Supplementary Figure S9 from Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

Supplementary Figure S9. (A and B) Representative dotplots and summary results showing the ability of TAM pretreated with blocking anti-CD64 F(ab')2 and anti-CD32 F(ab')2 fragments (5ug/ml) to kill PKH67+ A431 cells at a 1:1 E:T ratio in the presence of cetuximab (1ug/ml) in a 12 hrs FACS-based assay. Dead A431 tumor cells were defined as TO-PRO-3+PKH67+ cells. (C and D) Representative dotplots and summary results demonstrating the ability of HLA-A2neg TAM to stimulate of Ly95 cells responses (intracellular IFN-γ) after their preincubation with cetuximab-opsonized A549/HLA-A2+NY-ESO tumor cells at the indicated time points. Kruskal-Wallis multiple comparisons test. Data represented as mean ± SEM. (E) The expression of NY-ESO-1 protein in different batches of transduced A549 tumor cells. The expression of intracellular NY-ESO was evaluated by flow cytometry using NY-ESO-1 mAb (clone D1Q2U) that recognizes endogenous levels of total NY-ESO-1 protein. Non transduced A549 cells were used as a control. (F-H) Representative dotplots and cumulative data showing the ability of HLA-A2+TAM to stimulate of Ly95 cells responses (intracellular IFN-γ) after preincubation with A549/HLA-A2+NY-ESOhi (F) and A549/HLA-A2+NY-ESOlo tumor cells (G) in the presence or absence of cetuximab for 4 hrs. Wilcoxon matched pairs test for groups with (+) or without (-) Ab. * p<0,01. Kruskal-Wallis test for TAM groups co-cultured with A549/HLA-A2+NY-ESOhi or A549/HLA-A2+NY-ESOlo. Data represented as mean ± SEM. FcR+ effectors were freshly isolated for all experiments.