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. 2019 Jul 16;216(9):2170–2183. doi: 10.1084/jem.20181953

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© 2019 Moser et al.

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Figure 7. — B cell Itch limits antibody responses to immunization. (A) Experimental design for adoptive transfer and immunization. (B) Donor NP-binding GC B cells (CD45.2⁺NP⁺GL7⁺) from spleen of recipient mice were enumerated by flow cytometry after immunization (n = 3 for day 4, n = 6 for day 8, n = 10–12 for day 12, and n = 6 or 7 for day 20, two or three independent experiments, two-way ANOVA with Sidak post-test). (C and D) NP-specific IgG1 PCs were enumerated on day 12 and day 20 after immunization in the spleen (C) and BM (D) by ELISPOT (n = 10–12 for day 12 and 6 or 7 for day 20, three independent experiments, two-way ANOVA with Sidak post-test for spleen, Mann–Whitney test for BM). (E–G) Donor-derived NP-specific IgG1[a] was quantified by ELISA. NP-BSA with either a high or low conjugation ratio was used to capture total or high-affinity NP-specific antibody, respectively. (E) Low-affinity antibody = total − high. (F) High-affinity antibody. (G) Ratio of high/low affinity was enumerated within each mouse (for day 12, n = 10–12, for day 20, n = 6 or 7, three independent experiments, two-way ANOVA with Sidak post-test). (H) Chimeras were immunized with NP-OVA, and after 12 d, NP⁺GL7⁺ GC B cells were FACS-sorted. Vh186.2 gene was amplified using RT-PCR, and the amplicon was cloned and sequenced. Data are derived from sequences (WT, 18; Itch KO, 34) that were obtained from multiple mice (WT, 4; Itch LP, 10) and two independent experiments (two-way ANOVA and multiple t tests were performed). Error bars indicate SEM. *, P < 0.05; **, P < 0.01.