The Functional Evaluation And Immune Reconstitution Of IFN-γ~+/IL17~+/Foxp3~+ γδ T Cells After Allo-hsct

Objective:The expression of interferonγ（IFN-γ）⁺,interleukin-17（IL-17）⁺,and forkhead/winged helix family transcriptional repressor p3（Foxp3）⁺γδT cells subsets in peripheral blood of recipient under allogeneic hematopoietic stem cell transplantation（allo-HSCT）were analyzed.The clinical significance of immune reconstitution ofγδT cells subsets were investigated.To explore the relevant regulatory mechanism ofγδT cell subsets will help to understand the immune reconstitution ofγδT cell subsets after allo-HSCT.Methods:Peripheral blood samples of recipients were collected at the different time point（before transplantation,2 weeks,4 weeks,8 weeks,and 12 weeks after transplantation）.Peripheral blood samples of donors were collected before or after mobilization.Flow cytometry was used to detect the expression proportion of IFN-γ⁺,IL-17⁺and Foxp3⁺γδT cells,and the expression proportion of eomesodermin（Eomes）,retinoic acid receptor related orphan receptorγt（RORγt）and zinc-finger transcription factor-3（GATA3）.In vitro induction and culture ofγδT cells from recipients at 8-12 weeks after allo-HSCT,and then co-culture with leukemia cell lines to detect the killing effect ofγδT cells;Real time quantitative PCR was used to detect the relative m RNA expression levels of killer related granzyme B（grmb）,perforin（perf）,NK cell receptor member 2D（NKG2D）genes inγδT cells from recipients at 8-12 weeks after allo-HSCT and donor mobilizedγδT cells.Peripheral blood samples of healthy individual（HI）were served as control.Results:There was no significant difference between the proportion of CD3 T cells and totalγδT cells in PBMC before and after donor mobilization.After mobilization,theγδT cell subset with the function of secreting IFN-γ（γδT1）in donor were increased significantly than before mobilization and the HI group;and the expression proportion ofγδT cell subset with the function of secreting IL-17（γδT17）after mobilization was significantly reduced compared with the pre-mobilization group and HI group.Although the expression ofγδT1 subsets increased after transplantation,it was only increased significantly in 12 weeks after transplantation which compared to before transplantation.The expression proportion ofγδT17 was reduced after transplantation,the lowest was at2 weeks after transplanted,and at the 12^thweek post-transplant,it increased to the level of pre-transplant.No matter before and after mobilization or before and after transplantation,Eomes was significantly positively correlated withγδT1,so as to RORγt andγδT17.The expression of Foxp3⁺γδT cells（γδTregs）and IL-17⁺γδTregs in lymphocytes of peripheral blood did not change significantly before and after donor mobilization,but IFN-γ⁺γδTregs increased significantly after mobilization.The expression of IFN-γ⁺γδTreg was similar in the pro-mobilization group and the groups from pre-transplantation to12^thweek post-transplant,but was significantly higher than those in pre-mobilization group.There was no significant difference in the expression proportion of IL-17γδTregs before and after transplantation.In this study,three a GVHD cases were involved.In one a GVHD patient,before a GVHD treatment remission,the expression ofγδT1,γδT17,andγδTregs were decrease,while the patient’s a GVHD condition continued to improve,the expression proportion ofγδT cells and functional subsets increased to the level of recipients without a GVHD during the same period.We also detected the cytotoxicity ofγδT cells from peripheral blood of the recipient at8-12 weeks after transplantation,and found thatγδT cells have a certain cytotoxic effect on leukemia cell lines,especially the ALL cell line nalm-6.The gene expression levels of granzyme,perforin and NKG2D related to cytotoxic functions inγδT cells from recipients of 8-12 weeks after transplantation were higher than those in donor mobilized group.Conclusions:γδT1,γδT17,andγδTregs could obtain a certain immune reconstitution in8-12 weeks after allo-HSCT.Transcriptional factors Eomes and RORγt might play an important role in regulating the expression of functional subsets ofγδT1 andγδT17.The allo-HSCT mobilization might promoteγδTregs of donors to produce IFN-γ.The cytotoxicity function ofγδT cells in peripheral blood could reconstitute in early period after allo-HSCT.