The Role Of ZNF683 In Remodeling Immune Homeostasis After Haplo-HSCT

Background:Allogeneic HSCT(allo-HSCT)is a curative treatment of a variety of malignant and nonmalignant hematologic disorders.Nowadays,the successful establishment of haploidentical stem cell transplantation(haploHSCT)has achieved outcomes comparable to those of transplantation with identical silbling donors,providing a possibility for every patients who need to receive transplantation.However,the major limitation of haplo-HSCT has been intense alloreactivity resulting in unacceptably high incidences of graft failure,graft-versus-host disease(GVHD).Thus,the timely reconstitution of immune homeostasis driven by a donor-derived immune system is of utmost importance for the recovery and long-term survival of patients after alloHSCT.Although a lot of prior studies have found the association of post-HSCT immune reconstitution and the number of immune cell subsets,little is known about whether the functions of each subsets are altered.Moreover,this change of each subsets on immune homeostasis is still not in hand.The balance between immune tolerance and immune response orchestrates immune homeostasis.And T cells are the master regulators of immune homeostasis,which can exert adaptive immune responses,and maintenance of their tolerance is critical to prevent GVHD.Using single-cell sequencing to explore the distribution of donor-derived cells in recipients after HLA-matched sibling donor transplantation(MSDT)and Haplo-HSCT,our study identify the CD8 Teff were significantly expanded and highly expressed the transcription factor ZNF683 compared with the donor in haplo HSCT recipients.ZNF683,also known as Hobit,is a homolog of PRDM1 which is an important transcription factor that regulates the development of T cells and B cells.In mice,ZNF683 transcriptional module is also required for tissue-resident lymphocytes,including natural killer T cells and liver-resident NK cells,all of which share a common transcriptional program in mice.ZNF683 also controlled the maintenance of quiescent,fully differentiated NKT cells and regulated their immediate effector functions.However,the currently available results suggests ZNF683’s regulatory functions may different between in mice and human.ZNF683 is highly expressed in effector T cells in human.But more detailed investigations are necessary to elucidate how ZNF683 regulate Teff functions.Therefore,this study aimed to investigate the regulatory effect of ZNF683 on human primary T cells,and to preliminarily explore whether the CD8 Teff which highly expressed ZNF683 in recipients is one of the mechanisms for maintaining immune homeostasis after haploHSCT.Importantly,our study tried to provide a new theoretical and experimental support for establishment of immune homeostasis after hematopoietic stem cell transplantation.Objective:To explore the regulatory effect of ZNF683 in human primary T cells and to clarify whether the high expression of ZNF683 on CD8Teff in haplo-HSCT recipients is one of the mechanisms for maintaining immune homeostasis.Materials and Methods:In this study,8 patients were selected after hematopoietic stem cell transplantation with free of infection and recurrence,and survived at least for 16 month.PBMCs from the patients and their donor were performed 10×Genomics single-cell sequencing and CD3+CD4+T+and CD3+CD8+T cell subpopulations were sorted for RNA-seq.In order to explore the regulatory function of ZNF683 on T cells,we overexpressed ZNF683 in human primary T cells by lentiviral vectors,and used flow cytometry to detect the proliferation,apoptosis and cytokine secretion.Simultaneously using RNA-seq to explore the molecular mechanism of ZNF683 inhibiting T cell activation.Results:Our results identify that the donor derived cell subsets has completely reconstituted immune system in haplo-HSCT and MSDT by using scRNAsequencing.The distribution of recipients subsets in the MSDT is similar with that of the donor,while in the haplo-HSCT CD8 Teff in the recipient significantly expanded and highly expressed transcription factor ZNF683 compared to their donors.We further overexpressed ZNF683 in primary T cells from healthy donors,and found decreased proliferation and apoptosis,and concomitant lowered levels of IL2,IL-4,IL-10 and CD107a in ZNF683-OE group.At the same time,we found that ZNF683 was highly expressed on T cells in patients more than one year after transplantation,but the expression of ZNF683 was down-regulated when T cells were activated by antigen stimulation.Therefore,it is suggested that ZNF683 may be a checkpoint that maintains effector T cells in a relatively quiescence and long-lived state,and restimulation can recover effector T cell function by downregulating ZNF683.Conclusion:Our study shows that ZNF683 is an important regulatory molecule that inhibits T cell activation.Primary T cells with high expression of ZNF683 exhibit long-term survival and maintain a low-responsive state,suggesting that the significantly high expression of ZNF683 in CD8Teff may be the one of the key mechanisms of immune homeostasis.