Combined loss of IL-6 and IL-21 does not affect the virus-specific CD8 response but limits T_FH formation and the antibody response in influenza infection. Analysis of anti-influenza CD8 responses in WT and DKO animals. Mice were analyzed on day 10 after infection. (A) Splenocytes and cells from the bronchoalveolar lavage (BAL) were stained with α-CD8, α-CD44, and either NP-tetramer or PA-tetramer. Frequency distribution of splenic, virus-specific CD8⁺ T cells (tetramer stains: NP, black symbols; PA, gray symbols) is shown in a representative of two independent experiments using three to five animals of each genotype. (B) Frequency distribution of KLRG1/CD44 double-positive CD8⁺ T cells in spleen and bronchoalveolar lavage. Each symbol represents an individual animal. Bars and numbers show mean percentage with ± SEM. (C) WT, IL-6– or IL-21–deficient, and DKO mice were infected with HKx31 influenza virus and analyzed on day 10 after infection. Cells from the mLNs were stained for T_FH cells with α-CD4, α-CXCR5, and α–PD-1, and the percentage of PD-1/CXCR5 double-positive CD4⁺ T cells was measured. (D) Frequency of T_FH from WT and mutant mice analyzed on day 10 after infection. Representative example shown of two to six independent experiments, totaling 6 naive WT, 23 WT, 8 Il6^−/−, 8 Il21^−/−, and 19 DKO-infected mice, respectively. (E) CD4⁺PD-1⁺CXCR5⁺ T_FH cells and CD4⁺PD-1⁻CXCR5⁻ T cells were sorted from spleen on day 14 of HKx31 influenza infection. Bcl6 and Il21 expression was measured by RT-qPCR. (As expected, Il21 is not expressed in the DKO mice. This is a control only.) Bars and numbers show relative gene expression normalized to the housekeeping gene, Hmbs, ± SEM (n = 3). (F) IL-21–GFP reporter mice, on a WT or Il6^−/− background, were infected, and mLNs were harvested on days 6, 8, and 10 and stained for T_FH cells (CD4, TCR-β, CXCR5, and PD-1). The dot plot shows IL-21–expressing cells in the CD4⁺/TCR-β⁺ gate on day 10. (G) Time course showing total numbers of IL-21–expressing T_FH in the draining mLNs from days 6 to 10 of infection. Numbers show means ± SEM of five animals in each group. IL-21/GFP⁺ cells were also CD4⁺, TCR-β⁺, PD-1⁺, CXCR5⁺. (H) IgM and IgG HKx31-specific responses in WT, IL-6, IL-21R, and DKO mice. Serum IgM and IgG titers were measured by ELISA on day 14 of the influenza infection and are represented as the reciprocal of serum dilutions, giving an absorbance that was 50% of maximum value for the assay. Each symbol represents an individual animal. ***, P < 0.001; **, P = 0.001–0.001; *, P = 0.01–0.05. Bars and numbers show mean dilution ± SEM.