Fig. 4: Notch2 surface expression and signaling is enhanced in activated pre-Germinal Center B cells.

a Median Fluorescence Intensity (MFI) of Notch2 surface expression on purified splenic Follicular B (FoB) cells after α-IgM+α-CD40 stimulation. n = 5 mice (2 males (m), 3 females (f)), except for 5 h n = 2 mice (2 m) (unstimulated (us)): white dots; stimulated cells after different time points: blue dots. The horizontal bar and the error bars show the mean value and the standard deviation (SD). Data were log-transformed. Brown-Forsythe and Welch ANOVA with Dunnett´s T3 multiple comparisons test (*p = 0.037 (0h–5h); **p = 0.0014 (5h–14h); *p = 0.037 (14h-24h); **p = 0.021 (0h–48h); **p = 0.058 (0h–72h)). b, c Gating of Germinal Center B (GCB) (CD38^–CD95⁺), non-GCB (CD38⁺CD95^low), and activated FoB/memory B cells (CD38⁺CD95^high) is depicted in Supplementary Fig. 6b. b Fold induction of Notch2 cell surface expression Median Fluorescence intensity (MFI) in GCB cells (red), CD38⁺CD95^low cells (green), CD38⁺CD95⁺ cells (blue) from control/CAR mice, relative to its expression levels in CAR^– B cells (MFI set to 1, depicted by the dotted line). d7 n = 5 mice (2 m, 3 f), d14 n = 6 mice (2 m, 4 f). For subpopulation comparisons within each time point, a repeated measures (RM) two-way ANOVA with Sidak´s multiple comparisons test was applied (*p = 0.0119 d7 ***p = 0.0002, ***p = 0.0008, **p = 0.0046; d14 ****p ≤ 0.0001, ***p = 0.0004) and for comparisons between time points an ordinary two-way ANOVA with Sidak’s multiple comparisons test (*p = 0.0119). c Fold induction of Hes1 (MFI) in the indicated populations of Cre-reporter⁺ cells (red dots: GCB cells; green dots: CD38⁺CD95^low; blue dots: CD38⁺CD95⁺) relative to its expression levels in Cre-reporter^– B cells (MFI set to 1, depicted by the dotted line) n = 4 mice for each genotype: control/CAR: 2 m, 2 f; N2KO//CAR: 2 m, 2 f; N2IC/hCD2: 3 m, 1 f. After log transformation of data, a RM two-way ANOVA with Tukey´s multiple comparisons test was applied (left to right: **p = 0.0077, **p = 0.001, **p = 0.0017). d Overlays between Cre-reporter⁺ B cells (red) and total B220⁺ cells (black) are shown. Gates and percentages refer to the Cre-reporter⁺ GCB cells. e, f Histogram overlays depict the expression of the indicated markers in CAR⁻ cells (filled gray); reporter⁺ cells from control/CAR mice (black line); reporter⁺ cells from N2IC/hCD2 mice (blue line). d–f Analyses are representative for n = 14 control/CAR and n = 9 N2IC/hCD2 mice. g Gating and percentages of Irf4^highB220^low cells within splenic Cre-reporter⁺ lymphocytes: d7: n = 11 (6 m, 5 f) and n = 9 (6 m, 3 f); d14: n = 11 (5 m, 6 f) and n = 8 (4 m, 4 f) control/CAR (white dots) mice and N2IC/hCD2 (blue dots) mice, respectively. The horizontal bar and the error bars show the mean value and the SD. After log transformation of data, an ordinary two-way ANOVA with Tukey´s multiple comparisons test was applied (***p = 0.0002, ****p < 0.0001). d–g All FACS plots are representative for d7 post-immunization. h Immunofluorescence analysis of splenic sections, stained for (pre-) plasmablasts and plasma cells (Irf4^high; green), B cells (B220⁺; red) and marginal zone (MZ) sinus (Laminin; purple). Scale bar represents 50 µm, n = 3 mice per genotype. Source data from (a–c), (e), and (g) are provided as a Source Data file.