Figure 6. T-bet⁺ T_FH cells are induced during viral infection are a stable population functionally distinct from T-bet⁻ T_FH.

(A to C) Tbx21-Cre mice were intranasally infected with 50 TCID₅₀ influenza (PR8). Mediastinal lymph nodes were collected on indicated time points and analyzed by confocal microscopy and flow cytometry. (A) Cell numbers of CD4⁺CD44⁺RFP⁺ and CD4⁺CD44⁺RFP⁻ in B cell follicles. (B) Percent RFP⁺ of CD4⁺CD44⁺CXCR5^HiPD-1^Hi T cells analyzed by confocal microscopy. (C) Numbers of NP tetramer⁺CD4⁺CD44⁺CXCR5^HiPD-1^HiRFP⁺ T cells. (D) Tbx21^{tdTomato-T2A-creERT2}Rosa26^{Lox-STOP-Lox-YFP} mice were intranasally infected with 50 TCID₅₀ influenza (PR8); 7 days after infection mice were gavaged with 1 dose of tamoxifen. Mice were analyzed on day 14 after infection. Percent RFP⁺ of YFP⁺CD4⁺CD44⁺CXCR5^HiPD-1^Hi T cells. (E and F) Tbx21-Cre; Bcl6-YFP/Bcl6^WT; Foxp3^Thy1.1 mice were intranasally infected with 50 TCID₅₀ influenza (PR8). Mediastinal lymph nodes were collected on day 14. Indicated cell types were sorted and analyzed by RNA-seq. (E) Heatmap showing k-means clustering of genes differentially expressed in any pairwise comparison. Values are Z-score normalized by row. (F) Expression patterns of select IFN and IL-12 signaling, T_FH signature and cell migration-related genes. (A to C) The data are shown as mean ± s.e.m. (D) Each point represents an individual mouse with mean ± s.e.m. All data are representative of ≥ 2 experiments, n ≥ 3 mice per group. (E and F) RNA-seq analysis of gene expression was performed using three biological replicates.