Fig. 3. Hells expression in B cells is mandatory for efficient TD humoral immune responses and establishment of high-affinity MBCs.

a, b Mice were immunized i.p. with alum-adsorbed NP-CGG and boosted 6 weeks later. Serum was collected at day 7, 14, and 41 after the primary immunization, and 5 days after the boost. NP-specific IgM (a), and IgG1 (b) from Mb1Hells^KO and control animals (n for numbers of mice used is indicated in figures) were quantified by ELISA (arb.units, arbitrary units). c Representative Flow Cytometry (FC) plots of splenic IgG1-secreting cells 5 days after antigenic boost, and quantification of these cells (n = 8 for Mb1Hells^KO, n = 9 for controls). d Enumeration by ELISPOT assay of splenic IgG1-ASC and NP-specific IgG1-ASC, 5 days after antigenic boost. Each well is representative of a triplicate (n = 8 for Mb1Hells^KO, n = 9 for controls). e NP₄-BSA and NP₂₃-BSA-binding IgG1 were quantified by ELISA in the serum of the animals described in (b) (n = 8 for each genotype) and the NP₄/NP₂₃ ratio was calculated. f Representative FC plots of splenic NP-specific IgG1⁺ MBC 28 days after immunization with NP-CGG, and quantification of NP-specific IgG1⁺ MBCs per million B220⁺ B cells in the spleen, 7, 14, 28 and 43 days after immunization (n = 7 for each genotype). g Proportion of VH186.2 BCR sequences from single-cell sorted NP-specific IgG1 MBCs at day 28 post NP-CGG immunization that bear W33L or K59R substitutions in control (n = 7) and Mb1Hells^KO (n = 8) animals. The numbers of sequences (W33L or K59R/total) are indicated at the center of the pies. Experiments (a) to (f) were done twice, (g) was done three times, and data were pooled. Bar charts and error bars represent the mean ± SD. Unpaired two-tailed Welch’s t-test were performed for (a), (b) and (e), and unpaired two-tailed t-test were performed for (c), (d) and (f). Source data are provided in Source Data File.