Biological Activity Of CD8⁺T Cell And Its Effects On The Development Of DSS-Enteritis In MiR-15a/16^-/- Mice

miR-16 is one of the most tumor-suppressing miRNAs and is down-regulated in many types of tumors,including chronic lymphocytic leukemia,multiple myeloma,prestate cancer,pancreatic cancer,and ovarian cancer.In order to study the effect of miR-16 on anti-tumor immune function of the body,miR-15a/16^-/-gene knockout mice were introduced from the Jackson laboratory in the United States in the early stage of this research group.The phenotype analysis of the immune cells showed that there was no significant change in the function of macrophages from the spleen of miR-15a/16^-/-mice under the physiological condition.However,the frequency of macrophage was significantly decreased and the activity was changed in H22 hepatocellular carcinoma of these miR-15a/16^-/-mice.In this study,the phenotype and function of CD8⁺T cells were further measured of miR-15a/16^-/-mice,and the function of CD8⁺T cells in the mice was evaluated by DSS-induced mouse enteritis model.Our study can be divided into three parts.Part I.Analysis of the activity of CD8⁺T cells in miR-15a/16^-/-miceObjective:To determine whether the function of CD8⁺T cells in miR-15a/16^-/-mice has changed.Methods:Firstly,execute miR-15a/16^-/-mice?KO?and C57BL/6 mice?WT?of 8-10 weeks old in the physiological state.Secondly,isolate the spleen cells from these mice,the frequency of CD8⁺NKG2D⁺,CD8⁺CD44⁺,CD8⁺CD44⁺CD62L-,CD8⁺CD25⁺CD69⁺T cells in the spleen were detected by flow cytometry.Moreover,after the stimulation of PMA/Ionomycin,CD8⁺T cells were detected to secrete IFN-y by flow cytometry,and co-incubated with MC-38 cells,CD107a was labeled which was used to detect the killing activity of CD8⁺T cells by flow cytometry.Results:Compared with C57BL/6 mice?WT?,the frequency of CD8⁺NKG2D⁺T cells in the spleen of miR-15a/16^-/-mice?KO?was increased.Compared with C57BL/6 mice?WT?,the toxicity of CD8⁺T cells in miR-15a/16^-/-mice?KO?was increased.Compared with C57BL/6 mice?WT?,the frequency of CD8⁺CD25⁺CD69⁺T cells and CD8⁺CD44⁺T cells from the spleen cells of the experimental group in the miR-15a/16^-/-mice was increased,and the frequency of CD8⁺CD44⁺CD62L-T cells from the spleen cells of the experimental group KO mice was increased significantly compared with the control group WT.Conclusions:The knockdown of miR-15a/16 gene upregulated the frequency of CD8⁺T cells in the spleen of mice and enhanced their biological activity.Part ?.The effects of CD8⁺T cells in the miR-15a/16^-/-mice on DSS-induced enteritis.Objective:To determine whether CD8⁺T cells of the miR-15a/16^-/-mice are involved in the pathogenesis of DSS-enteritis mice.Methods:In the first place,the mice were fed with 2.5%DSS of miR-15a/16^-/-mice?KO?and C57BL/6 mice?WT?to induce enteritis,and to observe the disease activity index in mice,such as weight,blood stool,diarrhea.The frequency of CD8⁺NKG2D⁺T cells in the spleen and intestines of miR-15a/16^-/-mice?KO?and C57BL/6 mice?WT?by flow cytometry.The intestines of mice were taken out and to observe the intestinal inflammation.The intestinal tissues of mice were prepared by using H&E staining.To observe the pathological changes of the intestinal tract under the microscope.To detect the infiltration degree of CD8⁺T cells by immunohistochemistry.Results:Compared with C57BL/6 mice?WT?,we found the weight of miR-15a/16^-/-mice?KO?was decreased significantly,and the body mass index was decreased significantly,and the mouse disease activity index?DAI?was increased,and the mice had a severe hematopoietic condition,and the intestinal tract was significantly shortened.Compared with C57BL/6 mice?WT?,there were a large number of neutrophils,lymphocytes and other inflammatory cells in the intestinal tissue of miR-15a/16^-/-mice?KO?,and we also found that many CD8⁺T cells in the intestinal tissues of miR-15a/16^-/-mice?KO?mice.Moreover,compared with C57BL/6 mice?WT?,we found that the frequency of CD8⁺NKG2D⁺T cells from the spleen and intestines were increased significantly in the miR-15a/16^-/-mice?KO?mice,and the frequency of CD8⁺IFN-?⁺ cells from the spleen in the miR-15a/16^-/-mice?KO?mice was increased.Conclusions:miR-15a/16 gene knockout increased the incidence of enteritis in mice induced by DSS,and the intestinal tissues of miR-15a/16^-/-enteritis mice were infiltrated with a large number of CD8⁺T cells,and high expression of activated molecular NKG2D.Part ?.Effects of miR-16 on the biological activity of CD8⁺T cells in mice.Objective:To preliminary study on whether the activity of CD8⁺T cells in mice was regulated by miR-16.Methods:In the first place,execute C57BL/6 mice of 8-10 weeks old and prepare single cell suspensions.In the second place,CD8⁺T cells were sorted by magnetic beads and infected with miR-16 lentivirus or antago-miR-16 lentivirus.After 72 hours,the cells were collected and the frequency of CD8⁺NKG2D⁺T cells was detected by flow cytometry.At the same time,we should stimulate CD8⁺T cells with PMA/Ionomycin,the frequency of the secretion of IFN-y by CD8⁺T cells was detected by flow cytometry.Results:In vitro infection of CD8⁺T cells by lentivirus with miR-16 precursor sequence showed that the expression of GFP⁺CD8⁺NKG2D⁺T cells was down-regulated.And infecting with miR-16 antisense nucleotide sequence,the expression of GFP⁺CD8⁺NKG2D⁺T cells was up-regulated.Compared with the blank control group,the function of GFP⁺CD8⁺T cells which secreting IFN-y was weakened after miR-16 was overexpressed,and the function of GFP⁺CD8⁺T cells which secreting IFN-y was enhanced after knock-downing of miR-16.Conclusions:miR-16 may affect the function of CD8⁺T cells by regulating the expression of NKG2D.