Study On The Role Of PD1 And Tim-3 In T Cell Dysfunction In Fatal HFMD

Objective : Explore the expression of PD1,Tim-3 on the surface of peripheral blood T cells and its roles in the T cells about IFN-? secretion in children with fatal hand,foot and mouth disease(HFMD),providing a new idea to study the pathogenesis of fatal HFMD.Method:57 cases diagnosed hand,foot and mouth disease from March2017 to October 2017 in Guangxi were divided into mild group(n=18),severe group(n=25),fatal group(n=14)according to pathogenetic condition,whose of peripheral blood and clinical data were collected at the same time.Meanwhile,10 healthy children whose age and sex were matched were recruited as normal controls.Pharynx swab or anal swab collected from fatal and severe cases in the case group were detected by reverse transcription-polymerasechain reactionrt-pcrs(this part was completed by the Guangxi Center for Disease Prevention and Control).Flow cytometry was used to detect the expression of PD1,Tim-3 on CD4 / CD8 T cells of HFMD patients and normal children;The neutralizing antibody of PD1 and Tim-3 was used to block the PD1 and Tim-3pathway respectivly in vitro.And then the expression of IFN-? in CD4 and CD8 T cells were stimulated by PMA/ ionomycin.Flow cytometry was used todetect the intracellular expression of IFN-? in CD4 and CD8 T cells in the blocked group and the unblocked group.Result : Etiological findings in 57 patients included 11 cases with EV71-RNA positive,3 cases with CoxA16-RNA positive,,and 40 cases were other type.The positive rate of EV71-RNA in fatally ill group(72.7%)was significantly higher than that in severe group 4% and mild group 22.22%(P <0.05).The frequency of PD1 expressing on CD4 and CD8 T cells was15.95�7.29% and 18.96�6.84% respectively,significantly higher than that in mild group and normal group.In addition;The frequency of PD1 expressing on CD4 T cells in fatal group significantly higher than that in severe group(p <0.05);The frequency of Tim-3 expressing on CD4 and CD8 T cells was14.69�5.45% and 19.24�6.48% respectively,significantly higher than that in severe group,mild group as well as normal group.The frequency of co-expression of PD1 and Tim-3 on T cells in fatal group was significantly higher than that in severe group and mild group.The intracellular expression of IFN-? in CD4 T cells in the fatal group,the severe group and the mild group was5.64�3.96%,10.21�5.72% and 6.89�5.25%,respectively,which was significantly lower than that in normal control group,while the more profound decrease was seen in fatal group.The intracellular expression of IFN-? in CD8 T cells in the fatal,the severe and the mild group was 10.07�4.80%,29.96�11.45%and 40.15�14.88%,respectively.The intracellular expression of IFN-? in T cell of the fatal group and the severe group was significantly lower than that in the normal group,and the decrease in the fatal group was more significant than that in the severe group(all p < 0.05).The ratio of IFN-? expression in PD1 positiveT cell subgroup was significantly higher than that in PD1 negative T cellcounterpart(p < 0.05),while the ratio of IFN-? expression in Tim-3 positive CD8 T cell was significantly lower than that in Tim-3 negative counterpart in fatal group(p < 0.05).The intracellular expression of IFN-? in fatally ill CD4 and CD8T cells significantly increased after a single blockade of Tim-3 or in combine blockade of Tim-3 with PD1 pathway(p < 0.05),while there was no significant difference between the blockade and the unblockade in severe group and mild group.Conclusion:1.The expression of PD1 and Tim-3 on the surface of CD4 and CD8Tcells increased significantly in children with HFMD,as well as related to the pathogenetic condition.The expression of PD1 and Tim-3 in fatal cases was highest;2.Tim-3 negatively regulated CD4,CD8 T cell function.A single blockake of Tim-3 or in combination blockade of Tim-3 with PD1 pathway could be restored T cells function to secrete IFN-? in fatally ill children;3.Blockake of PD1 alone have minimal to no effect to restore the T cells function to secrete IFN-?,and the roles of PD1 about the dysfunction of HFMD T cells in fatally ill patients needs to be further studied.