Figure 2.

Effects of ADCC culture conditions on PD-L1 expression by NB cells. PD-L1 expression was analyzed on LA-N-1 cells by flow cytometry and data are shown by representative histograms (A-C) and rgMFI values (D) as described in Materials and Methods. Results show PD-L1 expression after 24 h incubation with ch14.18/CHO (A), ch14.18/CHO in combination with leukocytes (ADCC conditions, B) and ch14.18/CHO with leukocytes and anti-idiotype ganglidiomab (anti-Id) (specificity control, C). When the control histogram (isotype control) is not visible it is covered by the experimental histogram (PD-L1 staining). The effect of ADCC conditions on PD-L1 expression was also analyzed in the presence of IL-2 (D). Cells were stained with PE-labeled anti-human PD-L1 Ab. To distinguish between PD-L1-positive NB cells (PD-L1⁺/GD₂⁺/CD45⁻) and leukocytes (PD-L1⁺/GD₂⁻/CD45⁺), Alexa647-labeled anti-GD₂ and PE/Cy7-labeled anti-CD45 mAb were used. The PD-L1 expression level was quantified using relative geometric mean fluorescence intensity (rgMFI) according to the formula: MFI of PD-L1 by treated cells - MFI of PD-L1 by the respective untreated control. Ch14.18/CHO and rituximab served as controls. Data represent mean values ± SEM of at least 3 independent experiments. t-test, ^##P < 0.01 vs. ch14.18/CHO, ^###P < 0.001 vs. leukocytes, ^§§§P <0.001 vs. ADCC without IL-2, *P < 0.05 vs. ADCC without IL-2, **P < 0.01 vs. leukocytes, ***P < 0.001 vs. LA-N-1 incubated with IL-2.