Figure 2. Isolation, Expansion and Characterization of CCR4⁺CCR6⁺Th17 cells.

A- Frequency of IL-17 secreting CD4⁺ T cells in ex vivo PBMCs or in vitro cultured CD4⁺ memory T cells. Cells were stimulated with PMA/Ionomycin and stained for CD4 and intracellular IL-17. Result shown is representative of 6 independent experiments.

B- Characterization of CD4⁺IL-17⁺ T cells. FACS analysis of ex vivo isolated (top) and in vitro primed Th17 cells (bottom) for different surface markers (black lines) and isotype controls (filled histograms). Shown are representative histograms of 4 independent experiments.

C- Sorting of Th17 cells based on CD4 and IL-17 expression and post sorting analysis. In vitro primed Th17 cells were stimulated and sorted as described in Materials and Methods. CD4⁺IL-17⁺ and CD4+IL⁻17⁻ cells were collected as shown. The sorted cell populations were restimulated with PMA/Ionomycin to determine IL-17 and IFN-γ secretion in parallel. Shown is representative dot plot of 3 independent experiments.

D and E- Sorted CD4⁺IL-17⁺Th17 or CD4⁺IL17^neg T cells were γ-irradiated and co-cultured with autologous CD3⁺CD8⁺ T cells as described in Materials and Methods. Proliferation (D) and IFN-γ secretion (E) of CD8⁺ T cells are shown as the cumulative result of 3 independent experiments. *p<0.05.