice was much lower than that of WT mice,H&E staining showed that the liver injury in KO mice was very limited.Determination of AST and ALT levels in serum also demonstrated that liver injury in WT mice was more pronounced.4.Surface marker staining revealed reduced percentages and numbers of conventional T cells in the liver of KO mice after Con A treatment,whereas those of CD3+NK1.1+ NKT cells as well as CD3-NK1.1+ NK cells did not change.Intracellular cytokine staining demonstrated that the proportion and absolute numbers of IFN-γ+ T cells,NKT cells,and NK cells also decreased significantly,but the expression of TNF-α did not change significantly.5.After purification of mouse splenic conventional T cells and stimulation with Con A in vitro,it was found that IFN-γ expression was not defective.Conclusion: Our study suggests that although the proportion and absolute numbers of conventional peripheral CD8+ T cells in KO mice were greatly reduced,the mice showed enhanced ability to eliminate bacteria.RACK1 deficiency in T cells affects the percentages and numbers of peripheral γδT cells.Data from chimeric mice demonstrate that the increase of peripheral γδT cell numbers in KO mice is indeed the result of environmental external defects.Specific deletion of RACK1 in T cells not only leads to a significant reduction in the percentages and absolute numbers of conventional peripheral T cells,but also results in impaired CD3+CD1d-tetramer+ i NKT development.After the induction of Con A,RACK1 deficiency in T cells plays a protective role against liver injury to certain extent.In conventional peripheral T cells,RACK1 does not directly affect IFN-γ expression.The decrease in IFN-γ expression in after Con A injection may be due to changes in the microenvironment within the liver.