Increased PC differentiation of B2 cells upon CD40 stimulation. (A-B) Sorted splenic B2 cells (CD23⁺CD43⁻) from LMP1/CD30 and control (ctrl) mice were stimulated with anti-CD40 antibody for 3 days and analyzed for their B220/CD138 and CD43/CD23 surface expression. Sorting strategy and purity of the sorted B2 cells are shown in supplemental Figure 7A. Numbers in the fluorescence-activated cell sorting (FACS) plots indicate mean and standard deviation values of the percentages of gated populations in different FACS analyses. (C) CD23^lowCD43⁺ and CD23⁺CD43⁻ cells were gated as shown in panel B. B220/CD138 staining of CD23^lowCD43⁺ and CD23⁺CD43⁻ cells is shown as an overlay (n ≥ 3). (D) Histograms show overlays of CXCR4 and CD22 surface expression of CD23^lowCD43⁺ and CD23⁺CD43⁻ cells from ctrl and LMP1/CD30 mice. (E) Determination of phosphorylated STAT6 (pSTAT6) and pSTAT3 levels in nuclear extracts of splenic B cells stimulated with an anti-CD40 antibody for the indicated time points. Splenic B cells were purified by MACS using magnetic beads binding to CD43 to remove most of the B1 cells. Protein levels were analyzed and quantified using the WES separation system and software. Determination of p65 (F) and IRF4 (G) in cytoplasmic and nuclear extracts from the indicated genotypes. B cells purified by CD43 depletion were stimulated for 10 minutes with anti-CD40 antibody. Protein levels of p65 and IRF4 were analyzed and quantified using the WES separation system and software. For each mouse, the value after stimulation was standardized with the corresponding unstimulated value (n ≥ 5). (H) pJAK3 levels were analyzed in the cytoplasm of unstimulated splenic B lymphocytes. *P < .05, **P < .01, ***P = .001.