Lactic acid inhibits DC activation during antigen-specific autologous T-cell stimulation. (A) Purified CD8⁺ T cells were labeled with CFSE prior to stimulation with peptide-pulsed DCs in the absence or presence of lactic acid. After 1 to 6 days, T cells were stained with anti-CD3 and anti-CD25. For flow cytometric analysis, CD3⁺ cells were gated and analyzed after 24 hours up to 144 hours. (B) TADCs show an impaired IL-12 secretion compared with control DCs. MelIm-MCTSs and PC3-MCTSs were generated using the liquid overlay culture technique. On day 5, half of the medium was replaced with a monocyte suspension. After another 4 to 5 days, MelIm-TADC and PC3-TADC cocultures were matured for 72 hours with 10 ng/mL LPS. Control DCs were also matured for 72 hours. Supernatants were harvested and stored at -20°C. IL-10 and IL-12 (p70) were determined in the culture supernatants with commercially available ELISA kits (^*P < .05). (C) Lactic acid inhibits LPS-stimulated cytokine secretion by control DCs. DCs were generated in RPMI-1640 supplemented with GM-CSF and IL-4 and harvested on day 7 of culture. DCs (1 × 10⁶/2 mL) were transferred into 6-well plates and stimulated with 100 ng/mL LPS in the absence or presence of various concentrations of lactic acid. After 24 hours, supernatants were harvested, filtered, and stored at -20°C. IL-10 and IL-12 (p70) were determined in the culture supernatants with commercially available ELISA kits (^*P < .05). (B-C) Error bars indicate SEM.