Fig. 3: Reconstituted MZB cells functionally resemble wildtype MZB cells.

a , b Stimulation of purified B cells, with the indicated genotypes and treatment, with LPS for 48 h in vitro (w/o TAM: without tamoxifen treatment; +TAM (14d): day 14 after tamoxifen treatment). a Plots are pre-gated on living singlets, plasmablasts are gated as CD138⁺CFSE^low cells. b Cells were gated on living singlets (left) and consecutively separated in hCD2⁻ (black) and hCD2⁺ (red) cells. FACS plots in a, b are representative for two independent experiments. c–e Plasmablast differentiation in vivo after immunization with NP-LPS: c flow cytometric analyses show the percentages of plasmablasts (CD138⁺TACI⁺) in the spleen of unimmunised (UI) and immunized control and N2IC/creER^T2hom mice (3 days post immunization (PI) with NP-LPS). Cells were pregated on a large lymphocyte gate. d Summary and quantification of plasmablast percentages 3 days after NP-LPS immunization. Bar graph shows mean values and SD. Data points represent single mice from two independent experiments. ***p = 8.0E−04; *p = 0.040 (two-way ANOVA, Sidak´s multiple comparisons test). e Additional analysis of splenocytes from TAM-treated N2IC/creER^T2hom mice after immunization with NP-LPS: Indicated are the percentage of plasmablasts (CD138⁺TACI⁺) within hCD2⁺ cells and the percentage of hCD2⁺ cells in all TACI⁺CD138⁺ plasmablasts (red plot and histogram, respectively). Gray histogram: hCD2-staining of plasmablasts from immunized control animals (negative control). f, g Intracellular flow cytometric analysis of splenocytes from the immunized mice shown in (c). f Plasmablasts were identified as IRF4^highIgM^high. g Separation of plasmablasts in IgM⁺ and IgG1⁺ cells. FACS plots in c–e are representative for two individual experiments with n = 4 mice per group.