Identification And Distribution Of Immunoregulatory Nk1.1~-CD4~+ NKG2D~+ T Cells

Our group made transgenic mice before.We found that splenic NK1.1-CD4+NKG2D+cells frequency was enhanced in the transgenic mice,and we also found that NK1.1-CD4+NKG2D+cells can secrete TGF-P to play an immune regulation.In addition,we preliminary found that splenic NK1.1-CD4+NKG2D+cells frequency was enhanced in mice treated by DSS.In order to clarify whether the increased splenic NK1.1-CD4+NKG2D+T cells in mice treated by DSS are similar to pCD86:RAE-1? transgenic mice,that to play an immunoregulatory activity,we further analyzed the frequency and phenotypic changes of NK1.1-CD4+NKG2D+T cells in mice treated by DSS,and we also identified whether these cells played an immunomodulatory activity and we go to explore its molecular mechanisms.And then we want to observe the proliferation of NK1.1-CD4+NKG2D+T cells in vitro and observe its distribution in pCD86:RAE-1? transgenic mice.Our study can be divided into two parts.Part ?.Identification of the immunoregulatory NK1.1-CD4+NKG2D+T cellsObjective:To identify the immune regulatory functions about NK1.1-CD4+ NKG2D+ T cells.Methods:Firstly,we used 2.5%DSS to feed C57BL/6 mice in order to prepare colitis mice model.We detected the frequency of CD4+NKG2D+ T cells in intestinal and spleen of enteritis mice and wild type mice,respectively,by flow cytometry.We detected the expression of CD3,??T and NK1.1 in CD4+NKG2D+ T cells in spleen of colitis mice and wild type mice.We examined the expression of TGF-? in NK1.1-CD4+NKG2D+T cells.Secondly,we transferred NK1.1-CD4+NKG2D+T cells sorted by flow cytometry,into DSS-induced mice through the tail vein.We transferred NK1.1-CD4+NKG2D+T cells incubated with TGF-? antibody into another group of DSS mice to observe the incidence and weight changes of mice.Then we removed the mouse's intestinal tract,and observed the intestinal inflammation of mice by our naked eye.And we made the intestinal tissue into paraffin sectionsto.We observed the pathological changes of the intestinal tract in mice under a microscope by HE staining.Finally,we used gene chip technology to compare the differential expression of transcriptional mRNA and long non-coding RNA(LncRNA)between NK1.1-CD4+ NKG2D+ T cells and NK1.1+ CD4+ NKG2D+ T cells.Results:Compared with wild-type mice,the frequency of the CD4+ NKG2D+ T cells in the DSS-induced colitis mice was increased in the spleen and decreased in the intestine.The increased in CD4+NKG2D+T cells in the spleen mainly was NK1.1-cells,and most of them are CD3+ NK1.1-??-CD4+NKG2D+ cells.TGF-? is highly expressed in NK1.1-CD4+NKG2D+T cells.Compared with the normal group,DSS group,DSS + NK1.1-CD4+NKG2D+T cell group,DSS + NK1.1-CD4+NKG2D+T cell + TGF-? antibody blocking group mice all had different degrees of disease.And the DSS+NK1.1-CD4+ NKG2D+ T cells group was the lightest incidence,the smallest decline in weight and the lowest disease activity index.And the DSS +NK1.1-CD4+NKG2D+T cell group was the slightest intestinal damage by observed the intestinal tract of the mice.After HE staining,the pathological changes of the intestinal tract of DSS +NK1.1-CD4+NKG2D+T cells in the microscope were also the slightest.While at the same time,in the mice transferred NKI1.1-CD4+NKG2D+ T cells that incubated with TGF-p antibody group,these phenomena had obvious disappearance.We found that a total of 161 mRNA genes and 741 IncRNA genes(threshold = 2)were altered between the two subsets by gene chips.Conclusions:The increased of the CD4+NKG2D+T cells in the spleen of mice induced by DSS were mainly NK1.1-cells.NK1.1-CD4+NKG2D+T cells and NK1.1+CD4+NKG2D+T cells are two different subpopulations of cells.The increased NK1.1-CD4+NKG2D+T cells in the spleen of mice induced by DSS are regulatory T cells.NK1.1-CD4+NKG2D+T cells can inhibit the onset of DSS-induced enteritis in mice,and the mechanism is mainly through the production of TGF-? to play an immune regulatory function.Part ?.The proliferation and organizational distribution about NK1.1-CD4+NKG2D+T cellsObjective:To investigate the proliferation characteristics of NKl.1-CD4+NKG2D+T cells and their organizational distribution in pCD86:RAE-1? transgenic mice.Methods:Firstly,we sorted these NK1.1-CD4+NKG2D+T cells by flow cytometry.And then stimulated NK1.1-CD4+NKG2D+ T cells with IL-10,TGF-p,IL-6 and IL-15 alone or in combination with sRAE protein.and after that we observed the proliferation of NK1.1-CD4+NKG2D+T cells by MTS/PMS and CFSE.We detected the frequency of NK1.1-CD4+NKG2D+T cells in spleen,intestine,liver,lung and peripheral blood of pCD86:RAE-1? transgenic mice by flow cytometry.At last we detected the expression of chemokine receptor CCR9,CCR6,CCR8,CCR4 and CCR7 in NK1.1-CD4+ NKG2D+ T cells by flow cytometry.Results:Compared with the control group,recombinant sRAE protein could promote the proliferation of NK1.1-CD4+NKG2D+T cells.IL-6 and IL-15 can promote the proliferation of NK1.1-CD4+NKG2D+T cells alone,and IL-10,TGF-p and IL-15 in combination with sRAE protein also can promote the proliferation of NK1.1-CD4+NKG2D+ T cells.Compared with wild-type control mice,the frequency of NK1.1-CD4+NKG2D+ T cells were significantly increased in the spleen,intestine,liver,peripheral blood,but in the lung,the frequency was no significant difference.Furthermore,the expression of CCR9,CCR8 and CCR4 on NK1.1-CD4+NKG2D+ T cells cells were significantly increased in the transgenic mice,while the expression of CCR6 and CCR7 was not significantly different from that of wild-type mice.Conclusions:Recombinant sRAE protein can promote the proliferation of NK1.1-CD4+NKG2D+T cells.And IL-6 and IL-15 can promote the proliferation of NK1.1-CD4+NKG2D+T cells.IL-10,TGF-??IL-15 combined with recombinant sRAE protein can also promote the proliferation of NK1.1-CD4+NKG2D+T cells.The frequency of NK1.1-CD4+NKG2D+T cells were significantly increased in the spleen,intestinal,liver,peripheral blood,and these phenomenon can suggest that these cells can play an immunomodulatory role in the body.