Fig. 3.

IL-7 promotes expansion of IL-17–competent γδ T cells via selective STAT3 activation. (A) Staining for Ki67 (cells in cycle) in gated γδ²⁷⁻ (Left) and γδ²⁷⁺ (Right) LN cells ex vivo (gray line) and after 4-d culture with IL-7 (black line). Shaded histograms show Ki67 isotype staining. (B) Offset histograms of γδ²⁷⁺ (red) and γδ²⁷⁻ (blue) LN cells labeled with CFSE and then cultured for 4 d with IL-7. Shaded gray area represents γδ T cells stained ex vivo. (C) CFSE-labeled LN cells were cultured for 4 d with IL-7, activated with PMA + ionomycin and stained for intracellular IL-17 and gated on γδ cells. (D) Flow cytometric detection of intracellular pSTAT5 and pSTAT3 in gated γδ²⁷⁻ and γδ²⁷⁺ cells as labeled. Open and shaded areas indicate IL-7 treatment and controls, respectively. (E) Intracellular localization by ImageStream Flow Cytometry of pSTAT3 among two representative CD44^hi γδ²⁷⁻ (Upper) and CD44^lo γδ²⁷⁺ (Lower) LN cells after 30-min culture with IL-7 (BF, Bright field). (F) LN cells preincubated with a specific STAT3 inhibitor and subsequently cultured with IL-7 for 72 h. Representative plots (from n = 3 experiments). (G) Staining for Ki67 and intracellular IL-17 in gated γδ T cells cultured as in F; open and shaded areas indicate STAT3 inhibitor preincubation and controls, respectively. For all plots, numbers indicate percent of cells in relevant gate or quadrant.