Fig. 4.

TGF-β causes downregulation of CD16 on NK cells and limits ADCC capability in vitro. NK cells from human blood were isolated and cultured for 3, 7 or 14 days in the presence of 5 ng/ml IL-15 and 500 IU/ml IL-2 plus the indicated cytokines. (a) CD16 expression on NK cells (CD45⁺lin⁻CD56⁺NKp46⁺) across indicated timepoints. (b) representative flow cytometry plots and (c) histogram plots at day 7. Data are from a 5 biological replicates (n = 5). Error bars indicate mean ± SEM. Groups were compared using Mann–Whitney test, where P > 0.05 was deemed not significant, where ∗P < 0.05. (D) CD16 expression on NK cells (CD45⁺CD3⁻NKp46⁺) treated with TGF-βR inhibitor (LY2157299) cultured for 7 days. (e) representative flow cytometry plots and (f) histogram plots at Day 7. Data are from 3 biological replicates, where symbols denote different patients, with two technical replicates each (n = 3). Error bars indicate mean ± SEM. Groups were compared using Mann–Whitney test, where P > 0.05 was deemed not significant, where ∗P < 0.05, and ∗∗P < 0.01. (g) cytotoxicity of NK cells against bladder cell line UM-UC-3 in the presence of indicated conditions. Data are from 2 biological replicates, where symbols denote different patients, with 3 technical replicates each (n = 2). Error bars indicate mean ± SEM. Groups were compared using Mann–Whitney t test, where P > 0.05 was deemed not significant, where ∗P < 0.05, and ∗∗P < 0.01.