Fig. 5: Humoral immune responses in FlipFlop mice.

Mice were immunized with NP-KLH/alum by i.p. injection and analyzed. a, T_FH cells in immunized B6 and FlipFlop mice (day 10). Numbers indicate percentage of CXCR5⁺PD1⁺ T_FH cells (top). Bar graph shows T_FH (CXCR5⁺PD1⁺) cell numbers (mean ± s.e.m.) among TCRβ⁺CD44⁺ (bottom). (n = 6, representative of 3 independent experiments). Gate for TCRβ⁺CD44⁺ T cells is shown in Extended Data Fig. 6a. b, Characterization of T_FH cells. T_FH cells (TCRβ⁺CD44⁺CXCR5⁺PD1⁺) in FlipFlop and B6 spleens were analyzed on post-immunization day 10 (n = 4 per strain, representative of 2–3 independent experiments). c, Numbers of B220⁺ B cells in immunized B6 and FlipFlop spleens (day 10) (B6: n = 13, FlipFlop: n = 14, 7 independent experiments). Staining profile is shown in Extended Data Fig. 6b. d, Circulating IgM and IgG1 anti-NP30 antibodies (optical density (OD) at 405 nm) in serum from B6 (black line), FlipFlop (purple line), and TCRα^KO (gray line) mice at 1 week post-immunization, measured by ELISA. Pre-immunization serum was included as a negative control (B6 and FlipFlop: n = 4, TCRα^KO: n = 3, 3 independent experiments); mean ± s.e.m. e, High-affinity IgG1 production. Concentration of IgG1 anti-NP2 antibody (ng/ml) in serum over time after immunization, as measured by ELISA (n = 3–4/strain, 3 independent experiments); mean ± s.e.m. f, Immunofluorescence staining of spleen sections from immunized B6 and FlipFlop mice (day 10). Staining of GL7 (green and yellow arrow, GC), CD3 (red, T cells), and IgD (white, B cells) is shown (n = 3 per strain, representative of 3 independent experiments). g, Numbers of NP-binding GC B cells (NP⁺B220⁺GL7⁺Fas⁺) in the spleens of immunized B6 and FlipFlop mice (day 10). Staining profile of immunized splenocytes is shown in Extended Data Fig. 6b (B6: n = 12, FlipFlop: n = 13, 6–7 independent experiments). h, Induction of NP-binding GC B cell induction in mixed BM chimeras from Extended Data Fig. 6c. Mixed BM chimeras reconstituted with both B6 and FlipFlop BM cells were immunized with NP-KLH/alum and were assessed 10 days later for numbers of B6-origin and FlipFlop-origin NP-binding GC B cells. Staining profile and gate are shown in Extended Data Fig. 6d,e (n = 6 per strain, 2 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test); mean ± s.e.m. (c,g,h).