Bim-deficient mice accumulated abnormal numbers of AFCs but still only recruited high-affinity AFCs into the bone marrow. Wild-type and bim^−/− mice were immunized with NP-KLH. After 14 or 28 days, spleen and bone marrow were harvested and ELISPOT assays performed to determine the numbers of NP-specific AFCs. (A) The picture shows the total of all IgG1⁺ NP-specific AFCs (high and low affinity; ie, antibodies capable of binding to NP₂₀) and the high-affinity (ie, antibodies capable of binding to NP₂ but not NP₂₀) IgG1⁺ NP-specific AFCs from the spleen (note: wt input, 10⁶ cells; bim^−/− input, 10⁵ cells). (B) Frequencies of NP-specific IgG1⁺-secreting AFCs in the spleen (left, total cell number) and bone marrow (right, percentage) of wt, bim^−/−, and bcl-2 transgenic mice. The total column represents all AFCs (high plus low affinity; ie, binding to NP₂₀) and the high-affinity (antibodies binding to NP₂) AFCs are represented by the black proportion of the column. Affinity maturation is calculated as the ratio of NP₂/NP₂₀ cells and this is shown on top of each column. Data represent the mean of n = 3 to 9 mice; *P ≤ .008 (spleen) and P ≤ .04 (bone marrow) for both high-affinity and low-affinity anti-NP AFCs. (C) Splenic B cells from NP-KLH immunized wt (white columns) and bim^−/− (black columns) mice were cultured in simple medium (no added cytokines) for the indicated times, and ELISPOT assays were performed to enumerate the surviving NP-specific (high-affinity; ie, binding to NP₂) AFCs. The number of NP-specific IgG1⁺ AFCs at time 0 h was set as 100%, and the proportion surviving after the different times in culture is displayed. Data represent mean (± SD) from 4 cultures of 2 mice of each genotype. *P ≤ .02.