CATALYTIC-INDEPENDENT FUNCTIONS OF KDM6A IN B CELL MATURATION AND IMMUNE REGULATION

Kdm6a is an X-linked gene encoding a lysine demethylase involved in epigenetic regulation of gene expression. It catalyzes the demethylation of histone H3 lysine-27, counteracting the function of Polycomb Repressive Complex 2 (PRC2). Kdm6a plays a key role in transcriptional activation of genes that control cell differentiation during embryonic development and postnatal life. It is recurrently mutated in several solid and hematological cancers and is responsible for Kabuki syndrome. While Kdm6a has been extensively studied in early hematopoiesis, T-cell development, immunity, and plasma cell differentiation, its role in B-cell maturation and immunity has been largely overlooked. This study aims to elucidate the role of Kdm6a in B cell differentiation and its influence on adaptive B cell immune responses. To achieve this goal, I generated B-cell-specific Kdm6a gene-targeted mice using Cre/loxP recombination technology in vivo. In these conditional mutant mice, Kdm6a inactivation started in pro-B cells and reached completion in pre-B cells and later stages of B-cell lymphopoiesis. Kdm6a deficiency was dispensable for early B-cell differentiation but limited the re-entry of recirculating mature B cells into the bone marrow. Kdm6a-deficient mice exhibited a significant reduction in follicular B-2 B-cells in the spleen, peritoneal cavity lavages, and Peyer’s patches. This defect was linked to reduced expression of the Baff receptor in mutant B cells. Conversely, the fraction and absolute number of splenic marginal zone (MZ) B cells were increased in Kdm6a-deficient mice. Self-replenishing B-1 B cells in the peritoneal cavity remained unaffected by Kdm6a deficiency. Following immunization with T-cell-dependent antigens, B-cell-specific Kdm6a mutant mice exhibited a modest but consistent increase in the fraction of germinal center (GC) B cells in the spleen. A significant increase in GC B cells was also observed in the Peyer’s patches (PP) of Kdm6a-deficient mice. Flow cytometric analyses revealed an increased fraction of Cxcr4hi centroblasts at the expense of Cxcr4lo centrocytes in Kdm6a mutant mice. Kdm6a mutant GC B cells expressed higher transcript levels for genes promoting cell cycle progression, DNA replication, and mitosis. Conversely, genes associated with chemotaxis and trafficking, including Ccr6, Ccr7, Ebi2/Gpr183, Itga4, and Itgb7, were significantly downregulated upon Kdm6a inactivation. B-cell-specific Kdm6a deficiency led to a significant decrease in serum IgA titers, associated with reduced seeding of B cells in PP. Residual Kdm6a-deficient B cells reaching PP were preferentially recruited into the GC reaction. By 18–24 months of age, Kdm6a mutant mice accumulated an atypical population of Cd21loCd23loCd11b+Cd43+ B cells in the spleen, closely resembling aged B cells. Immunoblotting analyses ruled out major changes in global H3K27me3 levels in Kdm6a mutant B cells. Chromatin accessibility studies indicated that the genome-wide map of accessible regions at transcriptional start sites remained largely conserved upon Kdm6a inactivation. These findings align with the highly restricted subset of differentially expressed genes observed when comparing Kdm6a-proficient and -deficient GC and follicular B cells. To assess the significance of Kdm6a’s catalytic function, a comprehensive immunophenotypic analysis was conducted on B cells from germline mutants expressing a catalytically inactive Kdm6a protein. Notably, defects observed in B-cell-specific Kdm6a null mice, including impaired follicular/marginal zone B-cell differentiation, reduced GC B-cell pool size, contraction of the PP B cell pool, and diminished IgA serum titers, were normalized in Kdm6a catalytic-dead animals. In conclusion, this study establishes a non-redundant catalytic-independent role for Kdm6a in regulating follicular and marginal zone B-cell differentiation, homeostasis, and trafficking across primary and secondary lymphoid organs, as well as in the prevention of aged B-cell accumulation. The marked defect in B cell homing to Peyer’s patches, a major source of IgA+ B cells, combined with impaired expression of integrins Itga4 and Itgb7, necessary for IgA+ plasma cell homing to the intestinal lamina propria, highlights the essential contribution of Kdm6a to mucosal B-cell immunity. These findings provide insights into the mucosal immune defects observed in Kabuki syndrome patients.