Immunized Puma-deficient mice exhibited more antigen-specific CD38^high memory B cells compared with wild-type mice. Wild-type (Wt) and Puma-deficient (Puma^−/−) mice were injected intra-peritoneally with 100 μg NP coupled to KLH. After 14, 28, or 42 days, leukocytes were collected from the spleen, blood or bone marrow, then subjected to enumeration of AFCs by ELISPOT or stained with the indicated antibodies and analyzed by flow cytometry. (A-B) Viable IgG1⁺B220⁺ (but IgM⁻IgD⁻Gr1⁻Mac1⁻) B cells that can bind the immunizing hapten NP coupled to the fluorescent protein, allophycocyanin (APC), from spleen (A) and blood (B) were identified by flow cytometry. The total numbers of antigen-specific NP⁺IgG1⁺B220⁺ B cells in the spleen and their percentages in blood are presented in A and B, respectively. Data represent mean (± SD) of n = 3 at d14, n = 7-9 at d28 and at d42, n = 3 mice per genotype. (**P < .01, *** P < .005). (C-D) Frequencies of NP⁺IgG1⁺B220⁺ AFCs in the spleens (C, number) and bone marrow (D, percentage) of wild-type or Puma^−/− mice. The total column represents all AFCs (high plus low affinity for NP; ie, binding to NP20) and the high-affinity (antibodies binding to NP2) AFCs are represented by the black portion of the column. Affinity maturation is calculated as the ratio of NP2/NP20 cells and this is indicated at the top of each column. Data represent mean (± SD) of n = 3 at d14, n = 6 at d28, and at d42, n = 3 mice per genotype (P > .05). (E-F) NP⁺IgG1⁺B220⁺ B cells with a memory phenotype (CD38^high) were quantified by flow cytometry. The total numbers of memory B cells in the spleens (E) and the percentages of memory B cells in the blood (F) are indicated. Data represent mean (± SD) of n = 3 at d14, n = 4-9 at d28, and at d42, n = 3 mice per genotype. (**P < .01, *** P < .005).