TLo stimulation induces higher numbers of proliferating Th17 cells than THi stimulation. (A-B) Human CD4⁺ T cells were labeled with CFSE and cultured under THi or TLo conditions for 6 days. Proliferating IL-17⁺ (A) and IFN-γ⁺ (B) cells were assessed by intracellular flow cytometry. Plots are representative of 3 independent experiments. (C) The number of alive cells on day 6 was determined by cell counting using trypan blue, and the absolute number of IL-17⁺ cells was then calculated using the proportion of IL-17⁺ cells after intracellular flow cytometry (n = 3). Error bars represent SEM. P values were calculated using the Student t test: *P < .05. (D) Human CD4⁺ T cells were cultured under THi or TLo conditions for 3, 6, or 10 days. To generate day 10 cells, beads were removed at day 6 and cells were washed, replated, and cultured for 4 more days. Expression of IL-17 was determined by intracellular flow cytometry. Day 3 and 10 graphs: n = 3; day 6 graphs: n = 4. Error bars represent SEM. P values were calculated using the Mann-Whitney U test: *P < .05