Modulation of Cytokine Secretion Allows CD4 T Cells Secreting IL-10 and IL-17 to Simultaneously Participate in Maintaining Tolerance and Immunity
Fig 3
Ex vivo isolation of IL-10 or/and IL-17 secreting populations and generation of clones.
(A) CD4+ T cells were stimulated ex vivo with PMA/ionomycin. Cells secreting IL-10 and/or IL-17 were assessed using a two-color cytokine secretion assay combining the IL-10 and the IL-17 secretion assays from Miltenyi Biotec and sorted by flow cytometry to a high degree of purity. Dot plots show the cytokine secreting populations prior to and after cell sorting. (B-C) Clones were generated by limiting dilution culture and assessed for cytokine secretion after PMA/ionomycin stimulation by intracellular staining or by ELISA. (B) Dot plots are shown for representative IL-10+, IL-17+ and IL-17+/IL-10+ clones. (C) The levels of IL-10 and IL-17 secreted by representative clone of each population were assessed in the 24hrs culture supernatants after PMA/ionomycin stimulation by ELISA. Mean and SEM of duplicates are shown. (D) IL-17+ clones (n = 136), IL-10+ clones (n = 275) and IL-17+/IL-10+ clones (n = 17) were assessed for cytokine secretion by ELISA. Data are representative of three independent experiments. Statistical analyses were performed using the Mann—Whitney U test. *p < 0.05, ***p < 0.001.