Loss of Ten-Eleven Translocation 2 (TET2) reduces CCR6 expression and increases B1 B cell number in the peritoneal cavity

Abstract TET2 is an evolutionarily conserved dioxygenase that catalyzes the conversion of 5-methyl-cytosine to 5- hydroxymethyl-cytosine, promotes DNA demethylation and regulates transcription. TET2 has been reported to regulate germinal center formation and class switch recombination in B2 B cells, yet its role in B1 B cell subsets remains largely unexplored. To investigate the role of TET2 in B1 subsets, flow cytometry of cells from the peritoneal cavity (PEC), spleen and bone marrow of TET2−/− mice and wildtype littermate controls (n = 14/group) was performed. In addition, sort purified PEC B1a, B1b and B2 cells were analyzed for methylation status (n = 24) and RNA expression (n = 24). Results demonstrated increased B1a (p = 0.0162) and B1b (p = 0.0056) cells in TET2−/− mice in the PEC, their primary niche, but not in the spleen, while in the bone marrow only TET2−/− B1a cells were increased (p = 0.0021). RNAseq analysis of PEC B1a, B1b, and B2 cells from TET2−/− and wildtype mice revealed reduced chemokine receptor CCR6 expression in B1a (−4.879068141 fold change, 1. 8.03E-08 padj.) and B1b cells (−2.17044776 fold change, 0.34771255 padj.) from the TET2−/− mice. Further, methylation analysis showed significant hypermethylation of CCR6 in B1a and B1b TET2−/− cells compared to wildtype B1a and B1b cells. This was corroborated by gene pathway analysis of the RNAseq data which showed significant downregulation of chemotaxis and migration pathways in B1a and B1b TET2−/− cells. We conclude that loss of TET2 increases hypermethylation of CCR6 and suppresses CCR6 RNA expression in B1 cells which may impair trafficking out of the PEC to the spleen, but not necessarily the bone marrow, leading to PEC accumulation of B1 subtypes. Supported by grants from the NIH: 1R01HL 136098-01 (McNamara, C PI), R01 HL141123 (McNamara, C PI), T32 HL007284 (Dennis, E)