Figure 4

Ipilimumab triggers ex-vivo isolated NK cells to kill melanoma cells and to secrete TNF-α. Panel A, NK cells were analyzed for their cytolytic activity in an ADCC assay using Ipilimumab (0.2-2.0-20 μg/ml) and melanoma primary cell lines (MECA, MECO, MEMO, METR), as well as established cell lines (MeWo, C32 and FO-1) at different effector to target cell ratio (E:T = 40:1, 20:1, 10:1, 5:1, 2:1, 1:1). NK cells were incubated with target cells in medium alone (none) or in the presence of Rituximab used as isotype-matched control Ab. Results are expressed as % of ⁵¹Cr specific release and are the mean ± SD of experiments with 6 donors. Panel B, effect of the addition of anti-FcγRIIIA mAb (α-FcγRIIIA) on the ADCC triggered with Ipilimumab (at the indicated doses) using ex-vivo NK cells and CTLA-4⁺ MECO target cells. Results obtained by adding an anti-NCAM mAb (α-NCAM), as isotype-matched control Ab for the anti-FcγRIIIA mAb, to ADCC assay are shown. ***P < 0.0001, **P < 0.0002. Panel C, lysis of PHA-stimulated PBMC or MECO or C1R-neo CD20⁺ B cells using ex-vivo NK cells alone (none) or with Ipilimumab or Rituximab (at 20 μg/ml). Panel D, NK cells, ex-vivo isolated from 4 different healthy donors, were incubated with MECO at the E:T ratio of 1:1 alone or in the presence of Ipilimumab or Rituximab (2.0 μg/ml) for 24 h. Then, culture SN were harvested and analyzed by ELISA for the presence of TNF-α. Results are expressed as pg/ml of TNF-α/10⁵ NK cells. Statistical significance: ***P < 0.001, **P < 0.005.