Puma-deficient mice contained more antigen-specific CD38^high memory B cells and AFCs than wild-type mice after secondary immunization. Wild-type and Puma^−/− mice were injected intraperitoneally with 100 μg NP coupled to KLH at day 0 and day 42. Five days after the second injection (day 47), leukocytes were collected from the spleen or bone marrow and subjected to ELISPOT for enumeration of AFCs or stained with the indicated antibodies and then analyzed by flow cytometry to determine the numbers of memory B cells. Numbers of splenic NP⁺IgG1⁺B220⁺ B cells (A) and NP⁺IgG1⁺B220⁺ CD38^high memory B cells (B) of wild-type or Puma^−/− mice. (C) Frequencies of anti-NP Ig secreting AFCs in the bone marrow 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. Data represent mean (± SD) of n = 3 at d42 and at d47, n = 7 mice per genotype. (**P < .01, *** P < .005).