1. IL-17 produced by innate sources in the FGT is critical for potentiating Th17 responses primed by vaginal APCs. Vaginal cells from hormone cycle–matched WT (C57BL/6) or IL-17A−/− mice (n = 5) were pooled, pulsed with chicken OVA peptide, and cultured alone (TC) or cocultured at a 1:1 ratio with CFSE-stained OT-II Tg CD4+ T cells (TC+CD4) for 3.5 d.
IL-17 produced by innate sources in the FGT is critical for potentiating Th17 responses primed by vaginal APCs. Vaginal cells from hormone cycle–matched WT (C57BL/6) or IL-17A−/− mice (n = 5) were pooled, pulsed with chicken OVA peptide, and cultured alone (TC) or cocultured at a 1:1 ratio with CFSE-stained OT-II Tg CD4+ T cells (TC+CD4) for 3.5 d. (A) IL-17 levels in culture supernatants were measured by ELISA. (B) On day 2 of coculture, a cell stimulation mixture containing Golgi inhibitors and PMA plus ionomycin was added, and 18 h later, cocultures were stained with Abs against CD3, CD4, and IL-17 and analyzed by flow cytometry to identify IL-17+ cells and measure MFI. Upper box indicates CFSE−, IL-17–producing cells in culture. The lower box indicates CFSE+, IL-17–producing CD4+ T cells, with a lower cut-off that excludes 95% of isotype control CSFE+ cells. (C) The difference in MFI was compared between innate and Th17 sources of IL-17 (n = 3 independent experiments). Data were analyzed using an unpaired t test with 95% confidence interval (*p < 0.05). (D) Similar cocultures were conducted, with 250 ng/ml of rIL-17 added during the peptide-pulse stage (TCIL-17p+CD4) and washed away before coculture or added on day 1 of coculture (TC+CD4+IL-17) and remained present throughout the duration of the experiment as indicated on the x-axis. IL-17 levels in culture supernatants were measured by ELISA. Data in (A) and (D) are mean ± SEM of three individual wells per coculture condition. Data are representative of three independent experiments with similar results. Significance was calculated by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance).
Varun C. Anipindi et al. ImmunoHorizons 2019;3:
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