Lack Of IFN-γ Receptor Signaling Inhibits Graft-versus-Host Disease By Potentiating Regulatory T Cell Expansion And Conversion

Background and Objective:Allogeneic hematopoietic cell transplant(allo-HCT)is a potentially curative treatment for several hematologic malignancies,in which bone marrow or immune system is replaced via the intravenous infusion of healthy donor.Donor-derived immune cells identify and attack cancer cells in the patient producing a unique graftvs.-tumor(GVT)effect.However,alloreactive donor T cells can damage normal host cells thereby causing graft-vs.-host disease(GVHD),which results in substantial morbidity and mortality.To date,GVHD remains as the major obstacle for more successful application of allo-HCT.Graft-versus-host disease(GVHD)is a systemic disease in which donor immune cells attack recipient tissues directly or indirectly,mainly induced by donor T cells recognizing mismatched host major histocompatibility complex(MHC)and minor histocompatibility antigen(mi HA),after the implantation of donor hematopoietic stem cells in patients.Graft-versus-host disease(GVHD)is the main complication and the main cause of non-tumor recurrence death after allogeneic hematopoietic stem cell transplantation.It is a type of multi-organ syndrome,mainly involving skin,gastrointestinal tract,liver,lung and other tissues and organs.The high incidence of GVHD poses a huge barrier to MHCmismatched allo-HCT,severely limiting the clinical application of this important therapy.Therefore,there is still an urgent clinical need to explore new immunosuppressive regimens and transplantation regimens,which will help to further improve the outcome of allogeneic hematopoietic stem cell transplantation for leukemia patients.Regulatory T cells(Tregs)are a promising therapeutic target in autoimmune diseases and transplantation.Foxp3-expressing CD4+ T cells are broadly categorized to thymus-derived Tregs(t Tregs),peripherally-derived Tregs(p Tregs)and in vitroinduced Tregs(i Tregs).p Tregs result from conversion of conventional CD4+T cells(Tcons)or effector T cells(Teffs)during immune responses,and thus,play an important role in restraining antigen-specific immune responses and as a key regulatory subset maintaining immune tolerance in interplay with t Tregs.Treg therapies can promote favorable outcomes in allo-HCT,although the role of Tregs in promoting beneficial graft-versus-leukemia(GVL)effects while impairing toxic effects of graft-versus-host disease(GVHD)is still not well understood.Given the importance of IFN-g R signaling in the differentiation and functions of Tregs,we sought to understand the role of this pathway in the function of allogeneic Tregs in the setting of allo-HCT.Exploring strategies that favor the stabilization of Treg cells and the generation of inducible Treg cells will be the key to the successful development of Treg therapies.Methods:1.To compare the effect of lymphocytes infusion from wild-type or IFN-γ receptor knockout donors on graft-versus-host disease(GVHD)in an experimental allogeneic hematopoietic stem cell transplantation(allo-HCT)model,and through in-depth analysis of the engraftment of donor immune cells,To explore the possible mechanism of IFN-γ receptor knockout donor lymphocytes infusion attenuating graftversus-host disease(GVHD).2.To compare the effect of delayed infusion of lymphocytes from wild-type or IFN-γreceptor knockout donors on graft-versus-host disease(GVHD)in the Mixed chimera(MC)allogeneic hematopoietic stem cell transplantation model,and to confirm the experimental conclusion and mechanism again.3.To investigate the effects of IFN-γ receptor knockout donor lymphocytes infusion on graft-versus-tumor(GVT)and graft-versus-host disease(GVHD)in an experimental model of allogeneic hematopoietic stem cell transplantation(allo-HSCT)for leukemia.The optimized protocol successfully separated GVT from GVHD,thus effectively improved the therapeutic effect of allo-HCT.4.To investigate the effect of IFN-γR depletion on the function of human CD4+Foxp3+Treg cells in the in vivo environment,we also established a humanized xenograft GVHD model to further confirm the effect of IFN-γR signaling on Treg cells and ultimately regulation of GVHD.Results:1.In the experimental allogeneic hematopoietic stem cell transplantation(allo-HCT)model,the infusion of IFN-γ receptor knockout donor lymphocytes resulted in significant graft-versus-host disease(GVHD)compared with wild-type lymphocytes,and it was found that the increase of IFN-γ receptor knockout donor Treg cells may be the reason for the reduction of GVHD.2.Depletion of IFN-γR signaling promotes the expansion of donor pre-existing Tregs and the induction of conventional T cells into p Treg cells,thereby inhibiting the graftversus-host reaction.3.The rapid expansion of pre-existing Treg cells depleted of IFN-γ receptor not only suppressed GVHD,but also suppressed GVL effect after allo-HCT.If donor preexisting Tregs were removed prior to allo-HCT,p Tregs induced by IFN-γR1deficiency could effectively prevent persistent alloreactivity and GVHD,while preserving the GVL response induced by early GVHR.4.In the humanized xenograft GVHD model,it was further confirmed that loss of IFN-γR signaling could also promote the expansion of human pre-existing Tregs and the generation of inducible Tregs,and injection of IFN-γR deficient human CD4+T cells could effectively suppress GVHD.Conclusion:IFN-γ is a pleiotropic cytokine that plays a controversial role in regulatory T cell(Treg)activity.Here,we sought to understand how IFN-γ receptor(IFN-γR)signaling affects donor Tregs following allogeneic hematopoietic cell transplantation(allo-HCT).The suppressive effect of IFN-γ receptor signaling on donor Treg cells after hematopoietic cell transplantation was demonstrated using gene knockout mice and IFN-γR1 knockout human CD4 T cells by CRISPR/Cas9.We show that lack of IFN-γR signaling promotes Treg expansion and conversion of conventional T cells(Tcons)to peripheral Tregs(p Tregs)in both mouse and human.In mice receiving Treg-depleted allo-HCT,IFN-γR deficiency induced p Treg conversion was effective in preventing persistent GVHD while minimally affecting GVL effects.Thus,impairing IFN-γ R signaling in Tcons may offer a promising strategy for achieving GVL effects without refractory GVHD.