Figure 6.

Effector functions of antigen presenting cells are enhanced upon contact with irradiated tumor cells. (a) Phagocytosis and antigen uptake. Immature DCs were differentiated from primary human monocytes (PKH67-labeled) with 40 ng/mL IL-4 and 20 ng/mL GM-CSF for 5 days. Afterwards, DCs were co-incubated with irradiated HCC1937 cells (4 days after irradiation, PKH26-labeled) at the indicated target:effector ratios. Phagocytosis was allowed for 2 h and analyzed by flow cytometry. The percentage of double-positive DCs with ingested HCC1937 cell material is shown as means ± SD of 5 independent experiments. Group comparison was calculated by two-way ANOVA with Bonferroni-Holm correction. (b) DCs were incubated with 20 µM cytochalasin D 1 h prior to performing the phagocytosis assay at a ratio of 1:4 as in (a). (c) Priming of T cell proliferation. DCs were differentiated from primary human monocytes upon exposure to supernatants of irradiated HCC1937 cells or TNF (100 ng/ml) as in Figure 5A. After 7 days, DCs were co-incubated with CFSE-labeled T cells from an allogeneic donor at a ratio of 1:5 (DC:T cells) for additional 5 days before T cell proliferation was analyzed by flow cytometry. The percentage of proliferating T cells was calculated as the percentage of CD3⁺CFSE^lowCD4⁺ or CD3⁺CFSE^lowCD8⁺ on the basis of all CD3⁺CD4⁺ or CD3⁺CD8⁺ cells, respectively. Results were normalized on the corresponding 0 Gy samples and are displayed as data from 6 independent experiments. Group comparison was carried out by two-sided exact Wilcoxon rank test with Bonferroni-Holm correction.