The Effect Of PD-1/PD-L1 On CD4~+T Cells In Primary Immune Thrombocytopenia

Objective:CD4+T cells are associated with the onset of primary immune thrombocytopenia,PD-1 and PD-L1 costimulatory molecules provide a second signal for T cell activation.Through studying the expression of PD-1 and PD-L1 on CD4+T cells in peripheral blood of newly diagnosed patients with primary immune thrombocytopenia,After the PD-1/PD-L1 pathway was blocked and activated after cell culture in vitro,the changes of CD4+T cell-related cytokines were observed,and the effects of PD-1/PD-L1on CD4+T cell subsets in newly diagnosed primary immune thrombocytopenia were comprehensively analyzed.Methods:1)Forty newly diagnosed patients with primary immune thrombocytopenia were collected as the case group and 30 healthy subjects as the control group.The levels of CD4~+T cell-related cytokines in peripheral blood serum of the two groups were detected by ELISA for comparative analysis.ELISA was used to detect the changes of CD4~+T cell-related cytokines in peripheral serum of patients with primary immune thrombocytopenia treated with high dose dexamethasone;2)40 newly diagnosed patients with primary immune thrombocytopenia were collected as case group and 30 healthy subjects as control group.The expression of PD-1 on CD3~+CD4~+T cells and PD-L1 on HLA-DR~+CD11c~+DC~+cells in peripheral blood mononuclear cells of two groups of subjects were detected by flow cytometry.The levels of sPD-1 and sPD-L1 in the peripheral blood serum of the two groups of subjects were detected by ELISA and compared;3)40 newly diagnosed patients with primary immune thrombocytopenia were collected as the case group and 30 healthy subjects as the control group.Peripheral serum mononuclear cells were extracted and cultured.Anti-PD-1 antibody was added to block PD-1/PD-L signal pathway in vitro and sPD-L1 protein was added to stimulate PD-1/PD-L signal pathway in vitro.The changes of CD4~+T cell-related cytokines in the supernatant of the subjects were detected by ELISA.Results:1)IFN-?in peripheral serum of newly diagnosed patients with primary immune thrombocytopenia was(316.50±101.00)pg/ml before treatment,which was higher than that of healthy controlgroup(249.70±81.00)pg/ml(P<0.05).IL-4 was(292.22±89.03)pg/ml,lower than that of the healthy control group(361.48±110.20)pg/ml(P<0.05).The difference between the two groups was statistically significant.The level of TGF-?in the peripheral serum of newly diagnosed patients with primary immune thrombocytopenia before treatment was(2545.44±661.53)pg/ml,lower than that of healthy control group(3051.69±794.30)pg/ml(P<0.05),and the level of IL-17 was(10.89±2.92)pg/ml,higher than that of healthy control group(8.26±2.87)pg/ml(P<0.05);2)Comparing CD4~+T cell-related cytokine levels before and after treatment for newly diagnosed patients with primary immune thrombocytopenia treated with high dose dexamethasone:IFN-?was(316.01±49.00)pg/ml before treatment,and decreased to(264.27±97.62)pg/ml after treatment(P<0.05);IL-4 was(287.00±41.64)pg/ml before treatment and increased to(315.52±54.90)pg/ml after treatment(P<0.05).The level of TGF-?was(2439.41±654.08)pg/ml before treatment and(2962.53±594.23)pg/ml after treatment(P<0.05).The level of IL-17 was(10.85±3.00)pg/ml before treatment and decreased to(7.30±1.08)pg/ml after treatment(P<0.05);3)The percentage of PD-1~+CD3~+CD4~+T cells in newly diagnosed patients with primary immune thrombocytopenia was(26.79±8.91)%higher than that in healthy controls(12.06±2.84)%,the difference was statistically significant(t=8.715,P<0.05);The percentage of PD-L1~+HLA-DR~+CD11c~+DC cells(12.75±1.86)%was higher than that of healthy controls(4.90±0.80)%,the difference was statistically significant(t=21.65,P<0.05);4)The difference between sPD-1(109.96±24.41)pg/ml in peripheral serum of newly diagnosed patients with primary immune thrombocytopenia and that of healthy control group(86.05±16.07)pg/ml is statistically significant.sPD-L1 in ITP patients(60.69±10.57)pg/ml is higher than that of healthy control group(55.39±12.20)pg/ml,but the difference is not statistically significant(P=0.056);5)After extracting peripheral serum mononuclear cells for in vitro cell culture,only phosphate buffer saline(PBS)culture and CD3+CD28+PHA stimulation culture are added respectively for 48 hours,the changes of CD4~+T cytokines are observed.The concentrations of pro-inflammatory factors IFN-?(5.49±1.84)ng/ml and IL-17(8.18±1.16)ng/ml in cell culture supernatant extracted from ITP patients in PBS group were higher than those in healthy control group(2.34±0.81)ng/ml and(3.22±0.82)ng/ml,respectively,with statistically significant differences.The concentrations of anti-inflammatory factors IL-4(12.12±2.57)ng/ml and TGF-?(124.49±27.26)ng/ml were lower than those of healthy control group(14.39±1.69)ng/ml and(180.42±59.42)ng/ml,respectively,with statistically significant differences;6)The concentrations of pro-inflammatory factors IFN-?(6.44±1.87)ng/ml and IL-17(10.04±2.32)ng/ml in the supernatants of peripheral blood mononuclear cells extracted from ITP patients in CD3+CD28+PHA group cultured in vitro are higher than those of healthy control group(3.31±1.23)ng/mll and(5.59±1.09)ng/ml respectively.The difference was statistically significant.The concentrations of anti-inflammatory factors IL-4(15.81±4.64)ng/ml and TGF-?(143.91±46.88)ng/ml were lower than those of healthy control group(21.29±6.30)ng/ml and(209.74±52.29)ng/ml,respectively.The difference was statistically significant;7)IFN-?(14.04±2.01)ng/ml level in the cell culture supernatant of ITP group was significantly higher than that of the former(6.44±1.87)ng/ml/ml after anti-PD-1 antibody blocked the passage,and the difference was statistically significant.IL-4 was(15.81±4.64)ng/ml and(14.09±3.84)ng/ml before and after blocking respectively(P=0.076),TGF-?was(143.91±46.88)ng/ml and(152.13±39.93)ng/ml(P=0.401)before and after blocking respectively,IL-17 was(10.04±2.32)ng/ml and(10.87±1.65)ng/ml(P=0.068),before and after blocking respectively,the changes of IL-4,TGF-?,IL-17 levels before and after blocking were not statistically significant;8)After activating PD-1/PD-L1 pathway with sPD-L1 in cultured cells of ITP group,The changes of cytokines secreted by CD4~+T cells in culture cell supernatants before and after activation pathway were compared:IFN-?levels were(6.44±1.87)ng/ml/ml and(3.25±0.48)ng/ml/ml,IL-4 levels were(15.81±4.64)ng/ml and(47.62±9.00)ng/mll,respectively.The levels of TGF-?were(143.91±46.88)ng/ml and(512.65±175.68)ng/mll respectively,and the levels of IL-17 were(10.04±2.32)ng/ml and(6.28±2.25)ng/ml respectively.The differences before and after activation were statistically significant.Conclusion:1)The newly diagnosed patients with primary immune thrombocytopenia present abnormalities of CD4~+T cell subsets,showing that the cytokines secreted by Th1 and Th17 cells are dominant,the cytokines secreted by Th2and Treg cells are lower than that of the healthy control group,the cytokines secreted by Th1 and Th17 cells decrease,the cytokines secreted by Th2 and Treg cells increase,and the abnormalities of CD4~+T cell subsets are improved in patients effectively treated with high dose dexamethasone;2)PD-1 on CD4~+T cells and PD-L1 on DC cells are highly expressed in newly diagnosed patients with primary immune thrombocytopenia.The level of sPD-1 in peripheral serum of newly diagnosed patients with primary immune thrombocytopenia was significantly higher than that of healthy control group.The level of sPD-L1 was not significantly different from that of the healthy control group;3)In vitro experiment,after anti-PD-1 antibody blocked PD-1/PD-L1 pathway,the IFN-?level in PBMCs culture supernatant of ITP patients with high level was higher,while the changes of IL-4,TGF-?and IL-17 levels had no statistical significance before and after blocking,and the advantages of Th1 cells were more obvious.After sPD-L1 activated PD-1/PD-L1 pathway in vitro,the high levels of IFN-?and IL-17 in PBMCs culture supernatant of ITP patients have decreased,while the low levels of IL-4 and TGF-?have increased,thus improving the imbalance of CD4~+T cells.The change trend of cytokines related to CD4+T cells in ITP patients treated with glucocorticoid was similar,suggesting that activation of this pathway improved the imbalance of CD4+T cell subsets in ITP patients.