Figure 3. Panel A: Flow cytometry plots... | Rockefeller University Press

Figure 3.

Panel A: Flow cytometry plots show the gating strategy for identifying plasmablasts (PB, antibody-secreting precursor cells) and plasma cells (PC, terminally differentiated antibody-secreting cells) in germinal centers (GC) and GL7 low or negative CD95 positive cell populations. The plots are divided into wild-type (WT) and AIDR112H mice, with markers for B220 (B cell marker), CD138 (plasma cell marker), GL7 (germinal center marker), CD95 (Fas receptor), CD83/86 (activation markers CD83 and CD86), and CXCR4 (chemokine receptor C-X-C motif receptor 4). Panel B: Bar graphs quantify the percentages of CD138 positive (plasma cell marker expressing) cells in dark zone (DZ), light zone (LZ), GL7 low or negative CD95 positive cells, CD138 positive in CXCR4 high (high CXCR4 expressing) cells, and CD138 positive in CD83/86 positive (activation marker expressing) populations, comparing WT and AIDR112H mice. Panel C: Immunohistochemistry images of spleen germinal centers (GCs) stained for CD21/35 (complement receptors CD21 and CD35, shown in red), GL7 (germinal center marker, green), and IgD (Immunoglobulin D, blue). A bar graph quantifies the percentage of IgD negative GL7 negative CD21/35 positive germinal centers among total IgD negative CD21/35 positive germinal centers in WT and AIDR112H mice. Panel D: Flow cytometry plots and bar graphs show the NP-specific (nitrophenyl antigen-specific) response of germinal center (GC) and GL7 low or negative CD95 positive B cells in WT mice, with quantifications for NP positive (antigen-binding) cells and NP positive CD138 positive (antigen-specific plasma cell marker expressing) cells. Panel E: RNA sequencing (RNA-seq, transcriptome profiling) analysis comparing GL7 high versus GL7 low germinal center B cells. The left panel shows gene set enrichment analysis (GSEA) using defined gene signatures, and the right panel shows a GSEA network analysis highlighting gene sets positively enriched in GL7 high versus GL7 low cells.

AID ^R112H mice have reduced PC differentiation in GCs and accumulate GL7 ^lo/− tGC B cells. (A) Gating strategy of PB and PC in GC and GL7^lo/−CD95⁺ cell population. PB: B220⁺CD138⁺; PC: B220⁻CD138⁺. FACS plot for GC and GL7^lo/−CD95⁺ gating used in Fig. S2 E to display other surface markers stained on the same samples. (B) Quantifications for A, WT versus AID^R112H; CD138⁺ cells in DZ, **P = 0.0019; CD138⁺ cells in LZ, nonsignificant. GL7^lo/−CD95⁺ cells: ****P < 0.0001; CD138⁺ in CXCR4^hi: ****P < 0.0001; CD138⁺ in CD83/86⁺: P = 0.044, unpaired t test (n = 8, two experiments). (C) Immunohistochemistry of spleen GCs. Red, CD21/35; green, GL7; blue, IgD. Quantification of IgD⁻GL7⁻CD21/35⁺ GCs among total IgD⁻CD21/35⁺ GCs, P = 0.0051, unpaired t test (n = 6, two experiments). Scale bar, 80 µm. (D) NP-specific response of GC and GL7^lo/−CD95⁺ B cells in WT mice. FACS plots and quantification, GC versus GL7^lo/−CD95⁺. NP⁺: P = 0.0028; NP⁺CD138⁺: P = 0.0002, unpaired t test (n = 10, two experiments). (E) RNA sequencing comparing GL7^hi versus GL7^lo GC B cells. Left panel: GSEA using gene signature defined in work (Gómez-Escolar et al., 2022; Glaros et al., 2021); right panel: GSEA network analysis showing gene sets positively enriched in GL7.hi versus GL7.lo. Value and error bar: mean ± SD.