Distribution and phenotype of splenic B cells and humoral immunity in Rag1-mutant mice. (A) Representative examples (left) and absolute count (right) of mature CD19/IgM⁺ splenic B cells (error bars represent standard error of the mean [SEM]). (B) Hematoxylin and eosin–stained sections of spleen counterstained for B220 and CD3 concentrated within follicles (dark staining). Original magnification ×4. (C) Spleens were analyzed by flow cytometry for follicular (CD19⁺CD93⁻CD21⁺CD23⁺) and marginal zone (MZ) B cells (CD19⁺CD93⁻CD21⁺CD23⁻) with percentage and absolute number of CD19⁺ cells shown on the right. (D) Immunoglobulin serum concentration (mean ± SEM) in naïve mice (n = 6-10 per group). Mice were immunized with TNP-Ficoll, and TNP-specific IgM (E) and IgG3 (F) were measured by enzyme-linked immunosorbent assay (ELISA; n = 4). (G) Mice were immunized with T-dependent antigen (TNP-KLH), and TNP-specific IgG was measured by ELISA (n = 4). (H) Th17 responses to immunization with Pneumococcal whole-cell antigen (WCA) antigen were measured by antigen-specific total IgG. One-way (A,C-D) and 2-way (E-H) analysis of variance: *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. OD, optical density.