Figure 6. Three different signatures of memory B cells were identified by single-cell RNA-Seq analysis in vaccinated mice.

(A) The expression of CD73, CD80, and PD-L2 was determined on dLN EYFP⁺GL7^– memory B cells by flow cytometry 50 days after boost from AID-Cre-EYFP mice that were primed and boosted (day 30) with homologous and heterologous combinations of the aP and wP vaccines or injected with alum (ctr). Three doses of tamoxifen were administrated at days 7, 10, and 31. Representative flow cytometry plots are shown for aP:aP and wP:wP conditions. CD73⁺CD80⁺ and PD-L2⁺CD80⁺ memory EYFP⁺ populations in dLNs are shown in the graphs for all vaccine combinations. (B) Uniform manifold approximation and projection (UMAP) and clustering of 707 EYFP⁺ GC and 1,962 EYFP⁺ memory sorted B cells analyzed by scRNA-Seq from dLNs of AID-Cre-EYFP mice 50 days after boost injection, and tamoxifen administration on days 7, 10, and 31, from 2 aP:aP-, 3 aP:wP-, 3 wP:aP-, and 2 wP:wP-immunized mice. (C) Selected gene expression for the 6 different clusters (numbered 0–5) is presented in dot plot scale on normalized unique molecular identifier (UMI) counts. Frequencies (D) and absolute numbers (E) of EYFP⁺ memory B cells within clusters 0, 3, and 4 are shown for the 4 vaccine combinations. (F) Isotype profile determined by flow cytometry during cell sorting is shown for the EYFP⁺ memory B cells from clusters 0, 3, and 4. Means (±SEM) are shown in panels A, D, and E.