Fig. 5
From: CD30 influences germinal center B-cell dynamics and the expansion of IgG1-switched B cells
GC kinetics are accelerated in transgenic CD30 mice. A Treatment scheme: CD30//Cγ1-Cre mice were immunized with NP-CGG and analyzed at the indicated time points. B The graph summarizes the percentages of reporter+ GC B cells at different time points after immunization. UI: unimmunized controls; red dots represent CD30//Cγ1-Cre mice, and blue triangles represent CAR//Cγ1-Cre mice (controls). Reporter+ cells were gated as described in Supplementary Fig. 7a (N = 3–13 per group). C CD30 expression is increased in GC B cells from CD30//Cγ1-Cre mice (red dots) compared with controls (CAR//Cγ1-Cre mice; blue triangles). The FACS plots indicate the gating of GC B cells (CD95+CD38−) at different time points after immunization. The FACS plots were sequentially pregated on living single cells, lymphocytes, B cells (B220+) (as shown in Supplementary Fig. 5a) and reporter+ cells (Supplementary Fig. 7a). The histogram overlays show the CD30 expression in the GC B cells of the CD30//Cγ1-Cre mice (red lines) versus the controls (CAR//Cγ1-Cre mice; blue lines). The graphs on the right side summarize the median fluorescence intensities (MFIs) of CD30 in GC B cells at the indicated time points after immunization (N = 7–13 per group). B + C Rounds of immunization: day 4: 5; day 7: 5; day 11: 4; day 21: 2. D Images of GC B cells detected via immunohistofluorescence (upper row) or immunohistochemistry (lower row) of splenic sections 11 days after immunization. For immunofluorescence staining, B cells were stained with anti-B220 (red), T cells with anti-Thy1.2 (blue) and GC B cells with anti-GL-7 (green). In immunohistochemical staining, metallophilic macrophages lining the marginal zone sinus are stained with anti-Moma-1, and germinal center B cells are stained with PNA; both stains are red in color. The data are representative of 2 immunization rounds. E FACS plots demonstrating the gating strategy for Bcl6+CD95high (pre-GC and GC B cells) and Bcl6−CD95− non-GC B cells. The FACS plots were sequentially pregated on single cells, living cells, lymphocytes, B220+ cells and reporter+ cells (Supplementary Figs. 5a and 7a). The graphs below summarize the percentages of Bcl6+CD95+ GC B cells and Bcl6−CD95− non-GC B cells at the indicated time points after immunization (N = 3–7 per group). Rounds of immunization: day 7: 2; day 11: 3. B 2-way ANOVA with Sidak’s multiple comparisons test. C + E, Unpaired t tests, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001
