Fig. 4: Surface staining of CD21/35, CD23, CD5, and CD49d resolves six distinct populations of switched memory B cells in spleen and bone marrow.

a, b Flow-cytometric identification of distinct populations among IgG⁺ memory B cells gated as IgG1⁺/IgG2b⁺ CD19⁺IgM⁻IgD⁻CD138⁻GL7⁻CD93⁻ live small lymphocytes (cf. Supplementary Fig. 5) in bone marrow (BM) (a) and spleen (b) of female C57BL/6 mice. CD21/35 and CD23 resolve three subsets resembling clusters I, II, and IV as identified by transcriptional profiles. Expression of CD5 separates the CD21/35⁻CD23⁻ population to a subset resembling cluster VI while CD5− cells are divided by high and intermediate staining of CD49d into two subsets, which differ in expression of CXCR3 and CD11b and resemble clusters III and V. Dotplots represent one of eight mice immunized 3× with NP-CGG/IFA. c Boxplots represent the frequency of subsets by cytometry according to expression of CD21/35, CD23, CD5, and CD49d among IgG⁺ Bsm. A non-parametric ANOVA (Friedman test) was performed (Two-stage linear step-up procedure of Benjamini, Krieger, and Ykutieli) P < 0.0001, followed by a one-sided paired t-test between spleen and BM cluster IV and V, respectively, n = 8. Boxplot indicates median, first and third quartiles, whiskers: 1.5 IQR. Source data for Fig. 4c is provided as a Source Data file.