Evidence for an intrinsic B-cell deficiency. (A) Transfer of B cells. Wild-type (WT) and WASp-deficient (WASp–) mice were immunized by intraperitoneal injection of SRBCs, diluted 1:10. This immunization was repeated 3 weeks later. On day 3 in the secondary response, donor B cells were purified from WT or WASp– spleens. CFSE-labeled donor cells (10⁷) were adoptively transferred into WT recipients, which had received an SRBC injection 3 days earlier. One day later, spleens were taken out and cryostat sections prepared. Representative images of spleen sections are shown. CFSE-labeled transferred B cells are green. B- and T-cell areas are detected with PE-labeled anti-B220 (red) and Cy5-labeled CD3 (blue) antibodies, respectively. The graphs show quantitative determination of transferred B cells. The numbers of transferred B cells were counted in the B-cell areas as well as in the total spleen area. Each bar represents measurements from 4 mice. Error bars show one SD and asterisks indicate a significant difference of P < .01 using a 2-tailed t test. Presented results are representative of 2 similar experiments. (B) Transfer of T cells. WT CD4⁺ T cells (10⁷) were adoptively transferred into WT or WASP– mice. The images show that transferred CFSE-labeled CD4⁺ T cells localize to the T-cell area of WT and WASp– spleens 24 hours after injection. (C) One day after transfer of unlabeled CD4⁺ T cells, mice were immunized with SRBCs diluted 1:10. Spleens were analyzed on day 6 or 10 after SRBC immunization. Representative images of spleen sections are shown. B- and T-cell areas were stained with PE-labeled anti-B220 (red) and Cy5-labeled anti-CD3 (blue) antibodies, respectively, and GCs were detected with biotinylated PNA, followed by streptavidin-FITC (green). Quantitative determination of white pulp areas is shown in the right-hand graphs. The B-cell, T-cell, and GC area was measured on images of random sections. The percentages of B-cell, T-cell, and GC areas of the total spleen area are shown. Each bar represents mean values from measurements of 3 different spleens. Error bars show one SD and asterisks indicate a significant difference of P < .05 using a 2-tailed t test. Numbers of direct PFCs detecting IgM-secreting cells and indirect PFCs detecting IgG-secreting cells are shown at day 6 after immunization. Each bar represents the mean value from 3 mice. Error bars show one SD. Presented results are representative of 2 similar experiments.