Function Of The PLZF Gene In Early Development And Self-renewal Of T Cells In Mice

We found that the total number of thymocytes and early T cell progenitors?ETPs?in PLZF deficient mice were lower than those in wild type mice,but the decrease of thymocytes in PLZF deficient mice was not cell-intrinsic.Through PLZF-EGFP reporter mice and newborn mouse thymus kidney transplantation models,we found that PLZF is highly expressed in DN1?Lineage^-CD44⁺CD25^-?cells of thymus grafts in Rag2/?c^-/-recipient mice,and the loss of PLZF in donor cells can significantly affect the proportion of donor cells in DN1 cells of thymus grafts.To sum up,PLZF gene may not be necessary for the normal differentiation and development of T cells,but it may be involved in maintaining the self-renewal of T cell early progenitor cells.The purpose of this study is to explore the function of PLZF gene in the early development and self-renewal of mouse T cells,which mainly includes two parts:first,whether PLZF gene promotes the production of mouse hematopoietic stem cells;second,whether the deletion of PLZF gene is cell-intrinsic to the reduction of mouse thymocytes.The research contents are summarized as follows:1.In this study,wild-type and transgenic reporter mice for PLZF gene?PEG?were used as research materials.The results of gating analysis of hematopoietic stem cells?LSK,Lineage^-Sca-1⁺c-Kit⁺?by flow cytometry showed that LSK cells were divided into two groups:HSC(LSK Flt3^lo)and LMMP(LSK Flt3^hi).HSC cells have totipotency to develop into various lineages of blood cells,while LMMP cells are considered to be the precursor of T cells.The HSC cells of PEG mice express high levels of GFP,and the GFP levels of their LMMP cells are similar to those of C57BL/6 mice after they migrate to the thymus and initiate the developmental process of T cells.This indicates that PLZF gene is expressed in HSC cells of bone marrow and then silenced in LMMP cells.Based on the experimental results of bone marrow and thymocytes of PEG mice,we believe that PLZF is expressed in HSC.2.In this study,PLZF wild-type gene and deficient mice(PLZF^-/-)were used as research materials to make it clear that thymocytopenia in PLZF-deficient mice was not cell-intrinsic.The bone marrow cells of PLZF^+/+?CD45.1⁺?and PLZF^-/-?CD45.2⁺?mice were separated by immunomagnetic beads,and the enriched Sca-1⁺bone marrow cells were mixed at 1:1 as donor cells.The donor cells were injected into the recipient mice(Rag2/?c^-/-)which had been irradiated with sublethal dose in advance.Two months later,the thymus was taken for flow cytometry analysis.Rag2 and?c double gene deficiency makes T,B and NK cells of recipient mice can not develop normally and can only be derived from exogenous donor cells.The results showed that the proportion of thymocytes derived from PLZF^+/+and PLZF^-/-in the thymocytes of recipient mice after bone marrow transplantation was about the same,and both of them could develop normal T cells in the thymus.In addition,the spleen of recipient mice was taken for analysis at 2 and 6 months after transplantation,and it was found that the proportion of PLZF^+/+?CD45.1⁺?-derived cells in spleen B cells and T cells remained basically constant at about 50%,indicating that PLZF^-/-?CD45.2⁺?cells were not at a disadvantage in the competition.These results suggest that the decrease of thymocytes in PLZF deficient mice is not cell-intrinsic.3.The thymus of newborn C57BL/6 or PEG mice was removed,transplanted into the renal capsule of recipient mouse Rag2/?c^-/-,and one month later,the thymus graft was taken for analysis.We used flow cytometry to perform gate analysis on CD4^-CD8^-TCR?^-?TN?thymocytes differentiated DN1 cells?CD44⁺CD25^-TN?into donor cells??c⁺?according to the expression level of?c,and receptor cells??c^-?.By performing GFP fluorescence intensity analysis on donor cells derived from PEG mice,we found that PLZF was highly expressed in DN1 cells of thymic grafts,even higher than positive control iNKT cells.4.The thymus of newborn PLZF^+/+or PLZF^-/-mice was removed and transplanted under the renal capsule of recipient mice Rag2/?c^-.One month later,thymus grafts were taken for analysis.TN thymocytes were analyzed by flow cytometry and DN1 cells?CD44⁺CD25^-TN?were divided into donor cells??c⁺?and receptor cells??c^-?according to the expression level of?c.The results show that PLZF gene may not be necessary for the normal differentiation and development of T cells,but may be involved in maintaining the self-renewal of T cell early progenitor cells.