Figure 2. c-Rel gain enhances immune responses upon immunization.

(A) CD95^hiCD38^lo GCB and CD138⁺B220^lo plasma cell numbers 10–12 days after SRBC immunizations obtained from 4 independent experiments. Bars and numbers below graphs are medians. *P ≤ 0.05, ***P ≤ 0.001, paired t test. (B) Areas of splenic GCs quantified based on Bcl6 immunofluorescence in 5 CD19Cre^I/+ and 5 Rel^TG CD19Cre^I/+ mice 10 days after SRBC immunizations (CD19Cre^I/+, n = 61; Rel^TG CD19Cre^I/+, n = 53). Bars and numbers below graphs are means. **P ≤ 0.01, unpaired t test. (C) Percentages of Ig subtypes in splenic plasma cells obtained by intracellular flow cytometry 10–12 days after SRBC immunization. Displayed medians include data from 6 mice from 2 independent experiments. **P ≤ 0.01, paired t test. (D) Individual anti-SRBC IgG1 titers analyzed by flow cytometry 10 days after immunization of 2 independent experiments. Bars and numbers below graphs are medians. (E) NP-CG–immunized mice analyzed after 14 days: Individual percentages of CD95^hiCD38^lo GCB and IgG⁺NP⁺ GCB cells within all splenic B cells are plotted. Bars and numbers below graphs are medians. *P < 0.05, unpaired t test. (F and G) Specific IgG1 serum titers measured by ELISA and calculated by absorbance summation (61) following NP-CG immunizations. Titers for NP2 (high affinity) and NP23 (high and low affinity) (F) and the NP2/NP23 ratio (G) are displayed from 2 independent immunizations. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, multiple t tests. (H) Affinity maturation 14 days after NP-CG immunizations analyzed by sequencing of IgG1 VH186.2 rearrangements in GCB cells for 2 independently immunized cohorts with 2 mice per genotype: 4 CD19Cre^I/+ mice: immunization I, n = 126 sequences; immunization II, n = 148 sequences; 4 Rel^TG CD19Cre^I/+ mice: immunization I, n = 122 sequences; immunization II, n = 117 sequences. The percentage of sequences with the VH186.2 W33L mutation is shown. SPL, spleen; Imm, immunized; ND, not determined. See Supplemental Figures 5 and 6.