Humoral immunodeficiency in Sppl2a^−/− mice. (A) Percentage of CD19⁺ B cells among blood lymphocytes and representative IgM/IgD flow cytometric plots gated on CD19⁺ B cells from mice of the indicated genotypes. Numbers in top left corner are geometric mean fluorescence intensity of IgD, and other numbers are percentage of cells in each gate. ****, P < 0.0001. (B) Schematic of Sppl2a splice-donor mutation, the resulting skipping of exon 7, and the effect on the SPPL2A protein. (C) Surface IgD on GFP⁺IgM⁺ Sppl2a^−/− B cells from IL-7 bone marrow cultures transduced with a retroviral vector encoding wild-type SPPL2A and GFP (thick line) or empty vector encoding GFP alone (thin line), compared with Sppl2a^+/+ B cells transduced with empty GFP vector (shaded gray). (D) ELISA analysis of total serum IgM and IgG1 in unimmunized animals. (E) ELISA analysis of serum antibodies to B. pertussis and CGG 2 wk after immunization, against the ABA 4 wk after immunization, and against NP-Ficoll 6 d after booster immunization. Bars represent the mean, and each symbol represents a single mouse. Data are representative of more than five experiments with at least three animals per group in each (A), one experiment (B and C) or two (D), and one experiment with at least five to seven mice per group (E).