IL-6 and IL-21 are expressed early during an influenza infection. (A) CD4⁺ and CD19⁺ cells were isolated from mLNs on the indicated days after influenza infection. Il21 or Il6 mRNA expression was measured by qPCR. (B) Cells from the mLNs of naive and infected animals were stained with α-CD19, α-CD11c, α-CD11b, α-CD69, and α-CD86. Resting (G1) and activated follicular B cells (G2) were sorted. Bars and numbers show relative Il6 expression normalized to Hmbs ± SEM (n = 3) in each sorted population. (C) Time course of total cell numbers of activated and GC B cells, DCs, and macrophages in mLNs of infected mice. Numbers are means ± SEM of five mice. (D) Co-culture of WT naive CD62L⁺/CD4⁺ T cells, stimulated with α-CD3/α-CD28, and different numbers of CpG-preactivated B cells from control or IL-6–deficient mice. The left panel shows CD3/CD28-activated T cells with medium alone or with recombinant IL-6. After 4 d of co-culture, the CD4⁺ T cells were sorted, and Il21 mRNA expression was determined. The results are representative of three independent experiments. (A and D) Error bars represent SDs of triplicate assays. (E and F) WT, congenic Ly5.1⁺ B cells were injected into WT and DKO animals 2 d before influenza infection. 10 d after infection, cells from the mLNs were stained for T_FH and GC B cells as described for Figs. 2 C and 1 A, respectively. Figures show fold change of each animal’s T_FH or GC B cells compared with the mean percentage of T_FH or GC B cells from controls within each experiment. Results are from three to six independent experiments. (E) T_FH ratio comparing WT and DKO animals without or with rescue by WT Ly5.1 B cells. (F) GC B cell ratio comparing WT and DKO animals without or with rescue by WT Ly5.1 B cells. Each symbol represents an individual animal. Statistical analyses were performed using the two-sample Wilcoxon test, and all p-values are two-sided. Bars and numbers show fold change ± SEM.