FIGURE 2.

Age‐associated B cells (ABC) subsets and iABC develop independently of T cells. (a) Experimental Design: Uninfected Aged (18–20 M) and young (3–4 M) female B6 mice were sacrificed. CD23⁻CD21⁻ABC subsets in the spleen were analyzed by flow cytometry. Representative FACS plot of gated ABC (CD43⁻CD93⁻B220⁺CD19⁺CD23⁻CD21⁻) (left) and the number IgD⁺, IgM⁺IgD⁻ and IgD⁻IgM⁻ ABC (right) (n = 9–12 pooled from 5 to 6 separate experiments) (b, c) Experimental Design: Uninfected Aged (18–20 M) female B6 mice were sacrificed, Spleens were harvested, IgD⁺CD23⁻CD21⁻ABC, IgD⁻CD23⁻CD21⁻ABC and IgD⁺CD23⁺CD21⁻FOB were FACS sorted for RNA sequencing. (b) Venn diagrams showing the fraction of expressed genes shared between IgD⁺ABC and published ABC genes reported by Marrack (Rubtsov et al., 2011) and Cancro (Russell Knode et al., 2017) (top) and the same analysis with IgD⁻ABC (bottom). (c) GSEA plots assessing enrichment of ABC signature genes that are upregulated in sorted IgD⁺ (top) versus IgD⁻ (bottom) ABC (n = 2 pooled from 2 separate experiments). (d) Experimental Design: Aged (18–20 M) and young (3–4 M) female B6 mice were treated with anti‐Thy1.2 or isotype control Ab on 0 DPI and 7 DPI, infected with 0.3LD₅₀ (25 PFU) PR8 (IAV) and sacrificed at 14 dpi. Spleens were harvested and analyzed for iABC and GCB by flow cytometry. Representative FACS plots and cell numbers of iABC (Fas⁺GL7⁻) and GCB Fas⁺GL7⁺ gated on mature B cells (B220⁺CD19⁺) in anti‐Thy1.2 or isotype control Ab treated aged and young mice in the spleen (n = 8–10 pooled from 2 to 3 separate experiments). Error bars represent SEM and statistical significance determined by two‐tailed, unpaired Student's t‐test; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.