Researching Themis's Function With Listeria Monocytogenes Animal Model And Bone Marrow Reconstitution Experiment

T cell is an important subset of cells in the acquired immune system,fighting the infection of pathogenic microorganisms,removing autologous cancerous cells and maintaining immune steady-state.T cells are mature in the thymus and play a role in peripheral immune organs,but the regulating mechanism of T cell development and T cell anti-infection immunity remains unclear.It is reported in recent years that Themis is specifically expressed in T cells,and it was found that Themis was important in T cell development.The germline absence of Themis greatly affects the thymocyte development.Although the great progress has been made in the follow-up researches on Themis's function in T cell development,there are many questions to be solved.The molecular mechanism of Themis in T cell development is still studied in depth,and there exists nothing about its role in peripheral T cells,especially in anti-infection immunity.Therefore,clarifying the function and molecular mechanism of Themis in T cells will contribute to providing a new immune intervention target for clinic.Here,to study the function of Themis in anti-listeria infection in CD4+T cell,we used Listeria monocytogenes to infect the control mice and mice Themis-specifically knockout in peripheral T cells(Themisfl/fl dlck Cre).Cre begins to be expressed after the positive selection in T cell development in dlck Cre mice,so that the effect of Themis deficiency on thymocyte development is avoided.We sacrificed the mice at the 7th day post-infection and analyzed the splenic CD4+T cell.The results showed that Themis deficiency did not affect the activation of peripheral CD4+T cells and the production of cytokines in anti-listeria infection.It suggested that Themis did not affect fighting Listeria infection mediated by CD4+T cells.Moreover,to make clear the mechanism of Themis in thymocyte development and T cell function,and search proteins binding directly to and interacting with Themis,we plan to build the over-expressing Themis-3×FLAG mouse.However,before making the mice of the genotyping,we firstly identified whether the role of Themis-3 × FLAG is consistent with that of Themis WT.Here,we performed the bone marrow reconstitution experiment to identify the function of Themis-3×FLAG.The results showed that,compared with hematopoietic stem cells of Themis-/-mice transduced with empty plasmid,whether over-expressing Themis WT or Themis-3×FLAG could rescue the deficiency of T cell development caused by the deletion of Themis,but in over-expressing Themis-3 xFLAG mice,the percentage of CD4+T cells of spleen and lymph nodes and splenic CD8+T cells was less than that of over-expressing Themis WT mice.This suggested that 3 xFLAG tag may have a partial effect on Themis's function,so the further experiments are needed to be carried out to identify its function.