LMP1 signaling substitutes CD40 in germinal center formation and production of class-switched and high-affinity antibodies. (A) Flow cytometry to identify germinal center (GC) B cells (CD95⁺PNA^high) in the spleens of wt CD40^+/+ and CD40/LMP1⁺//CD40^−/− mice at the indicated days after immunization with 100 μg NP-CGG. Cells are gated on B220⁺; numbers indicate the mean percentages (± SEM) of GC B cells of 2 to 5 mice analyzed per group. (B) Immunohistologic analysis of GC in the spleen, 14 days after immunization with 100 μg NP-CGG. Cryosections were stained with anti-IgM specific for B cells (red) and PNA specific for germinal center B cells (blue). Original magnification, ×50. (C) NP-specific antibody response 14 days after immunization with 100 μg NP-CGG. NP-specific immunoglobulin concentrations for the indicated isotypes were analyzed for 5 mice from both wt CD40^+/+ (♦) and CD40/LMP1⁺//CD40^−/− (▲). (D) Affinity maturation of NP-specific IgG1 antibodies at days 7, 14, 21, and 28. The ratio of antibody binding to low-density hapten (NP3-BSA) versus high-density hapten (NP17-BSA) is plotted for 2 to 5 mice per group.