Figure 2. Direct TGF-β is required for influenza-specific Tfh differentiation.

2 × 10⁵ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO) Stg cells were adoptively transferred into C57BL/6 congenic recipients infected with WSN-GP33/66 the following day. (A–D) On day 8 p.i, Stg cells in the MLN, spleen, and lung were stained with antibodies against the indicated proteins to distinguish Th1 and Tfh cells. Cells were also stimulated with GP₆₆-peptide for 6 hr to assess IFN-γ and IL-2 production by intracellular cytokine staining and flow cytometry. (E) Stg cells (blue, highlighted by white arrows) located within MLN PNA⁺ GCs (green) were assessed using immunofluorescent microscopy and their numbers were enumerated using Imaris software. Graphs in A–D are representative of one of five independent experiments (n = 4–5 mice/group/experiment). Panel E shows representative microscopy images and the cumulative data from two independent experiments with 8 total mice/group were graphed. *p < 0.05, **p < 0.005, and colored asterisks correspond to the color in the stacked graphs.

DOI: http://dx.doi.org/10.7554/eLife.04851.005

Figure 2—figure supplement 1. Direct TGF-β restricts anti-viral T_H1 precursor formation but does not impact overall effector CD4 T cell differentiation during LCMV.

(A) Stg chimeric mice (1 × 10⁶ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO)) were infected with LCMV Armstrong and 3 days p.i., spleens were collected and analyzed by flow cytometry for the indicated proteins. (B–D) Stg chimeric mice (1 × 10⁴ CD44^lo TGF-βRII^+/+ CD4-cre⁺ (WT) or TGF-βRII^f/f CD4-cre⁺ (KO)) were infected with LCMV Armstrong and the phenotype of Stg cells was assessed 8 days p.i. Data are representatives of five independent experiments with 15–20 total mice/group. *p < 0.05.

DOI: http://dx.doi.org/10.7554/eLife.04851.006

Figure 2—figure supplement 2. Expression of PSGL1 and Ly6C distinguish between influenza-specific Th1 and Tfh CD4 T cells.

2 × 10⁵ Stg CD4 T cells were adoptively transferred into congenic C57BL/6 recipient mice, which were infected with influenza WSN-GP33/66 i.n. the following day. 8 days p.i., lymphocytes in the MLN were assessed for the indicated proteins. (A and C) Representative example of Stg cells in the MLN. In the histograms, red lines denote PSGL1^hi Ly6C^hi Stg cells, blue is PSGL1^hi Ly6C^lo Stg cells, and green is gated on PSGL1^lo Ly6C^lo Stg cells. (B) Stg cells from the MLN, spleen, lung, and airways. (C) includes FoxP3⁺ Tregs in the shaded gray histograms. Data depict an individual mouse representative of more than 30 total mice from 10⁺ independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.04851.007