T_H cell specific cytokines and monocyte-T_H cell contact regulate the formation of specialized DC_Th subsets. (A,B) DC_Th1 (open columns), DC_Th2 (hatched columns) or DC_Th17 (filled columns) were formed in the presence of isotype control (Iso) or the indicated neutralizing antibodies for 6 days. (A) MFIs of DC-associated molecules are shown after excluding T_H cells. Data are from 5 independent experiments and > 10 donors (mean and SEM), *P < .05; **P < .01; ***P < .001. (B) On day 6 of culture, each DC_Th coculture was stimulated with LPS in the presence of the indicated isotype control (Iso) or neutralizing Ab for 36 hours. Supernatants were subsequently collected for measurement of IL-1β, IL-6, IL-10, IL-12p70, IL-23p19, and TNF-α by ELISA. Data are from 5 independent experiments and > 10 donors (mean and SEM), *P < .05; **P < .01; ***P < .001. (C) CD14⁺ monocytes were cultured for 6 days with IL-2 (Mono), IL-2 and allogeneic T_H cells (DC_Th), or IL-2 and allogeneic T_H cells on top of a 0.4 μm transwell membrane and CD14⁺ monocytes below the transwell insert (Transwell). MFIs of cells below the transwell insert are shown. Dashed lines indicate the average MFIs of isotype control Abs used for staining from all culture conditions. Data are from 1 experiment and 3 donors (mean and SEM). (D) CD14⁺ monocytes were cultured for 6 days with IL-2 and isotype control Ab (Mono, hatched column) or T_H cells, IL-2 and the indicated isotype control (open columns) or neutralizing Ab (filled columns). MFIs are shown after excluding T_H cells. Dashed lines indicate the average MFIs of isotype control Ab used for staining from all culture conditions. Data are from 3 independent experiments and 6 donors (mean and SEM), *P < .05.