Fig. 6: N2IC/hCD2⁺ B cells differentiate into Marginal Zone B cells with the correct surface phenotype and splenic localization.

a Cre-reporter⁺ lymphocytes were separated into B220^lowIrf4^high (gate with the black line) and B220⁺ cells (gate with the red line). b Histograms show overlays of the Bcl6 expression in Cre-reporter⁺B220⁺ (red line) and Cre-reporter⁺B220^lowIrf4^high cells (filled gray). c Gating of Marginal Zone B (MZB) cells (CD21^highCD23^low) and Follicular B (FoB) cells (CD23^highCD21^low) in N2IC/hCD2 mice. The left plot is gated on total B220⁺ cells, and the right plot on hCD2⁺B220⁺Irf4^mid cells. a–c FACS plots are representative for d7 post-immunization (p.i.). n = 7 control/CAR and n = 4 N2IC/hCD2 mice. d Graphical summary of the percentages of CD21^highCD23^low MZB cells within Cre-reporter⁺ cells from (c) at indicated time points. d7 n = 12 mice (6 males (m), 6 females (f)), d14 n = 9 mice (5 m, 4 f) and d30 n = 7 mice (6 m, 1 f). An ordinary one-way ANOVA with Tukey´s multiple comparison test was performed (*p = 0.0119, ***p = 0.0009). e Histogram overlays of the expression of indicated markers within Cre-reporter⁺ MZB cells (solid blue line) and total splenic Cre-reporter⁻ B cells (dotted blue line) from N2IC/hCD2 mice at d7 post-immunization (p.i.), compared to total MZB (filled histogram dark gray) and FoB cells (filled histogram bright gray) from unimmunized (UI) N2IC/hCD2 animals. Analyses are representative for n = 9 immunized N2IC/hCD2 mice and n = 4 UI N2IC/hCD2 mice. f Chromogenic immunohistochemical analysis for the localization of hCD2⁺ cells at the indicated time points. Splenic sections were stained for hCD2 (blue) and for basement membranes of endothelial cells lining the marginal zone (MZ) sinus with Laminin (brown). Scale bar represents 100 µM and applies to all images. n = 3 mice per time point. Source data from (d) are provided as a Source Data file.