Figure 1
Figure 1. DC-mediated activation of human IL17-producing T cells. T cells were culture alone (T alone) or with immature DCs (T + IDC), cytokine-matured DCs (T + Cyt DC), lypopolyscharide-matured DCs (T + LPS DC) or poly I:C–matured DCs (T + PIC DC). At 5 days later, the T cells were examined for the production of IL17 using PMA/ionomycin in the presence of GolgiStop for 5 hours. The cells were labeled with aqua LIVE/DEAD dye to distinguish dead cells, fixed, permeabilized, and simultaneously stained for the presence of IL17, IL2, and IFNγ. (A) Representative plot from one of the experiments showing the frequency of the IL17 + T cells in the cultures (top panel). Middle panel shows the phenotype of IL17-producing cells (data gated on CD3+ T cells). Bottom panel shows the cytokine profile of the IL17-producing cells in the various conditions. Data gated on CD3+IL17+ cells and analyzed for the expression of IL2 and IFNγ. The numbers represent the percentage of Th17 cells expressing IL17 alone or with IL2 or IFNγ. (B) Panel on the left is a bar graph showing the percentage of IL17 cells that make IL17 with or without IFNγ. Panel on the right shows the percentage of IL17 cells that make IL17 with or without IL2. The graphs represent data from 7 different healthy donors. *P < .05 when compared with T cells alone. **P < .05 when compared with T cells alone as well as T cells cultured with immature DCs. (C) Expansion of IL17-producing T cells by DCs. T cells were cultured with DCs as above for 5 days. After 5 days, IL2 was added to the DC–T-cell cocultures, and the T cells were cultured for an additional 7 days. On day 12 of culture, the T cells were stimulated with PMA and ionomycin to assess the frequency of IL17-producing T cells. Figure shows frequency of IL17-producing cells after coculture with cytokine-matured as well as LPS- and poly I:C–matured DCs. (D) T cells cultured alone (T alone) and those from cocultures with Cyt-DCs as in panel C were stimulated with PMA/ionomycin for 5 hours, and the expression of RORC was analyzed by TaqMan. (E) T cells cultured alone (T alone) and those from cocultures with Cyt-DCs as in panel C were stimulated with PMA/ionomycin for 5 hours, and supernatants were assayed for the presence of IL17 by ELISA. Error bars in panels D and E indicate SD.

DC-mediated activation of human IL17-producing T cells. T cells were culture alone (T alone) or with immature DCs (T + IDC), cytokine-matured DCs (T + Cyt DC), lypopolyscharide-matured DCs (T + LPS DC) or poly I:C–matured DCs (T + PIC DC). At 5 days later, the T cells were examined for the production of IL17 using PMA/ionomycin in the presence of GolgiStop for 5 hours. The cells were labeled with aqua LIVE/DEAD dye to distinguish dead cells, fixed, permeabilized, and simultaneously stained for the presence of IL17, IL2, and IFNγ. (A) Representative plot from one of the experiments showing the frequency of the IL17 + T cells in the cultures (top panel). Middle panel shows the phenotype of IL17-producing cells (data gated on CD3+ T cells). Bottom panel shows the cytokine profile of the IL17-producing cells in the various conditions. Data gated on CD3+IL17+ cells and analyzed for the expression of IL2 and IFNγ. The numbers represent the percentage of Th17 cells expressing IL17 alone or with IL2 or IFNγ. (B) Panel on the left is a bar graph showing the percentage of IL17 cells that make IL17 with or without IFNγ. Panel on the right shows the percentage of IL17 cells that make IL17 with or without IL2. The graphs represent data from 7 different healthy donors. *P < .05 when compared with T cells alone. **P < .05 when compared with T cells alone as well as T cells cultured with immature DCs. (C) Expansion of IL17-producing T cells by DCs. T cells were cultured with DCs as above for 5 days. After 5 days, IL2 was added to the DC–T-cell cocultures, and the T cells were cultured for an additional 7 days. On day 12 of culture, the T cells were stimulated with PMA and ionomycin to assess the frequency of IL17-producing T cells. Figure shows frequency of IL17-producing cells after coculture with cytokine-matured as well as LPS- and poly I:C–matured DCs. (D) T cells cultured alone (T alone) and those from cocultures with Cyt-DCs as in panel C were stimulated with PMA/ionomycin for 5 hours, and the expression of RORC was analyzed by TaqMan. (E) T cells cultured alone (T alone) and those from cocultures with Cyt-DCs as in panel C were stimulated with PMA/ionomycin for 5 hours, and supernatants were assayed for the presence of IL17 by ELISA. Error bars in panels D and E indicate SD.

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