Fig. 5: In B cells, IκBε sets the proliferation threshold in response to TLR or T-dependent antigen stimulations.

a Sorted splenic B1 cells were labeled with CFSE and cultured in the presence of LPS or CpG. Cell division was measured by CFSE dilution at 72 h. Percentages of cells that underwent at least one division and cell numbers at 72 h are shown. Each symbol represents one mouse (n = 3). b Sorted peritoneal Nfkbie^+/+ B1a, Nfkbie^−/− B1a IgMhi, and Nfkbie^−/− B1a IgMlow cells were labeled with CFSE, cultured in the presence of LPS or CpG, and analyzed as in a. Each symbol represents one mouse. Nfkbie^+/+ (n = 4), Nfkbie^+/− mice (n = 5), and Nfkbie^−/− mice (n = 5). c Sorted peritoneal Nfkbie^+/+ B1a, Nfkbie^−/− B1a IgMhi, and Nfkbie^−/− B1a IgMlow cells were cultured in complete medium without any stimulant for 24 h. Spontaneous cell death was assessed by sytox staining. Each symbol represents one mouse. Nfkbie^+/+ (n = 4), Nfkbie^+/− mice (n = 5), and Nfkbie^−/− mice (n = 5). d Analysis of phosphorylated p65 (Ser536) in sorted Nfkbie^−/− and Nfkbie^+/+ B1a B cells at T0 and after treatment with LPS for 15 min and 3 h. Each symbol represents one mouse (n = 3). e Analysis of intracellular and nuclear localization of cRel in sorted B1a Nfkbie^−/− and Nfkbie^+/+ B cells at T0 and after treatment with LPS for 15 min, 3 h, and 18 h. To show nuclear localization of cRel, cytoplasm was removed from cRel-stained cells with nuclear isolation media (HBSS media with 1 mM EDTA and 0.5% NP-40). Each symbol represents one mouse (n = 3). f Sorted Nfkbie^−/− and Nfkbie^+/+ B1a B cells were stimulated with LPS and treated for 72 h with serial dilutions of IT-901. At 72 h viable cell number was determined by cell count and trypan blue exclusion assay. The percentage of inhibition was calculated relative to vehicle-treated cells number. 0 stands for vehicle (DMSO). Each symbol represents one mouse (n = 3). g Sorted Nfkbie^−/− and Nfkbie^+/+ B1a B cells were stimulated with LPS and treated for 72 h with serial dilutions of IT-901. Apoptosis was evaluated by Annexin V–PE and sytox staining. Percentages of double-negative (i.e., Annexin V-negative and sytox-negative) living cells are shown. 0 stands for vehicle (DMSO). Each symbol represents one mouse (n = 3). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.