LMP1 signaling induces cytokine-independent class-switch recombination to IgG1. (A,B) CD43- and IgG1-depleted splenic B cells were labeled with CFSE and cultured in the presence of the indicated stimuli for 5 days. Cells are gated on living (PI⁻) cells. Numbers indicate means of percentages plus or minus SEM of IgG1⁺ cells of 4 independent experiments. (A) CD40/LMP1⁺//CD40^−/− B cells showed CSR to IgG1 after stimulation with agonistic anti-CD40 antibody (α-CD40) only, whereas in wt CD40^+/+ B cells CSR was dependent on IL4. (B) Anti-IL4 antibody (15 μg/mL) was added daily to the cultures. The percentages of IgG1-positive cells were not affected in anti-CD40–stimulated CD40/LMP1⁺//CD40^−/− B cells, whereas CSR was inhibited by anti-IL4 antibody in wt CD40^+/+ B cells stimulated with anti-CD40 and IL4. (C) Mixed B-cell cultures. IgG1-negative B cells of Ly5.1⁺ CD40^+/+ and Ly5.2⁺ CD40/LMP1⁺//CD40^−/− mice were isolated by depletion of CD43⁺ and IgG1⁺ cells and cocultured in a 1:1 ratio for 5 days with the indicated stimuli. Treatment with anti-CD40 antibody induced CSR in only Ly5.2⁺ CD40/LMP1⁺//CD40^−/−, but not in Ly5.1⁺ CD40^+/+, B cells. In contrast, stimulation with anti-CD40 and IL4 led to equal amounts of IgG1⁺ B cells in both cell types. This indicates that CSR of CD40/LMP1⁺//CD40^−/− B cells reflects an intrinsic effect of the LMP1 signaling domain and not an increased release of cytokines. Numbers indicate means of percentages of IgG1⁺ cells of 3 independent experiments. Further evidence that LMP1 signaling induces cytokine-independent CSR to IgG1 is given in Figure S5.