THE ROLE OF THE M6A METHYLTRANSFERASE METTL3 IN AN IN VITRO MODEL OF ANTIGEN-SELECTED GERMINAL CENTER B CELLS

N6-methyladenosine (m6A) represents the most abundant modification introduced into messenger RNA. In mammals, m6A regulates gene expression by controlling different facets of mRNA metabolism including splicing, translation and stability. Emerging evidences have assigned to m6A a critical role in the regulation of cellular and humoral immunity. To date, the function of m6A in mature B cells, especially those recruited into T-cell dependent immune responses, remains unexplored. Here, we show that the Mettl3 gene, encoding for the catalytic subunit of the m6A methyltransferase complex, is expressed at relatively stable levels throughout B-cell development and increases in mature B cells activated in response to the engagement of the CD40 receptor. Given the crucial role played during a T-cell dependent immune response by CD40 signaling in sustaining the transient expansion of antigen-selected B cells in the light zone of the germinal center (GC) and, together with Interleukin-21 (IL-21), their terminal differentiation into antibody-secreting plasma cells (PCs), we asked to which extent these processes are under m6A methylation control. To answer these questions, we disrupted by CRISPR/Cas9 technology the Mettl3 gene in primary in vitro-induced germinal center-like B (iGB) cells, which were stimulated for 8-to-13 days with membrane-bound CD40-ligand (mCD40L). Efficient targeting of the Mettl3 gene in iGB cells exerted distinct phenotypes on CD40-activated B cells starting from the fourth day of the culture, when IL-4 was replaced with IL-21 in the culture medium. Using two independent sgRNAs to disrupt the Mettl3 gene, we observed a consistent significant growth delay of Mettl3-mutant iGB cells, which was restricted to the first 48 hours of mCD40L + IL-21 stimulation. The proliferation defect of Mettl3-mutant iGB cells caused their rapid counter selection when placed in competition with Mettl3-proficient counterparts. The growth impairment of CD40-activated Mettl3-mutant iGB cells was associated with limited upregulation of c-MYC expression, a defect in cell-cycle progression at the G1-to-S transition, due to reduced expression of E2F family members, and defective activation of the nutrient-sensitive mTORC1 signaling pathway. During the first 48 hours of mCD40L + IL-21 stimulation, Mettl3-mutant cultures suffered also from a significant defect in the transcriptional activation of genes encoding for master regulators of PC differentiation, including Irf4, Blimp1, Pou2af1 and Xbp1. The growth and developmental defects of Mettl3-mutant iGB cultures were normalized in the following 48 hours, due to the exhaustion of the proliferative burst of control B cells, while mutant ones completed the missing cell divisions, prior to undergoing terminal differentiation and/or senescence. Sequencing of the Mettl3 gene, retrieved from day-8 Mettl3-mutant iGB cultures, indicated a strong counter selection of iGB cells carrying loss-of-function alleles. This result was confirmed in terminally differentiated CD138+ plasmablasts isolated from day-13 in Mettl3-mutant iGB cultures, which displayed METTL3 protein levels comparable to those of wild-type cells. Altogether, these results establish for the first time a strict requirement for a full complement of METTL3 molecules, for optimal mitogenic B cell response to CD40 engagement, for initiating PC differentiation and for PCs persistence. In light of these results, our work assigns to METTL3 a crucial supportive function in B cells for the processes of clonal selection and antibody affinity maturation occurring in the GC during T-cell dependent immune responses. To shed further light on the importance of METTL3 function in developmental decision-making processes, we employed an in vitro model of reversible immortalization of multipotent murine hematopoietic progenitor cells (HPCs), by expression of an estradiol-inducible form of the HoxB8 (H8) transcription factor. Application of CRISPR/Cas9 technology to H8-HPCs ensured efficient Mettl3 gene targeting in these cells. We could show that interference with Mettl3 expression led to the counter selection of highly proliferating Mettl3-mutant H8-HPCs. Moreover, Mettl3-targeted H8-HPCs expressed prematurely the myeloid-associated marker CD11b and displayed accelerated myeloid differentiation upon estradiol withdrawal from the culture medium. Altogether, these results support a view whereby METTL3 sustains self-renewal of multipotent HPCs and prevents inappropriate and premature differentiation of these cells into the myeloid lineage.