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. 2018 Apr 2;215(4):1079–1090. doi: 10.1084/jem.20170369

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Figure 3. — Innate-like CD8⁺ and DN αβT cells produce IL-17 independently of STAT-3 but require the transcription factor RORγt. (A) DNA was isolated from at least 30,000 CD4⁺, CD8⁺, DN, and iNKT cells from CD4-Cre × Stat3^fl/fl (STAT-3 KO) or Stat3^fl/fl (WT) mice. Data shows copy number using TaqMan primers from ABI spanning intron4–exon4, (intact Stat3 region control, Mm00369932_cn) and intron20–exon20 (deleted region, Mm00369911_cn). (B–E) CD44^hiCD62L^lowαβ TCR⁺ γδ TCR⁻ cells (B–D) or naive T cells (E) were sorted from WT or STAT-3 KO mice and stimulated for 72 h with IL-1β and/or αCD3. Brefeldin A was added the last 6 h in culture. After incubation, cells were harvested, fixed, permeabilized, and stained for intracellular cytokines, IL-17 and IFN-γ, for flow cytometric analysis. (B) Representative dot plots showing IL-17 and IFNγ production in T memory cells from WT (top) STAT-3 KO (bottom) mice. Frequencies of the cell populations are within the plots. (C) Bars represent the mean frequency of IL-17⁺ cells in T memory cells after culture. Symbols represent individual mice and data are representative of five independent experiments. (D) Bars represent the fold increase ± SEM in IL-17 protein in supernatants after culture. Background levels ranged from 10 to 50 pg/ml. Total n = 2; each n was a single experiment consisting of cells pooled from three mice. (E) Representative flow plots showing IL-17 and IFN-γ production in naive T cells after 72 h of culture using Th0 (top) or Th17 (bottom) skewing condition. Data are representative of at least four independent experiments. (F) CD44^hiCD62L^lowαβ TCR⁺ γδ TCR⁻ cells from WT or RORγt^−/− mice were sorted and stimulated with IL-1β and αCD3 for 72 h. Brefeldin A was added the last 6 h of culture. Cells were harvested, fixed, stained for intracellular IL-17, and analyzed by flow cytometry. Bars represent the mean frequency ± SEM of live cells that were IL-17⁺. Symbols represent technical replicates from cells pooled from four mice from a single experiment that was independently performed two times.