Roles And Mechanisms Of Regulatory γδT Cells On Graft-versus-host Disease Regulation

IntroductionGraft-versus-Host disease (GVHD) is one of the major complications and leading causes of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) including acute and chronic GVHD. In HLA-matched related allo-HSCT the aGVHD rate was30%-60%while in HLA-matched unrelated allo-HSCT aGVHD rate was40%-90%. Currently MTX combined with CSA is the common strategy of aGVHD prevention but severe and refractory aGVHD rate and mortality is still high. T cell-depletion is effective for aGVHD prevention but the rate of graft failure and leukemia relapse became high. Though novel therapies such as new monoclonal antibodies, immunomodulatory chemotherapy, cellular therapies and photochemotherapy were appeared in clinical trials, graft-versus-leukemia effect was reduced and leukemia relapse rates as well as infection posttransplantation increased. cGVHD is an increasingly frequent cause of morbidity and mortality of allo-HSCT. Anasetti C et al reported that in allogeneic peripheral blood stem cell transplantation and allogeneic bone marrow transplantation the2year cGVHD rates were53%and41%respectively. cGVHD has negative impacts on patients’ health-related quality of life and organ function and become the major cause of late death, still lacking with regard to an optimal therapeutic strategy. Corticosteroids are considered the standard of care for first-line treatment of cGVHD, but only a minority of the patients responds to them durably. Management of steroid-refractory cGVHD is not well defined. Novel immunosuppressants, agents targeted to immune cells or inflammation cytokines, regulatory T cells or mesenchymal stem cells etc might play important roles in cGVHD therapy.Regulatory γδT cells (γδTreg) is a novel subset of lymphocytes with immuosuppressive function. Like common regulatory T cells (common Treg), these cells express high level of transcription factor Foxp3. Ex vivo studies found that these cells had similar biological characterization with common Tregs and its major function is to inhibit peripheral blood lymphocyte proliferation stimulated by anti-CD3and anti-CD28monoclonal antibodies via cell-cell contact dependent. They also secrete high levels of inhibitory cytokines such as TGF-β1and IL-10. Recently Li et al found that γδTregs were also implicated in the pathogenesis of systemic lupus erythematosus. These findings further demonstrated the immunosuppressive features of particular γδTregs. Moveover, γδTregs can be induced, expanded and enriched in vitro under the condition of isopentenylpyrophosphate (IPP) or zoledronic acid (ZOL) combined with cytokines IL-2, IL-15and TGF-β1which lays the foundation for further studying γδTreg biological characterizations. According to the current study, γδTregs and common Tregs had similar biological characterization. So we have great interests whether γδTregs can negatively regulate GVHD after allo-HSCT.In the current study we optimized the system of γδTreg induction, expansion and enrichment and further studied the mechanisms for the optimization. Then we studied the biological characterization of γδTregs induced in our culture system and further studied their GVHD regulation and correlated mechanisms. Finally the correlation of γδTreg percentage in peripheral blood and GVHD was analyzed in our study. Our research will provide experimental evidences for establishing a novel strategy for GVHD prevention and treatment. It will also lay the foundation of application in rejection after solid organ transplantation and autoimmune diseases.Our study includes3parts:part1to establish culture system of ySTregs; part2to investigate roles and mechanisms of ySTregs on Graft-versus-host Disease regulation in vitro and in animal models; part3to explore the regulation role of ySTregs on GVHD after allo-HSCT in clinic.Pat1Establishment of γδTreg induction and culture systemAim:γδTreg cells is a novel subset of lymphocytes with immunosuppressive function. The aim of the study is to establish an effective induction and culture system of ySTreg cells in vitro laying the foundation on further studying biological characterization of γδTregs.Methods:Human peripheral blood mononuclear cells (hPBMCs) were isolated from adult healthy volunteers and cultured with IL-2, IL-15, TGF-β1and ZOL. On day2, decitabine (0-2.0μmol/ml) was added to aliquots of hPBMCs. On day10, percentage of γδTregs was detected by FACS and ySTregs were enriched by Magnetic bead sorting (MACS). Then molecular mechanisms of γδTreg induction by decitabine was investigated by western blot, molecular clone, transcription factor chip, DNA bisulfite sequencing and promoter/enhancer reporter gene assay.Results:After10days’ induction, percentage of ySTregs were36.18%and59.87%in groups without decitabine and with0.5μg/ml decitabine, respectively. After MACS isolation,42.1%and62.8%y8Tregs were enriched in2groups (p<0.05). In groups without decitabine and with0.5μg/ml decitabine, the mean fluorescence intensity of Foxp3was52.6and182respectively (p<0.05). After decitabine induction, NF-κB expression in γδ T cells was up-regulated. We also found that upstream enhancer (-5031to-4792) and CNS3(3824to3938) of Foxp3gene demethylation induced by decitabine increased the enhancer activity which played important roles in γδTreg induction. Conclusion:For the first time we reported that0.5μmol/l decitabine,26.0U/ml IL-2,50ng/ml IL-15and8.5ng/ml TGF-β1synergized to induce stable γδTregs and successfully established the γδTreg induction and culture system. Decitabine increases the induction of γδTregs via NF-κB up-regulation and upstream enhancer as well as CNS3of Foxp3gene demethylation. Our culture system has obtained the national patent authorization. Some of our results have been published in the famous international journal ’Leukemia’.Part2Roles and mechanisms of γδTregs on Graft-versus-host Disease regulation in vitro and in animal modelsAim:In part1we established the induction and culture system of γδTregs. The aim of this part is to investigate the roles and mechanisms of γδTregs induced in our system on graft-versus-host disease in vitro and in animal models.Methods:We compared hPBMCs proliferation rate stimulated by anti-CD3/anti-CD28and cocultured with common γδTregs and decitabine-induced γδTregs respectively using CFSE-based assay. To clarify hPBMCs proliferation and underlying mechanisms we performed ELISA and Flowcytomix to measure levels of cytokines TNF-α, INF-γ, IL-10, IL-4and TGF-β1in supernatant of CFSE-based assay. Suface markers of γδTregs were also evaluated by FACS. To confirm the results in vitro we tested the functional ability to prevent GVHD in vivo. GVHD was induced in NOD/SCID mice. Animals were co-injected with either decitabine-induced γδ Tregs or common γδTregs or y8T cells at a ratio of1:1. Survival time and GVHD manifestations of the transplanted mice were evaluated.Results:1) CFSE-labeled hPBMCs proliferation was significantly reduced when cocultured with γδTregs for5days. The inhibition rates were significantly higher in decitabine-induced γδTreg group than in common γδTreg group.2) Compared with common γδTregs, decitabine-induced γδTregs have more stability in vitro.3) Compared with common γδTregs, decitabine-induced γδTregs express higher levels of HLA-DR, Foxp3and ICOS.4) Compared with common γδTregs, decitabine-induced γδTregs secreted higher levels of IL-10and TGF-β1.5) Transplantation of human PBMCs alone induced lethal GVHD with average survival time18.2±2.3days while the survival time was32.0±2.2and45.5±4.3days in mice co-injected with common y8Tregs and decitabine-induced y8Tregs, respectively.Conclusion:Our findings for the first time reveal that decitabine-induced γδTregs provide enhanced protection from GVHD via improved Foxp3stability, ICOS up-regulation, higher levels of cytokine secretion and higher levels of Foxp3expression. The xenogeneic GVHD model has been applied for national patent. And some of our results has published in international journals ’Annals of Hematology’and’Leukemia’.Part3Regulatory roles of ySTregs on GVHD after allo-HSCT in clinicAim:To further explore the regulatory roles of γδTregs on GVHD after allo-HSCT in clinic.Methods:Patients who underwent allo-HSCT in Bone Marrow Transplantation Center, the First Affiliated Hospital, School of Medicine Zhejiang University from January2000to December2012were selected. Among them, patients who underwent allo-HSCT from June2012to December2012were selected for aGVHD cohort. Patients who underwent allo-HSCT for1year or longer were selected as cGVHD cohort including non-cGVHD patients and cGVHD patients.10ml peripheral blood was drawn from all the selected patients for y8Treg percentage and CD4/CD8ratio analysis. We also collected peripheral blood from10healthy volunteers as normal control.Results:In the aGVHD cohort,39patients were selected in whom21patients developed aGVHD. We found the percentages of γδTregs in peripheral blood within3months post allo-HSCT were very low or even deficient. Thus γδTregs have not been reconstituted within3months posttransplantation and are not associated with aGVHD. In the cGVHD cohort,41patients were selected in whom26patients developed cGVHD and15patients had no cGVHD complication. Our result showed no statistical difference of γδTreg percentage between healthy volunteers and cGVHD cohort with a trend of elevated percentage of γδTregs in cGVHD cohort. We found a statistical difference of γδTreg percentage between non-cGVHD and cGVHD cohorts (P=0.044) with obviously elevated y8Treg percentage in non-cGVHD cohort. Furthermore, γδTreg percentage was positively correlated with CD4/CD8ratio in cGVHD cohort.Conclusion:We found for the first time that γδTreg cells played important regulatory roles in cGVHD after allo-HSCT in clinic via change of CD4/CD8ratio.