Low-strength T-cell stimulation favors Th17 responses. (A-D) Human CD4⁺ T cells were cultured under THi or TLo conditions for 6 days. (A-B) THi and TLo cells were restimulated with PMA/ionomycin for 5 hours, and expression of IL-17 and IFN-γ were determined by intracellular flow cytometry. Plots of 1 example of 24 independent experiments are shown (A). (B) Percentages of IL-17 and IFN-γ single and double producers; data of 24 independent experiments are shown. (C) THi and TLo cells were restimulated with PMA/ionomycin for 24 hours, and cytokines were measured by ELISA (n = 3). (D) Expression of IL-17 and ROR-γt in TLo cells was determined by intracellular flow cytometry. One representative of 3 independent experiments is shown. (E) Mouse CD4⁺ T cells were cultured under THi or TLo conditions, and expression of IL-17 and IFN-γ were determined by intracellular flow cytometry (n = 6). (F) Human CD4⁺ T-cells were cultured under TLo conditions in the presence (“TLo”) or absence (“w/o cytokines”) of IL-1β, IL-23, and TGF-β for 6 days. Expression of IL-17 and IFN-γ were determined by intracellular flow cytometry. Results are representative of 3 independent experiments. Horizontal bars represent the median value; error bars represent SEM. P values were calculated using the Wilcoxon test: *P < .05; ***P < .0001.