Production of antibodies by WASp-deficient mice is reduced and delayed, but class switching in vitro is enhanced. (A) Individual antibody-secreting cells were enumerated from spleens of immunized wild-type (WT) and WASp-deficient (WASp–) mice, using the hemolytic plaque assay. Mice were immunized by intraperitoneal injection of SRBCs, diluted 1:10 (results presented in large graphs) or 1:100 (small graphs). Numbers of direct PFCs detecting IgM-secreting cells and indirect PFCs detecting IgG-secreting cells are shown as a function of days after immunization (DaI). Each bar represents the mean value from at least 4 mice. Error bars show one SD and asterisks indicate a significance level of P < .05 using a 2-tailed t test. Presented results are representative of 2 similar experiments. AD indicates antigen dose. (B) Antibody response to the T-cell–independent antigen dextran. The 6 WASp– mice produced much less, if any, dextran-specific antibodies compared with 6 WT mice. Serum antibody titers were measured 6 or 9 days following intraperitoneal injection of 2 μg dextran. The dextran-specific immunoglobulins were analyzed in serum dilutions using ELISA. The level of significance was calculated using a 2-tailed t test and the bracket indicates P < .01. (C) Numbers of IgG2b- and IgG1-producing B cells. WT and WASp– B cells were cultured with LPS or IL-4 plus anti-CD40 mAbs for 4 days. IgG-producing cells were identified after intracellular staining and flow cytometry. The percentage of IgG2b- and IgG1-producing cells is indicated above the brackets in the histograms (left). The graphs to the right indicate the percentage of IgG2b- and IgG1-producing cells from 4 WT and 4 WASp– mice. Brackets indicate a significance level of P < .01 using a 2-tailed t test. The isotype-matched controls had less than 0.5% stained cells. Presented results are representative of 2 similar experiments.