Extended Data Fig. 6: Ablation of Hhex in GC B cells impairs memory B cell differentiation following T_H2-type immunization.

a, Representative FACS plots of the percentage of S1pr2-Tomato⁺ cells among splenic B220⁺IgD^loGL7⁻CD38⁺NP⁺ cells in Hhex Het and KO mice at day 30 post NP-CGG in alum immunization. Data are representative of 2 independent experiments with 3 mice per group. b, Percentage of B cells that are GC B cells (B220⁺IgD^loGL7⁺CD95⁺NP⁺S1pr2-Tomato⁺) and MBCs (B220⁺IgD^loGL7⁻CD38⁺NP⁺S1pr2-Tomato⁺) in Hhex Het and KO mice at day 30 post NP-CGG in alum immunization. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. c, Ratio of NP⁺ MBCs to NP⁺ GC B cells in Hhex Het and KO mice at day 30 post NP-CGG in alum immunization. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. d, Representative FACS plots of CD44 and CD62L expression on MBCs (B220⁺IgD^loGL7⁻CD38⁺S1pr2-Tomato⁺) at day 12 post NP-CGG in alum immunization. Data are representative of 2 independent experiments with 3 mice per group. e, Percentage of CD44⁻CD62L⁻ (left), CD44⁺CD62L⁻ (middle), and CD44⁺CD62L⁺ (right) MBCs at day 12 post NP-CGG in alum immunization. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. f, Frequency of GC B cells (B220⁺IgD^loGL7⁺CD95⁺S1pr2-Tomato⁺) and MBCs (B220⁺IgD^loGL7⁻CD38⁺S1pr2-Tomato⁺) in Hhex Het and KO mice at day 12 post NP-CGG in alum immunization that have W33L mutation in the canonical NP-responding gene VH186.2. Data are pooled from 2 independent experiments with 3 mice per group. g, Representative FACS plot (left) and percentage (right) of CD138⁺ cells that are CD45.2⁺S1pr2-Tomato⁺ at day 5 post challenge with NP-CGG in alum. Equivalent numbers of splenic CD45.2⁺ T and B cells from Hhex Het and KO NP-CGG in alum immune mice were transferred to naïve CD45.1⁺ recipients one day prior to challenge. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. h, Percentage of CD138⁺ cells (left) and GC B cells (right) that were CD45.2⁺S1pr2-Tomato⁺ at day 5 post challenge with NP-CGG in alum when normalized to the percentage of MBCs present in the transferred cells. Data are pooled from 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean.