Marginal zone B cells receive robust Notch2 signals within the B cell follicle in the absense of S1pr1 signaling.

Marginal zone (MZ) B cells have innate-like response characteristics due to a unique ability receive and integrate Notch2 signals in the spleen. There is evidence showing MZ B cells receive these signals both in the marginal sinus and the B cell follicle. The relative strength and conservation of these Notch signals is not yet clear. Therefore, we generated mice in which S1pr1 is deleted in mature B cells. These mice have MZ B cells by phenotype but these cells are retained in the follicle and unable to transit to the marginal zone. In this experiment we examined the impact of Notch2 signaling in these cells and cre expressing control animals using in vivo anti-Notch2 antibody blockade followed by RNA-sequencing. S1pr1-floxed mice were mated to Mb1cre mice to conditionally delete S1pr1 in B cells. S1pr1 conditional knockout (S1pr1FL/FL.Mb1Cre/+ mice and S1pr1+/+.Mb1Cre/+ control animals were treated with anti-Notch2 blocking antibodies or isotype control antibodies for 48 or 96 hours prior to sacrifice. Marginal zone B cells (CD19+, CD93-, CD1d+, CD23-) and follicular B cells (CD19+, CD93-, CD1d-, CD23+) were purified by FACS sorting and RNA-sequencing library generation.