NEMO lysine mutant mice reveal ROS-RelA signaling required for optimal CD40-induced humoral immunity

Oxidative and genotoxic stresses activate NF-κB signaling through intracellular pathways distinct from those initiated by membrane receptors, but their physiological roles remain incompletely defined. We created the NEMODK mouse model with germline arginine substitutions at NEMO (NF-κB essential modulator) lysines 270 and 302, key sites for post-translational modifications selectively induced by these intracellular stresses to enable NF-κB signaling in vitro. NEMODK mice exhibited B cell-intrinsic defects in the generation of germinal center B cells and humoral responses. Mechanistically, NEMODK B cells displayed a CD40-specific NF-κB signaling defect, unrelated to acute canonical and delayed noncanonical NF-κB activity, which was accompanied by the lack of sustained NEMO monoubiquitination downstream of mitochondrial reactive oxygen species generation without relying on nuclear DNA damage. This specifically reduced persistent RelA activity and a failure to mount transcriptomic and epigenetic changes critical for homotypic B cell aggregation, cell proliferation, class switch recombination and antibody secreting cell generation. Thus, CD40 induces a uniquely sustained NEMO modification and persistent RelA transcriptional activity to efficiently generate class-switched antibodies.