Minimal participation of MBCs from IM WT prime after 28 days of mucosal boosting. (A) Gating strategy for experiment featured in Figs. 8 and S5. (B) Vaccination schedule using S1pr2-Cre tdTomato fate tracking mice. Tamoxifen was administered in mice 14 days after prime at time of optimal GC B cell production, and mice were boosted on day 28. Mice were culled 2 wk after boost, with LILN and RILN, respectively, CLN, and lungs collected for analysis. The data are compiled from three separate experiments. (C) Frequencies of GC B cells (Fas⁺GL7⁺IgD⁻B220⁺) in LILN, RILN, CLN, and lungs as measured by cell staining and flow cytometry. (D) For RILN and CLN cells that had measurable frequencies of GC B cells, the proportion of Ad-WT^IM prime-derived (tdTomato⁺) cells (median group response) were indicated in pie charts. (E) Frequencies of o-RBD⁺ B cells (IgD⁻B220⁺) in LILN, RILN, and CLN. (F) Proportion of o-RBD⁺ B cells that were derived from the Ad-WT^IM prime (tdTomato⁺) cells (median group response) were indicated in pie charts. (G) Frequencies of o-RBD probe–specific spleen and lung PCs that were WT spike probe–reactive (CD19⁻CD138⁺IgD⁻IgM⁻o-RBD⁺WT-S⁺). Data were produced from the experiment featured in Fig. 1. Mann–Whitney tests were completed to test for statistically significant differences between groups (*P < 0.05). For data in C and E, a t test was performed. **P < 0.01. On violin plots, the dashed black line represents the group median and dots represent individual mice. Data shown are pooled from three independent experiments (n = 2–3 per group per experiment).