Regulation of B cell proliferation and cell cycle entry by the NAPDH oxidase and the adaptor protein Bam32

Ligation of the BCR is an important factor determining B cell fate decision. Signals initiated at the BCR complex may promote proliferation, differentiation, or death. Downstream signaling cascades must be carefully regulated in order to modulate not merely which response the cell chooses, but also the strength of that response. Adaptor molecules help coordinate these enzymatic cascades by organizing signaling components in spatially and temporally distinct groupings. Secondary messenger molecules help modulate signaling cascades.;Reactive oxygen species are one class of secondary messenger molecule which have been gaining attention as modulators of signaling cascades in non-phagocytic cells. We tested their role in normal mouse B cells. In B cells missing the catalytic component of the NADPH oxidase, BCR stimulation resulted in enhanced cell cycle entry and proliferation. This correlated with a more rapid decrease in levels of the cell cycle inhibitor, p27Kip1. Additionally, the T-cell-independent-type 2 (TI-2) antibody responses of gp91 phox KO mice were stronger than WT, suggesting that BCR-induced ROS may help modulate the size of the humoral response to TI-2 immunization via by restraining the initial proliferative burst.;Mice deficient in the adaptor molecule Bam32 display a phenotype opposite that of gp91phox KO mice. Bam32 KO mice mount less robust TI-2 antibody responses, and their B cells proliferate poorly to BCR ligation. Fewer Bam32 KO B cells enter the cell cycle after BCR crosslinking, and they progress to S phase more slowly than WT B cells. Of note, Bam32 KO B cells generate much more BCR-dependent ROS than WT B cells. We hypothesized that the increase in ROS negatively regulated BCR-dependent proliferation in Bam32 KO B cells. Bam32/gp91phox KO DKO B cells also proliferated poorly to BCR stimulation.;However, we demonstrated that loss of NOX rescued cell cycle entry in Bam32 KO B cells. These data suggest that the lag in BCR-dependent cell cycle entry seen in Bam32 KO B cells may be due to increased ROS, but Bam32 helps regulate signals acting in late G1 to promote S phase entry and thus proliferation. TI-2 antibody responses in DKO mice were also reduced, comparable to Bam32 KO mice, which supports the hypothesis that the robust TI-2 response seen in gp91phox KO mice is due to enhanced B cell proliferation.