Rela is required for the generation of GC-derived plasma cells. (A–C) rela^fl/flCγ1-Cre, rela^fl/+Cγ1-Cre, and Cγ1-Cre mice were immunized with SRBC (A) or NP-KLH (B and C) and analyzed 14 or 28 d later. (A) CD138 and eGFP expression by splenic mononuclear cells from mice of the corresponding genotypes were analyzed by flow cytometry. Numbers below gates indicate the percentage of CD138^hieGFP⁺ or CD138^hieGFP⁻ cells. (Right) Data are cumulative from two experiments with 5–7 mice per genotype, with each symbol representing a mouse. Data are shown as mean ± SD. Statistical significance was determined by Student’s t test (*, P < 0.05). (B) Serum response to NP-KLH immunization before (Pre), 12 d after primary (Primary), and 7 d after secondary (Boost) immunization with NP-KLH (top). Ratio of anti-NP₉ to anti-NP₂₅ IgG1 antibodies (bottom). Data are cumulative from two experiments with 5 mice per genotype, with each symbol representing a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01). (C) Elispot analysis for NP-specific IgG1 ASCs in spleen and BM on days 14 and 28 after NP-KLH immunization. One representative Elispot experiment per genotype is shown for day 14 spleen and day 28 BM. Control, unimmunized mouse. (Bottom) Data from 3–5 independent experiments/genotype/tissue/time point are summarized. Data are shown as mean ± standard deviation. Statistical significance was determined by Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Purified B cells from rela^fl/flCD19-Cre and CD19-Cre mice were stimulated in vitro with LPS and analyzed 3 d later for CD138 and B220 expression by flow cytometry. Numbers besides gates indicate the percentage of CD138^hiB220^low cells. Representative example (left) and pairwise representation of individual experiments (right) for CD138^hiB220^lo expression of LPS-stimulated B cells, with each line representing an independent experiment (n = 6). Statistical significance was determined by Student’s t test (**, P < 0.01). (E) Representative Western blot analysis (left) of purified B cells ex vivo (day 0) and stimulated for 3 d with LPS for expression of BLIMP1 and IRF4 protein, and mean relative density of 3 experiments (right) compared with CD19-Cre B cells. Data are shown as mean ± standard deviation. Statistical significance was determined by Student’s t test (***, P < 0.001). (F) Purified B cells from rela^fl/flCD19-Cre and CD19-Cre mice were stimulated in vitro with LPS+IL-4 and analyzed 4 d later for CD138 and IgG1 expression by flow cytometry. Numbers within gates indicate the percentage of CD138^hiIgG1⁻ and CD138⁻IgG1⁺ cells. Representative example (left) and pairwise representation of individual experiments (right), with each line representing an independent experiment (n = 3). Statistical significance was determined by Student’s t test (**, P < 0.01). Experiments shown in C–E were performed with two different RELA-conditional mouse lines (see experimental procedures) that gave similar results. (G) Purified B cells from rela^fl/flCD19-Cre and CD19-Cre mice (p65^fl/+ cohort) were stained with CFSE on day 0, stimulated with LPS+IL-4 and analyzed by flow cytometry on days 2–4 for IgG1 expression to determine the fraction of class-switched cells in relation to cell cycle as determined by CFSE dilution. Numbers indicate the percentage of cells in the corresponding quadrants. One out of three independent experiments is shown.