Fig. 3.

IL-2 signaling is required for γδ T cell IL-17 production. a Lung cells isolated from naive mice were plated with anti-CD25, anti-CD122, or isotype antibody for 2 h then stimulated with IL-2 or vehicle for 25 min. Cells were fixed, permeabilized, and stained with antibodies specific for CD3, CD4, TCRγδ, and pSTAT5, as well as a live/dead marker. γδ T cells were gated as in Fig. 1 and the expression of pSTAT5 is presented. One experiment representative of two biological replicates is shown. b pSTAT5 expression in γδ T cells from four combined independent experiments is shown. Data shown are individual biological replicates +/− s.e.m. and each dot represent a mouse. Statistical significance was evaluated by student’s t-test. c Lung cells isolated from naive mice were stimulated with IL-2 or vehicle for 20 h with different doses of Ruxolitinib, a JAK1/2 inhibitor. Culture supernatants were obtained at 15–20 h and assayed for IL-17 by ELISA. d In a simultaneous experiment with C, the toxicity of Ruxolitinib was tested. Lung cells were stimulated with a low dose of PMA and ionomycin (P:I) for 20 h with different doses of Ruxolitinib. Culture supernatants were obtained and assayed for TNF by ELISA. IL-17, and TNF production is shown from four combined independent experiments. Each experiment was performed with lung cells obtained from 14−20 mice. Each dot represents an independent biological replicate. Data shown are individual biological replicate +/− s.e.m. Statistical significance was evaluated by student’s t-test