Supplementary Figure 2: Analysis of the B cell immune response in MD4 host mice and flow-cytometry gating strategies.

a, Flow cytometry plots and quantification of PP cells derived from wild-type and MD4 mice showing the frequency of GC, SED and T_FH cell populations. Data are pooled from two independent experiments with three mice in each experiment for GC and SED staining (n=6); one and two wild-type (n=3), two and three MD4 mice (n=5) for T_FH staining analysis. Line represents mean * P<0.05, two-tailed Student’s t test. ns, not significant. b, Representative LSFM images of a PP derived from MD4 mice that were transferred with B1-8^hi GFP⁺ cells and did not receive antigen. Scale bar, 300 μm. Two independent experiments with two mice in each experiment (n=4) showed similar results. c, Representative flow cytometry analysis and gating strategy of adoptively transferred B1-8^hi GFP⁺ and B1-8^lo DsRed⁺ cells in GCs of recipient MD4 mice nine days after NP-CT administration. d, Representative gating and analysis of naïve, GC and SED B cell proliferation by EdU incorporation in wild-type mice (upper panels). Mice were intravenously injected with EdU on day 9 after immunization with NP-CT and analyzed after additional 2.5 hours. Representative EdU analysis gated on these populations is shown with naïve as negative control (lower panels). Similar analysis was held following adoptive transfer of B1-8^hi GFP⁺ and B1-8^lo DsRed⁺ B cells, five days following NP-CT administration as shown in Fig. 2g.