Effects Of RhTPO On CD4⁺CD25⁺CD127^low Treg Cells And IL-35 Expression In Patients With Primary Immune Thrombocytopenia

BackgroundPrimary immune thrombocytopenia（ITP）is a common autoimmune hemorrhagic disease with platelet destruction mediated by antibodies in the blood system.With further research into its complex pathogenesis,more and more studies have shown that immune tolerance dysfunction caused by abnormal T lymphocyte subsets and related cytokines is also the key to the pathogenesis of ITP.It has been found that recombinant human thrombopoietin（rh TPO）combined with high dose dexamethasone can correct the disorder of immune regulation of T lymphocyte subsets in patients with ITP and regulate immunomodulatory cytokines to improve the therapeutic effect.However,it has not been reported that rh TPO can induce immune tolerance through regulatory T cells（Treg）and IL-35 in China.In this study,we detected the proportion of peripheral blood CD4⁺CD25⁺CD127^lowTregs cells and plasma IL-35 levels in patients with ITP before and after high-dose dexamethasone（HD-DXM）combined with rh TPO and before and after each single drug treatment,to explore the immunomodulatory mechanism of rh TPO in ITP therapy,and to evaluate the target of immune tolerance induced by rh TPO,so as to provide theoretical basis for this drug therapy and provide a new intervention target for ITP immunotherapy.ObjectiveTo compare and analyze the changes of peripheral blood CD4⁺CD25⁺CD127^lowTreg cells ratio,plasma IL-35 level and platelet count（Plt）in patients with ITP before and after three treatments（dexamethasone,rh TPO and dexamethasone combined with rh TPO）,to explore the molecular mechanism of immune regulation of rh TPO in ITP patients and to evaluate the target of immune tolerance induced by rh TPO.MethodPeripheral blood was collected from 62 ITP patients before and after treatment and from 20 healthy controls.The ratio of Treg cells in isolated peripheral blood mononuclear cells（PBMC）was determined by flow cytometry（FCM）.Plasma IL-35 expression was detected by enzyme-linked immunosorbent assay（ELISA）.Result1.Compared with the healthy control group,the levels of CD4⁺CD25⁺CD127^lowTreg cells,IL-35 and Plt in ITP group（dexamethasone,rh TPO and dexamethasone combined with rh TPO）decreased significantly before treatment（P<0.001）.2.The percentage of CD4⁺CD25⁺CD127^lowTreg cells in peripheral blood of three groups after treatment was remarkably increased compared with that before treatment（P=0.040;P=0.004;P<0.001）.3.Intra-group comparison after treatment,plasma IL-35 level in rh TPO and dexamethasone combined with rh TPO group was significantly inproved compared with that before treatment（P<0.001）.While there was no statistical difference in plasma IL-35level in dexamethasone group before and after treatment.（P=0.892）.4.The plt count after treatment in three groups was significantly upper than that before treatment（P<0.001）.5.Comparison between the three groups of patients after treatment,the ratio of CD4⁺CD25⁺CD127^lowTreg cells in peripheral blood of dexamethasone combined with rh TPO group was significantly higher than that of dexamethasone group and rh TPO group（P=0.014;P=0.004）,but there was no significant difference between dexamethasone group and rh TPO group（P=1.000）.6.Comparison between the three groups of patients after treatment,plasma IL-35levels in dexamethasone combined with rh TPO group and rh TPO group were observably greater than those in dexamethasone group（P<0.001;P<0.001）,but there was no significant statistical differences between the dexamethasone combined with rh TPO group and rh TPO group（P=1.000）.7.Comparison between the three groups of patients after treatment,Plt count of dexamethasone combined with rh TPO group was significantly upper than that of dexamethasone group and rh TPO group（P=0.040;P=0.030）,but there was no significant difference between dexamethasone group and rh TPO group（P=1.000）.8.There was no significant difference in the total effective rate among the three groups after treatment（χ~2=4.551,P=0.103）,but the total effective rate of dexamethasone combined with rh TPO group was the highest（90.48%）,while that of dexamethasone and rh TPO group was 62.50%and 68.00%,separately.9.The Plt count of ITP patients before treatment was significantly positively correlated with the proportion of CD4⁺CD25⁺CD127^lowTreg cells and IL-35 level（r=0.787,P<0.001;r=0.840,P<0.001）,IL-35 level was positively correlated with the proportion of CD4⁺CD25⁺CD127^lowTreg cells（r=0.713,P<0.001）.Conclusion1.By up-regulating Treg cells,rh TPO can effectively regulate the expression of IL-35,mediate the recovery of platelet autoantigen immune tolerance in ITP patients,and reduce platelet destruction.2.Dexamethasone combined with rh TPO has a synergistic effect in the treatment of ITP patients.Compared with dexamethasone alone,the expression levels of CD4⁺CD25⁺CD127^lowTreg cells,IL-35 and Plt can be significantly increased,and the clinical efficacy of ITP patients can be improved.3.There was a positive correlation among the proportion of Treg cells,IL-35 level and Plt count in patients with ITP before treatment.