Reduced AID activity in AID^+/− B cells. (A) AID mRNA (graph, AID^+/+ and AID^+/−; n = 5) and protein (AID^+/+, AID^+/−, and AID^−/−), as measured by real-time PCR and Western blotting. Error bars represent the mean ± SD. (B, left) IgG1 switching (pseudocolor plot) profiles in wild-type and AID^+/− B cells activated ex vivo, as determined by flow cytometry. (right) Mean switching to IgG1, IgG3, and IgG2a. Values represent the mean ± SD (n = 5). Antibodies were B220-PercP-Cy5.5 and IgG1-PE. Dead cells were gated out using DAPI. (C) Flow cytometric measurement of cell proliferation and IgG1 switching on CFSE-labeled B cells activated for 96 h with LPS + IL-4. The graph represents percentages of γ1 switching at each cell division (n = 2). Error bars represent the mean ± SD. (D) The percentage of intra-switch deletions as determined by Southern blotting on hybridomas generated from AID^+/+ and AID^+/− lymphocytes activated for 96 h in the presence of LPS + IL-4. Intact Sμ domains result in a 6.9-kb band, whereas smaller bands (stars) represent intra-switch deletions. (E) Analysis of AID gene expression by single-cell RT-PCR (137 bp) from sorted LPS + IL-4–activated B cells. (F) Sμ mutation frequency in AID^+/+, AID^+/−, and AID^−/− activated B cells. Pie chart segments are proportional to the number of sequences carrying an equal number of mutations (indicated on the periphery). The total number of sequences is portrayed in the middle of each chart, and mutation frequency (calculated as the total mutations per total base pairs sequenced) and p-values are shown below the charts.

Paucity of translocation-bearing clones and CSR-induced DNA breaks in AID^+/− mice. (A) Igh-cMyc chromosomal translocations in BALB/c-Bcl-xL AID^+/+ and BALB/c-Bcl-xL AID^+/− mice (three mice per time point) were amplified by PCR 25 or 35 d after pristane injection using nested primers (arrows) specific for cMyc intron 1 and Igh Cμ, Cγ2b, and Cα constant domains. PCR products were analyzed by Southern blotting using cMyc- and Igh-specific probes. (B) PCR products appearing multiple times were sequenced and counted once if they represented a single clone. (C) Igh FISH in metaphase spreads from AID^+/+H2AX^−/− and AID^+/−H2AX^−/− B cells activated for CSR. (left) The loss of the red VH signal with an intact green Cα signal (micrograph) denotes a chromosome 12 break. (right) Total number of breaks observed for each strain (metaphases analyzed are indicated on top of each bar).

Happloinsufficiency in plasma cell tumor development in AID^+/− mice. (A) Photomicrographs of plasma cell tumors arising in BALB/c-Bcl-xL AID^+/+ and BALB/c-Bcl-xL AID^+/− mice show no significant variations in plasma cell morphology. Bar, 20 μm. (B, left) Incidence of plasma cell tumors in the presence of Bcl-xL as determined by the histological appearance of foci, with each containing 50 or more atypical plasma cells (red squares, plasma cell tumors in 36 out of 41 AID^+/+ mice injected with pristane; black circles, plasma cell tumors in 58 out of 96 AID^+/− mice). (right) Plasmacytoma development in AID^+/+ (n = 16) and AID^+/− (n = 39) mice in the absence of Bcl-xL.

Reduced AID activity in AID^+/− mice during the immune response. (A) Measurement of IgG1 levels in GCs from AID^+/+ and AID^+/− mice. Values represent the mean ± SD (n = 5). (B) Somatic hypermutation as determined by sequencing the JH4 intron from AID^+/+, AID^+/−, and AID^−/− GC cells. (C) ELISA analysis of serum from five AID^+/+ and AID^+/− mice immunized with NP-CGG. IgG1 antibodies displaying low affinity for NP were captured in this assay using NP₃₀-BSA, whereas high affinity antibodies were monitored with NP₇-BSA, as described in Results and discussion. Horizontal bars represent the mean.