N⁶-Methyladenosine Directly Regulates CD40L Expression in CD4⁺ T Lymphocytes

Simple Summary The tight regulation of the expression of cytokines, surface receptors and co-stimulatory and co-inhibitory molecules is necessary for a well-functioning immune system. There are various cellular processes that regulate the expression of these immune regulatory proteins, for instance at the RNA transcript level. Epitranscriptomic regulation, involving RNA binding proteins and modifications, can determine the turnover and translation of mRNA transcripts. N-6-methyladenosine (m(6)A) is the most abundant RNA modification in eukaryotic cells and it was demonstrated that m(6)A is involved in the early differentiation of CD4(+) T lymphocytes. However, the function of m(6)A in CD4(+) T cell activation and function is still incompletely understood. Here, we demonstrate that m(6)A regulates the activation of CD4(+) T lymphocytes via the regulation of CD40 ligand expression, a key co-stimulatory molecule expressed on the cell surface of CD4(+) T cells. The discovery of this novel function of m(6)A and its regulatory proteins contributes to our general understanding of CD4(+) T cell activation, gene expression regulation and autoimmune disease pathogenesis. T cell activation is a highly regulated process, modulated via the expression of various immune regulatory proteins including cytokines, surface receptors and co-stimulatory proteins. N-6-methyladenosine (m(6)A) is an RNA modification that can directly regulate RNA expression levels and it is associated with various biological processes. However, the function of m(6)A in T cell activation remains incompletely understood. We identify m(6)A as a novel regulator of the expression of the CD40 ligand (CD40L) in human CD4(+) lymphocytes. Manipulation of the m(6)A 'eraser' fat mass and obesity-associated protein (FTO) and m(6)A 'writer' protein methyltransferase-like 3 (METTL3) directly affects the expression of CD40L. The m(6)A 'reader' protein YT521-B homology domain family-2 (YTHDF2) is hypothesized to be able to recognize and bind m(6)A specific sequences on the CD40L mRNA and promotes its degradation. This study demonstrates that CD40L expression in human primary CD4(+) T lymphocytes is regulated via m(6)A modifications, elucidating a new regulatory mechanism in CD4(+) T cell activation that could possibly be leveraged in the future to modulate T cell responses in patients with immune-related diseases.