Cell contact, GM-CSF, and TNF-α mediate DC_Th differentiation. (A, B) CD14⁺ monocytes were cultured for 6 days with IL-2 (Mono), IL-2 and autologous T_H cells (DC_Th), or IL-2 and autologous T_H cells on opposite sides of a 0.4 μm transwell membrane (Transwell) for 6 days. Cell surface expression of DC associated molecules is shown. (A) Representative FACS plots are shown from 4 independent experiments and > 10 donors. Isotype controls for straining (Isotype) are shown as dashed lines. (B) MFIs are shown. Dashed lines indicate the average MFIs of isotype control Ab used for staining from all culture conditions. Data are from 4 independent experiments and > 10 donors (mean and SEM), **P < .01, ***P < .001 compared with Mono. (C) CD14⁺ monocytes were cultured for 6 days with IL-2 and isotype control Ab (Mono Iso) or autologous T_H cells, IL-2 and the indicated isotype control or neutralizing Ab. Filled columns indicate rat IgG1 Ab, while open columns indicate mouse IgG1 Ab. Mono Iso (hatched columns) represent combined values of rat IgG1 and mouse IgG1 isotype control Ab. 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 4 independent experiments and > 10 donors (mean and SEM), *P < .05, **P < .01, ***P < .001.