Mechanism Of Mettl14-Mediated M~6A Modification In Regulating The Suppressive Function Of Treg Cells: Implication For Allograft Transplant Rejection

Organ transplantation has provided effective treatment options to increase the survival and quality of life with end-stage organ failure.Long-term graft survival is essential in determining the success of organ transplantation.However,allograft rejection mediated by allogeneic antigens remains the leading cause of graft loss after transplantation.The suppression function of regulatory T cells（Tregs）plays a vital role in regulating allograft survival.It has been reported that Tregs exhibit distinct functions in different environments,which may be due to the regulation of the Tregs-specific transcription factor FoxP3 by multiple physiological signals or protein modifications.Therefore,exploring the upstream regulatory mechanisms of FoxP3 is essential for maintaining Tregs function after transplantation.Epigenetics refers to the study of sequence-independent heritable DNA changes,which serve as a critical bridge between environment and phenotypes.RNA modification is a type of epigenetic modification,and the N6-methylation（m⁶A）is the most abundant post-transcriptional modification on eukaryotic m RNAs.Recently studies have reported that m⁶A modifications play a role in regulating the proliferation of Tregs and can influence the development and progression of autoimmune diseases.However,whether m⁶A modifications are involved in Tregs function in transplantation medicine has not been reported.The objective of our research is to explore the critical role of m⁶A modification in regulating Tregs function after transplantation and to identify the specific molecular mechanisms.The primary investigations of this dissertation are as follows:1.Tregs were isolated from the both allograft tolerance（AT-Tregs）and allograft rejection（AR-Tregs）model,and the levels of m⁶A modification and related methyltransferases expression was detected using dot-blot,western blot and q RT-PCR.The results revealed that the expression of m⁶A and related methyltransferases was significantly lower in AR-Tregs compared to AT-Tregs.Subsequently,we used the heterogenic Treg-specific Mettl14 knockout mice(Foxp3-Mettl14^f/+cKO,cKO)to performed islets allograft transplantation.We found that islet grafts from cKO mice lost the ability to regulate blood glucose and led to graft rejection,whereas islets graft from littermate controls（Ctrl）maintain the survival of islets allografts.2.On 7 days post-transplantation,we evaluated the proportion of Tregs in secondary lymphoid organs and graft by immunofluorescence and flow cytometry in cKO and Ctrl mice.The results showed that Mettl14 deficiency decreased the proportion of Tregs in spleen and draining lymph nodes of cKO mice after transplantation compared to the group of Ctrl.Additionally,the proportion of Tregs around the grafts was significantly reduced in cKO mice,while the proportion of TH1 and TH17 was increased,and the grafts were infiltrated by inflammatory T cells.We also assessed the expression of the anti-inflammatory cytokines IL-10 and TGF-β,which are markers of functional Tregs,in Ctrl and cKO mice using ELISA assays on 7 days post-transplantation.The results indicated that Mettl14 deletion inhibited the expression of IL-10 and TGF-β.More importantly,adaptive transfer assays and T cell proliferation assays showed that Mettl14 deficiency disrupted the suppressive function of Tregs after transplantation.3.On 7 days post-transplantation,Tregs were isolated from Ctrl and cKO mice,and subjected to RNA-seq and Me RIP-seq analysis.The expression profile data revealed that Sema4D may be a direct downstream target of Mettl14-mediated m⁶A modification in Tregs.IGV and Me RIP-q PCR analysis showed that Mettl14-mediated m⁶A modification directly interacted with the 3’UTR region of Sema4D m RNA.The m RNA decay assay and fluorescein reporter plasmid assay demonstrated that the m⁶A-Mettl14-YTHDF2molecular axis regulated the stability of Sema4D m RNA.Moreover,we observed that Mettl14 deficiency promoted Sema4D expression,leading to decreased phosphorylation levels of p-PAK,p-STAT5 and p-JAK,as well as disruption of FoxP3 the expression and Tregs function.In conclusion,this dissertation elucidated that the METTL14-mediated m6A modification is essential for the suppressive function of Tregs in transplantation,and further confirmed that m⁶A might be a potential regulatory mechanism for modulating Tregs function.Moreover,our findings provide a theoretical basis for considering Sema4D as a biomarker for predicting transplant rejection.