METTL3 inhibition reduces N⁶-methyladenosine levels and prevents allogeneic CD4⁺ T-cell responses

Alloreactive CD4⁺ T cells play a central role in allograft rejection. However, the post-transcriptional regulation of the effector program in alloreactive CD4⁺ T cells remains unclear. N⁶-methyladenosine (m⁶A) RNA modification is involved in various physiological and pathological processes. Herein, we investigated whether m⁶A methylation plays a role in the allogeneic T-cell effector program. m⁶A levels of CD4⁺ T cells from spleens, draining lymph nodes and skin allografts were determined in a skin transplantation model. The effects of a METTL3 inhibitor (STM2457) on CD4⁺ T-cell characteristics including proliferation, cell cycle, cell apoptosis and effector differentiation were determined after stimulation of polyclonal and alloantigen-specific (TEa; CD4⁺ T cells specific for I-Eα_52-68) CD4⁺ T cells with α-CD3/α-CD28 monoclonal antibodies and cognate CB6F1 alloantigen, respectively. We found that graft-infiltrating CD4⁺ T cells expressed high m⁶A levels. Administration of STM2457 reduced m⁶A levels, inhibited T-cell proliferation and suppressed effector differentiation of polyclonal CD4⁺ T cells. Alloreactive TEa cells challenged with 40 μm STM2457 exhibited deficits in T-cell proliferation and T helper type 1 cell differentiation, a cell cycle arrest in the G0 phase and elevated cell apoptosis. Moreover, these impaired T-cell responses were associated with the diminished expression levels of transcription factors Ki-67, c-Myc and T-bet. Therefore, METTL3 inhibition reduces the expression of several key transcriptional factors for the T-cell effector program and suppresses alloreactive CD4⁺ T-cell effector function and differentiation. Targeting m⁶A-related enzymes and molecular machinery in CD4⁺ T cells represents an attractive therapeutic approach to prevent allograft rejection.