Evaluation of mechanisms underlying ADCC mediated by M-DC8⁺ DCs.

(A) Freshly isolated M-DC8⁺ DCs were preincubated for 1 hour at 4°C with 50 μg/mL (■), 100 μg/mL (░), or 200 μg/mL (▪) of either an anti-FcγRIII (CD16), or an anti-FcγRII (CD32), or an isotype-matched control antibody. In addition, M-DC8⁺DCs were preincubated with a combination of an anti-FcγRIII (CD16) and an anti-FcγRII (CD32) antibody, each at 200 μg/mL (). In this experiment an isotype-matched antibody at 400 μg/mL was used as control (). DCs were cocultured either with ⁵¹Cr-labeled COLO 205 cells in the presence of 17-1A (10 μg/mL) at an E/T ratio of 40:1 (A) or with⁵¹Cr-labeled SK-BR-3 cells in the presence of Herceptin (10 μg/mL) at an E/T ratio of 10:1 (B). After 18 hours of incubation, chromium release was measured. Columns represent means ± SE of results obtained from 4 different donors. For each donor the mean of triplicate determination was taken. Asterisks indicate a statistically significant difference (P < .05) between the inhibition of ADCC in the presence of anti-FcγRIII (CD16) or anti-FcγRII (CD32) antibody or a combination of both anti-FcγR antibodies and the isotype-matched control antibody. (C) ⁵¹Cr-labeled COLO 205 cells were incubated with M-DC8⁺ DCs at an E/T ratio of 40:1 in the presence of 10 μg/mL 17-1A antibody with or without a neutralizing anti–TNF-α antibody at a concentration of 10 μg/mL. After 18 hours of incubation, chromium release was determined. The results of 2 different donors are presented as mean ± SE of triplicate wells.