The Investigation Of BATF And Its M~6A Methylation In Allogeneic T Cell Immune Responses

CHAPTER I.Ablation of BATF alleviates transplant rejection via abrogating the effector differentiation and memory responses of CD8⁺T cellsObjectives:Allogeneic CD8⁺T cells are prominently involved in allograft rejection,but how their effector differentiation and function are regulated at a transcriptional level is not fully understood.The basic leucine zipper ATF-like transcription factor（BATF）is a key transcription factor that drives the effector program of allogeneic CD8⁺T cells.Whether and how BATF regulated allogeneic CD8⁺T cell responses in the transplantation model is not clear.In this chapter,we investigated the role of BATF in allogeneic CD8⁺T cell responses.Methods:The BATF expression levels in splenic and graft-infiltrating CD8⁺T cells were determined by flow cytometric analysis in a skin transplantation model.Skin allograft survival was monitored in B6.Rag1^–/–mice that were reconstituted with wild-type（WT）CD8⁺T cells or BATF^–/–CD8⁺T cells followed by Balb/c skin transplantation.BATF^–/–and WT CD8⁺T cells were mixed in a 1:1 ratio and adoptively transferred into B6.Rag1^–/–mice one day before skin transplantation.The CD8⁺T cell numbers,states,and related transcription factors expressions were determined by flow cytometry on day 11 post skin transplantation.On day60 post initial transplantation,these mice were re-transplanted with Balb/c skins.The cell states of the CD8⁺T cells were determined by flow cytometry on day 67post-initial transplantation.Results:BATF is highly expressed in graft-infiltrating CD8⁺T cells and its ablation in CD8⁺T cells significantly prolonged skin allograft survival in a fully MHC-mismatched transplantation model.Compared with WT CD8⁺T cells in the rejection response,BATF^–/–CD8⁺T cells displayed a dysfunctional phenotype,evident by their failure to differentiate into CD127^–KLRG1⁺terminal effectors,impaired proliferative capacity,and production of pro-inflammatory cytokines/cytotoxic molecules,and diminished capacity to infiltrate allografts.In association with the failure of effector differentiation,BATF^–/–CD8⁺T cells largely retained TCF1 expression and expressed significantly low levels of T-bet,TOX,and Ki67.At the memory phase,BATF-deficient CD8⁺T cells displayed impaired memory responses upon allogeneic antigen re-stimulation.Conclusions:BATF is a critical transcriptional determinant that governs the terminal differentiation and memory responses of allogeneic CD8⁺T cells in the transplantation setting.Targeting BATF in CD8⁺T cells may be an attractive therapeutic approach to promote transplant acceptance.CHAPTER II.STM2457 prevents allogeneic CD4+ T cell responses by reducing BATF and its related transcription factors levelsObjectives:Alloreactive CD4+ T cells play a central role in allograft rejection.N6-methyladenosine（m6A）RNA modification is involved in gene expression,and its methylation is installed by the Methyltransferase-like（METTL）3-METTL14 complex.However,little is known about the m6 A methylation of the effector program in alloreactive CD4+ T cells.In this chapter,we investigated whether m6 A RNA modification plays a role in the allogeneic T-cell effector program by using a METTL3 selective inhibitor STM2457.Methods:The levels of m6 A RNA methylation in CD4+ T cells from spleens,draining lymph nodes,and skin allografts were determined in a skin transplantation model.The effects of the METTL3 inhibitor STM2457 on CD4+ T cell proliferation,cell cycle,cell apoptosis,and effector differentiation were determined after stimulation of polyclonal and alloantigen-specific（TEa;TCR transgenic）CD4+ T cells with α-CD3/α-CD28 m Abs and the cognate CB6F1 alloantigen,respectively.Results:Graft-infiltrating CD4+ T cells displayed high levels of m6 A RNA methylation.METTL3 inhibitor STM2457 at 40 μM reduced m6 A levels,inhibited T-cell proliferation,and suppressed T helper（Th）1/Th2 cell differentiation.Alloreactive TEa cells challenged with 40 μM STM2457 demonstrated deficits in T-cell proliferation and Th1 cell differentiation,exhibited a cell cycle arrest in the G0,and displayed elevated cell apoptosis.Moreover,this T-cell disfunction was associated with the diminished expression levels of BATF and its related transcription factors.Conclusions:METTL3 inhibitor STM2457 suppresses allogeneic CD4+ T-cell effector function and differentiation by reducing m6 A levels and modulating the expression of BATF and its several related transcriptional factors that promote the T-cell effector program.METTL3 inhibitor STM2457 may represents an attractive therapeutic approach to prevent allograft rejection in CD4+ T cells.