Low-affinity B cell clones are unable to seed GCs in the NALT independent of competition. (A) MD4 mice were adoptively transferred with GFP⁺ B1-8^hi or Rosa26^tdTomato/+ B1-8^lo B cells. NALT and MedLN were removed and imaged by TPLSM on day 7 following i.n. NP-OVA + MPLA immunization. Scale bars, 200 µm. n = 4 for each time point; two independent experiments. (B–D) MD4 mice were injected with GFP⁺ B1-8^hi or Rosa26^tdTomato/+ B1-8^lo B cells. NALT and MedLN were collected and analyzed by flow cytometry on day 3, 5, and 7 following i.n. NP-OVA + MPLA immunization. Representative plots for GC B cells on day 5 are shown (B). Flow cytometry quantification for GC B cell percentage of B1-8^hi or B1-8^lo B cells. Day 3, n = 5–6; day 5, n = 4–6; day 7, n = 5–7; six independent experiments. Unpaired two-tailed Student’s t test; data represent mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ns, not significant (C and D). (E and F) MD4 mice were injected with Rosa26^tdTomato/+ B1-8^hi or B1-8^lo B cells mixed with CD45.1⁺ OT-II T cells. LNs, BM, and spleen were collected and analyzed by flow cytometry on day 14 following i.n. NP-OVA + MPLA immunization. n = 5–6; two independent experiments. Unpaired two-tailed Student’s t test; data represent mean ± SEM; ns, not significant. Flow cytometry quantifications for total transferred cells (E) and GC B1-8^hi or B1-8^low B cells (F) are shown. (G and H) WT mice were injected with Rosa26^tdTomato/+ B1-8^hi or B1-8^lo B cells. LNs were analyzed by flow cytometry 5 days after i.n. NP-Ficoll + MPLA immunization. n = 6; two independent experiments. Unpaired two-tailed Student’s t test; data represent mean ± SEM; **, P < 0.01; ns, not significant.