Fig 1.
Ex vivo secretion of IL-10 and IL-17 in human circulating CD4 memory T cells.
(A) Ex vivo–isolated CD4+ T cells were stimulated with PMA/ionomycin during 6hrs, stained with anti-CD45RA, -IL-10, -IL-17, -IFN-γ and -IL-2 mAbs and analyzed by flow cytometry. A dot plot, gated on total memory (CD45RA−) cells, is shown for one donor, and the proportions of memory CD4+ T cell secreting IL-10 or/and IL-17 ex vivo are summarized for all donors (n = 8). (B-C) Dot plots illustrating the co-secretion of IFN-γ and IL-2 in the indicated subpopulations are shown for one donor and the proportion of IFN-γ (n = 8) and IL-2 (n = 6) co-secreting cells in the indicated subpopulations is summarized. Statistical analyses were performed using the Mann—Whitney U test. **p < 0.01, ***p < 0.001, ns, Non-significant.
Fig 2.
Circulating CD4 T cells secreting IL-10 ex vivo, alone or with IL-17, include FOXP3- cells and FOXP3+ Helios- pTreg.
Ex vivo–isolated CD4+ T cells were stimulated with PMA/ionomycin, stained with anti-CD45RA, -IL-10, -IL-17, -FOXP3, and -Helios mAbs and analyzed by flow cytometry. (A) Dot plots depicting FOXP3 expressing cells within cytokine secreting populations are shown for one donor. The proportion of FOXP3 expressing cells within each cytokine secreting populations and their mean fluorescence intensity (MFI) are summarized for all donors (n = 8). (B) The proportions of cells co-secreting IL-2 or IFN-γ within the indicated populations are summarized for all donors (n = 4). (C) The proportions of Helios expressing cells within FOXP3+ Treg in the indicated populations are summarized for all donors (n = 8). Statistical analyses were performed using the Mann—Whitney U test. *p < 0.05, **p < 0.01, *** p < 0.001, ns, Non-significant.
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.
Fig 4.
IL-10 and IL-17 secretion levels of clonal CD4 T cells vary depending on the mode of stimulation.
Pools of IL-10+ clones and IL-17+/IL-10+ clones (left side) or pools of IL-17+ clones and IL-17+/IL-10+ clones (right side) were stimulated with anti-CD3 and anti-CD28 or with PMA and ionomycin (A), or with anti-CD3 and anti-CD28, alone or in combination (B), or with PMA and ionomycin, alone or in combination (C). The levels of IL-10 and IL-17 secreted were assessed in the 24hrs culture supernatants, by ELISA. Data shown are mean with SEM of duplicates and representative of four independent experiments. Statistical analyses were performed using the Mann—Whitney U test. *p < 0.05, ***p < 0.001, ns, Non-significant.
Fig 5.
IL-10 and IL-17 secretion levels of clonal CD4 T cells vary depending on time after stimulation.
IL-10+ clones (n = 137), IL-17+ clones (n = 144) and IL-17+/IL-10+ clones (n = 83) were stimulated with anti-CD3/28, rested for 9 or 14 days, then restimulated. The levels of IL-10 and IL-17 secreted were assessed in the 24hrs culture supernatants, after stimulation, by ELISA. Statistical analyses were performed using the Mann—Whitney U test. *p <0.05, **p<0.01, ***p < 0.001.
Fig 6.
Secretion of IL-10 but not of IL-17 is increased in the presence of sodium butyrate.
Pools of IL-10+ clones and IL-17+/IL-10+ clones (A) or pools of IL-17+ clones and IL-17+/IL-10+ clones (B) were stimulated with anti-CD3 and anti-CD28 alone or in combination or with PMA and ionomycin, alone or in combination, in the absence or in the presence of sodium butyrate. The levels of IL-10 (A) and IL-17 (B) secreted were assessed in the 24hrs culture supernatants, by ELISA. Data are shown as mean with SEM of duplicates. Data are representative of three independent experiments. Statistical analyses were performed using the Mann—Whitney U test. **p < 0.01, ***p < 0.001, ns, Non-significant.