Figure 3. Aicda^GV/GV B cells do not undergo CSR ex vivo.

(A-F) Naïve splenic B cells from mice of the indicated genotypes were stimulated for 72 or 96 hrs with LPS plus IL-4 (A,B), LPS (C,D), or B cell activating factor (BAFF), retinoic acid (RA), IL-4, TGFβ, IL-5, and LPS (E,F). Class switch recombination (CSR) to IgG1 (B), IgG3 (D), or IgA (F) was quantified at 72 and 96 hrs post-stimulation. Representative CSR gates shown in (A), (C), and (E) (gated on live cells). (G) Retroviral rescue of CSR in Aicda^GV/GV B cells. Aicda^GV/GV B cells were infected with retroviruses expressing GFP only (empty vector, EV), wild type AID (AID^WT) or catalytically dead AID^H56R/E58Q (AID^CD), stimulated with LPS plus IL-4, and IgG1 CSR was quantified as a frequency of live infected cells (GFP⁺) at 72 and 96 hrs following stimulation. (H) Expression of AID^G133V ex vivo. Whole cell extracts were prepared from WT and Aicda^GV/GV purified naïve splenic B cells stimulated for 96 hrs with LPS plus IL-4, and immunoblotted with anti-AID antibodies; loading control, HSP90; quantification shown in Figure S4C. (I) Expression of AID^G133V in vivo. Following immunization with SRBCs, splenic GC B cells were sorted at d7, whole cell extracts were prepared and immunoblotted with anti-AID antibodies; loading control, α-Tubulin. Data in (A-F, H) are from, or representative of, 4 independent experiments with 1–6 mice per genotype, (G) are from 2 independent experiments with 3 mice, (I) are from one experiment with 2–3 mice per genotype. Aicda^GV/GV, Aicda^G133V/G133V. Error bars represent the mean ± std. dev. ****p < 0.0001; ns, not significant, p ≥ 0.05. All comparisons p < 0.001 in (B) unless noted; all comparisons p < 0.0001 in (D,F) unless noted. p-values calculated using a one-way ANOVA with Tukey’s multiple comparisons test with (G), or without pairing (B,D,F).