| IntroductionAllogeneic hematopoietic stem cell transplantation(allo-HSCT)is an effective therapy for hematologic malignant diseases.Acute graft-versus-host disease(aGVHD)is one of the key factors that hinder the success of transplantation.At present,the main strategy for prevention and treatment of aGVHD is immunosuppressive drugs,but it leads to increased risk of infection after transplantation.Removal of T cells in vitro and in vivo is another common strategy,but accompanied by graft failure,and increased relapse risk.In recent years,more and more immune cell subsets have been found to play an important role in the regulation of aGVHD.Therefore,adoptive immunocellular transfusion has a good clinical prospect for prevention and treatment of aGVHD.Regulatory γδT cell(γδTreg)is a newly discovered γδ T cell subset,which has biological properties similar to Foxp3+ αβ Treg cell,and exhibit negative immune regulation.We have established a high-efficient induction system of γδTreg in vitro and found that γδTreg can negatively regulate aGVHD in a xenograft model without affecting the GVL effect,but the mechanism is not yet clear.The inhibition of T cell proliferation and activation by γδTreg through intercellular contact is one of the mechanisms.Further elaboration of the mechanism by which γδTreg negatively regulate of aGVHD will lay the foundation for its clinical application.As antigen presenting cells,dendritic cells(DCs)are important in the pathophysiology of aGVHD.DCs are activated after conditioning,and the activated DCs present alloAg to donor T cells,and further make T cell activation and proliferation through the delivery of costimulatory molecular signal.This is the key event at the initial stage of aGVHD.In recent years,the induction of tolerogenic DCs(tolDCs)is one of the main research interests in the area of aGVHD treatment.There is a bidirectional tolerogenic feedback loop exists between αβTreg and tolDCs,and it leads to the maintenance of immune tolerance after transplantation.Therefore,we hypothesize that the induction of tolDCs is of the mechanism by which γδTreg negatively regulate of aGVHD.In the first two parts of this study,we studied the induction of tolDCs by γδTreg in vitro and in vivo.We investigated the immune regulation effects of γδTreg on the function of DC including maturation,phagocytosis,T cells activation,adhesion,migration,and explored possible mechanisms.Furthermore,we demonstrate that γδTreg can negatively regulate aGVHD by induction of tolDCs in vivo in a xenograft mouse model.On the other hand,the current studies on Foxp3+ γδTreg are based on γδTCR+mixed cells with γδTreg enrichment,but this could not fully reflect the real biological function of Foxp3+ γδTreg.There is still no cell surface marker with different expression between Foxp3+ γδTreg and Foxp3-γδT that could be used for purification of Foxp3+ γδTreg,which greatly limits the further study of the biological function of Foxp3+ γδTreg and the corresponding clinical application.Therefore,in the third part of this study,we explored the transcriptomic differences between Foxp3+ γδTreg and Foxp3-γδT by using single-cell RNA sequence,and tried to find a cell surface marker with different expression between these two subsets that could be used for purification of Foxp3+ γδTreg.Our study includes 3 parts:part 1 to study the induction of tolDCs by γδTreg in vitro and the related mechanisms;part 2 to investigate the regulation of aGVHD byγδTregs through the induction of tolDCs in vivo in a xenograft mouse model;part 3 to explore a cell surface marker that could be used for purification of γδTreg.Part 1 Induction of tolDCs by γδTreg in vitro and the related mechanismsAim:To investigate the immune regulation effects of γδTreg on the function of DC including maturation,phagocytosis,T cells activation,adhesion,migration,and explored possible mechanisms.Methods:Human peripheral blood mononuclear cells(hPBMCs)were isolated from healthy adult volunteers for DC and γδTreg culture,iDC or mDC cocultured withγδTreg for 48h,and then γδTreg was removed by MACS.The mean fluorescence intensity(MFI)of costimulatory molecules on DC were evaluated by flow cytometry;the endocytosis function was detected by using FITC-Dextran;and the mixed lymphocyte reaction assay,cell adhesion assay,and migration assay were also adopted to evaluate the functions of DC.Transwell or blocking antibodies was added in some of the cocultures.Results:γδTreg could inhibit the maturation of iDC promoted by LPS.The MFIs of CD80,CD86,CD40 and HLA-DR were significantly down-regulated.The immunosuppressive effects of γδTreg on iDC maturation were dependent on ICOS/ICOSL binding.γδTreg could also significantly inhibit the endocytosis and adhesion fuction of DC.The immunosuppressive effects on adhesion fuction were also dependent on ICOS/ICOSL binding.Moreover,γδTreg significantly down-regulated the expression of CXCR4 of DC but had no effect on CCR7,and this resulted to an impaired migration function toward CXCL12.The ability of iDC and mDC to activate T cells was also significantly reduced after coculture with γδTreg.Conclusion:γδTreg induced tolDCs in vitro by inhibiting iDC maturation,and down-regulating endocytosis,adhesion and migration fuctions of DC.The immunosuppressive effects of γδTreg on DC were partly dependent on ICOS/ICOSL binding.Part 2 The regulation of aGVHD by γδTregs through the induction of tolDCs in vivoAim:In part 1,we have demonstrated that γδTreg can induce tolDCs in vitro.In this part,we aimed to investigate the regulation of aGVHD by γδTreg through the induction of tolDCs in vivo in a xenograft mouse model.Methods:The humanized mouse aGVHD model was constructed by transfuion of PHA-activated human PBMCs and iDC to irradiated NOG mice.γδTreg cells were transfused simultaneously in some of the mice.GVHD symptoms,survival time,and the organ damage were evaluated.The MFIs of DC costimulatory molecules and the adhesion and migration fuctions of DC from spleen and peripheral blood were observed on the 7th day after transplantation.Results:1)In an allograft mouse model,the MFIs of DC costimulatory molecules from aGVHD group was significantly higher than control group(no aGVHD).2)γδTreg transfusion has a protective effect on aGVHD.In the γδTreg transfusion group,the symptoms of aGVHD were reduced,the survival time was prolonged and the organ damage was less.3)The immunosuppressive effect of γδTreg on splenic DCs was different from peripheral blood DCs.γδTreg significantly down-regulated the costimulatory molecules of peripheral blood DCs,while had no effect on splenic DCs;on the contrary,γδTreg significantly inhibited the adhesion and migration functions of splenic DCs,while had no effect on peripheral blood DCs;Conclusion:γδTreg transfusion has a protective effect on aGVHD.γδTreg induced tolDCs in vivo in a xenograft aGVHD mouse model.The immunosuppressive effect ofγδTreg on splenic DCs was different from peripheral blood DCs.Part 3 Screening of cell surface markers for purification of γδTregAim:To explore the difference of transcriptomes between Foxp3+ γδTreg and Foxp3-γδT in order to screen of cell surface markers for purification of γδTreg.Methods:Several cell surface markers related to Treg function or T cell activation were detected in Foxp3+ γδTreg and Foxp3-γδT by flowcytometry.we explored the transcriptomic differences between Foxp3+ γδTreg and Foxp3-γδT groups by using single-cell RNA sequence,and the differentially expressed genes were further validated by single-cell qPCR.By using these methods,we tried to find a cell surface marker with different expression between these two subsets that could be used for purification of Foxp3+ γδTreg.Results:We did not detect any different expression of cell surface markers CD127、CD45RA、CD49d、CTLA-4、GITR、ICOS、CD27、CD69 between Foxp3+γδTreg and Foxp3-γδT.Foxp3 was transient in the γδTreg induction system.Foxp3 expression increased rapidly,reached the highest value in about 10 days,kept at top value for about 15 days,and decreased to<5%40 days after the initial stimulation.The transcription level of Foxp3 mRNA at single cell level showed a continuous distribution.Totally 278 differentially expressed genes were screened out by single cell RNAseq analysis.By using protein localization database,35 differentially expressed genes for membrane proteins were further selected.The 35 selected genes were then validated by high-throughput single-cell qPCR,and 8 cell surface markers finally pass the validation.Conclusion:We for the first time reveals transcriptomic differences between Foxp3+γδTreg and Foxp3-γδT groups by using single cell RNAseq method,and obtained 8 potential cell surface markers for purification of Foxp3+ γδTreg. |
