IL-21–producing CD8⁺ T cells promote isotype switching in B cells in vivo during influenza virus infection. (A) WT or IL-6 KO mice were infected with a sublethal dose of H1N1 PR8 virus. 7 d (d 7) or 14 d (d 14) after infection, relative IL-21 mRNA levels in CD8⁺ T cells from lung or spleen/MLN (S/M) were determined. For 7 d, WT and IL-6 KO lungs, n = 5 and WT S/M, n = 3; for 14 d, WT lungs and S/M, n = 3. (B) CD8⁺ T cells from lungs or S/M of mice infected with influenza virus, as in A, for 14 d were assayed for the number of IL-21–producing cells (number of spots) and the total area of spots (area) per well by ELISPOT assay (n = 3). (C) IL-21 KO mice as host mice received WT CD8⁺ or IL-21 KO CD8⁺ T cells and were infected with a sublethal dose of H1N1 PR8 virus. After 14 d, lung tissue was immunostained and imaged. Percentage of GL-7⁺ cells relative to the number of B220⁺ cells in lungs of these mice was determined. (D) Percentage of GC B cells (PNA^hi FAS^hi IgD⁻) within the B220 population in lungs from influenza virus infected (day 14) IL-21 KO mice that had received WT CD8⁺, WT CD4⁺, IL-21 KO CD8⁺, or IL-21 KO CD4⁺ T cells (n = 4). (E–I) Sera from IL-21 KO recipients as described in C were analyzed for influenza-specific IgM (E; P = 0.5703), IgG (F; P = 0.0012), IgG1 (G; P = 0.2278), IgG2c (H; P < 0.0001), and IgG3 (I; P = 0.0002) levels by ELISA. (J) Sera from IL-21 KO recipients as described in C were i.p. transferred to WT mice 1 d after infection with a lethal dose of influenza virus. (WT-CD8, blue, n = 9; IL-21 KO-CD8, red, n = 10). Kaplan-Meier survival curve is shown. P = 0.05. (K) Confocal microscopy images of lungs from influenza virus–infected WT mice (day 12) showing the staining for B220, GL-7, and CD8 individually or as a merged image in lymphoid structures. Bars, 50 µm. Error bars represent the mean ± SD. *, P < 0.05, as determined by Student’s t test. (B and C) One-way ANOVA (A and D), two-way ANOVA (E–I), or log-rank test (J). Results are representative of two to three experiments.