| NKG2D is a activation receptor expressed on the surface of NK, CD8+T, y8T, NKT cells, and also can expressed on the surface of few CD4+T cells that have different functional states. Our previous work found the number of CD4+NKG2D+T cells in DSS-induced colitis mouse and Crohn’s patients were increased significantly. In order to depth study the increased of CD4+NKG2D+T in DSS-induced colitis mouse and Crohn’s patients, and the function and phenotypic characteristics of CD4+NKG2D+T subsets. Finally, preliminary study the specific of the increased CD4+NKG2D+T subsets.The research was divided into two parts.Part Ⅰ:Regulatory NK1.1-CD4+NKG2D+T cells inhibited the pathogenesis of DSS-induced colitis mouseObjective:In order to clarify NK1.1-CD4+NKG2D+T cells whether involved in the pathogenesis of DSS-induced colitis mouse, so we compared regulatory NK1.1-CD4+NKG2D+T cells with Treg, NK1.1+CD4+NKG2D+T cells in the biological characteristics. And explore the function of regulatory NK1.1-CD4+NKG2D+T cells in the pathogenesis of DSS-induced colitis.Methods:In order to manufacture colitis model, we used 2.5% dextran sulphate sodium (DSS) solution feed mices. Make use of flow cytometry to detect the frequency of NK1.1-CD4+NKG2D+T, CD8+NKG2D+T, NK1.1+NKG2D+T cells in mouse spleen and intestinal tissue, and the expression of CD28, CD62L, CD44, CD25, FasL, CD223 (LAG-3), CD69, CD39, CTLA-4 on DSS-colitis mouse and Wild type. Using intracellular staining detect the secretion of IFN-γ, IL-17, IL-10, TGF-β, granzyme B, perforin within the cytoplasm of the NK1.1-CD4+NKG2D+T cell subsets and the nuclear transcription factor Foxp3, T-bet, ROR-γt. Using flow cytometry to find the expression of 2B4, NKG2A, NKp46, Ly49D, Ly49H, KLRG1 on the surface of NK1.1-CD4+NKG2D+T, NK1.1-CD4+NKG2D+T cells. Adoptive input NK1.1-CD4+NKG2D+T cells by means of tail vein injection for the purpose of observed whether NK1.1-CD4+NKG2D+T participate in the development of the disease. Immunofluorescence staining detect the expression of RAE-1 on the surface of intestinal epithelial cells and DC cells in DSS-colitis mouse. RAE-1 expression level associated with infiltration of CD4+NK1.1-NKG2D+T cells in colon. Real Time-PCR analyzed the transcriptional profiling of NK1.1-CD4+NKG2D+T, NK1.1-CD4+NKG2D+T cells. CD107a marker was detected the cytotoxic activity of CD4+NKG2D+T cells to B16-MICA cells.Results:Compared to the control group, in spleen of DSS-induced colitis mice, NK1.1-CD4+NKG2D+T cells’s frequency were increased, but CD8+NKG2D+T cells, NK1.1+NKG2D+T cells’s frequency were reduced. In contrast, in the intestinal tissues, the frequency of NK1.1-CD4+NKG2D+T cells were decreased, the frequency of CD8+NKG2D+T, NK1.1+NKG2D+T cells were increased. After the adoptive input NK1.1-CD4+NKG2D+T cells can delay the onset of colitis in mice. NK1.1-CD4+NKG2D+T cells from DSS induced colitis mice expressed FasL, and secreted TGF-β.There were no production granzyme B and peforin in the cytoplasm of NK1.1-CD4+NKG2D+T cells. Meanwhile, the NK1.1-CD4+NKG2D+T cells produced low-level of IFN-y,Il-17 and showed no cytotoxic activity. Major CD4+NKG2D+T cells were verified as the effector-memory T cells, they highly expressed CD69, CD39, CD28 and low-level expressed CD25, CD223 (LAG-3), CTLA-4. NK1.1-CD4+NKG2D+T cell was a distinctive subset from the Treg cells, no express CD25 and GITR. NK1.1-CD4+NKG2D+T cells negatively expressed the NK cell’s marker, such as 2B4, NKG2A, NKp46, Ly49D, Ly49H, KLRG1,on the contrary, NK1.1+CD4+NKG2D+T cells highly expressed 2B4, NKG2A, NKp46, Ly49D, Ly49H, KLRG1. And low-level transcription of T-bet, RORγt, Foxp3 was also observed in NK1.1-CD4+NKG2D+T cells. Finally, Real Time-PCR showed difference between NK1.1+CD4+NKG2D+T with NK1.1-CD4+NKG2D+T cells.Conclusion:There was a subet of NK1.1-CD4+NKG2D+T cells in the DSS induced colitis mice. The T cell subset secreted TGF-β and sFasL to mediate regulatory function. NK1.1-CD4+NKG2D+T cell was a distinctive subset from the Treg and NK1.1+CD4+NKG2D+T cells,and inhibiting the onset of colitis.Part II:Study of biological characteristics and the distribution of CD4+NKG2D+T cells in IBD and colon cancerObjective:To study the frequency change and function of CD4+NKG2D+T cells in peripheral blood, include Crohn’s, ulcerative colitis, colorectal cancer patients. And further evaluation of its function.Methods:Firstly, variation of NKG2D expression on CD4+T, CD8+T, NK+ cells were infused into the peripheral blood of Crohn’s, ulcerative colitis, colorectal cancer, healthy human. CD4+NKG2D+T cells were checked for their expression of a multiple cell markers including CD28, CD69, CD39. Secretions of TGF-p, IL-17and IFN-γ of CD4+NKG2D+T cells from both Crohn’s and colorectal cancer patients were measured by the flow cytometric intracellular staining method. At last, the proportion of CD56 in CD4+NKG2D+T cell weere detected.Results:Patients with Crohn’s was significantly higher expressed CD4+NKG2D+T than ulcerative colitis, but CD8+NKG2D+T, NK+NKG2D+T frequencies were no significant difference. CD4+NKG2D+T cells from Crohn’s patients secreted IFN-y, IL-17, expressed CD69, CD39. Meanwhile, the CD4+NKG2D+T cells produced low-level of TGF-βand showed low-level expression of CD28. At the same time, NKG2D expression on CD4+T cells was up-regulated in colorectal cancer versus healthy control. CD4+NKG2D+T cells from colorectal cancer patients secreted TGF-β, expressed CD69, CD39, CD28. Meanwhile, the CD4+NKG2D+T cells produced low-level of IFN-y, IL-17. The proportion of CD56 accounted for CD4+NKG2D+T cell in Crohn’s were raised compared with cancer.Conclusion:There was a subset of CD4+NKG2D+T cells in the Crohn’s and colorectal cancer patients. The T cell subset in the Crohn’s secreted IFN-y, IL-17 to mediate inflammatory function. The T cell subset in the colorectal cancer secreted TGF-β to mediate regulatory function. CD4+NKG2D+T cells were divided into CD56+CD4+NKG2D+T, and CD56-CD4+NKG2D+T cell subsets, and the two subsets mediated different functions in different diseases. |
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