Figure 6

(A) Flow cytometric analysis of ADCP. SKCO-1 target cells were stained green with CMFDA and are present in the right lower quadrant of the dot plots. Macrophages were stained with anti-CD11b and CD14 conjugated with PE. They appear in the left upper quadrant of the dot plots. The left hand dot plot is from a representative 1-h culture of macrophages and target cells (SKCO-1) in the presence of IgG isotype control. The middle and right plots are from representative 1-h cultures of macrophages and target cells in the presence of uhPR1A3 and ghPR1A3, respectively (5 μg ml⁻¹). The effector : target ratio used was 5 : 1. (B) Effect of increasing concentrations of uhPR1A3 and ghPR1A3 on phagocytosis. Tumour targets were pre-incubated with an isotype control antibody (IgG, 10 μg ml⁻¹) or the variants of hPR1A3 at concentrations of 0.1–10 μg ml⁻¹. ADCP was determined by flow cytometric analysis as the percentage of targets in the upper right hand quadrant (see Figure 6C). The four graphs represent responses from four separate donors. (C) Effect of FcγR blocking on ADCP. Flow cytometry was used to calculate the percentage of tumour cell engulfment by cultured macrophages in the presence of 10 μg ml⁻¹ of uhPR1A3. Fab₂ fragments were used to block either FcγR I (CD64), FcγR II (CD32) or FcγR III (CD16) (each antibody concentration was1 μg ml⁻¹). The effector : target ratio used was 3 : 1, and the targets were SKCO-1. (D) Fluorescent images of macrophages phagocytosing SKCO-1 using the Ikoniscope. The macrophages (red) have been stained with anti-CD14 and anti-CD11b primary antibodies followed by goat anti-mouse-HRP and Tyramide 647. The target cell line (SKCO-1) was stained green with CMFDA (Celltracker probe). The left hand panels show microscope composite images viewed with FITC (green), Cy5 (for tyramide 647) and DAPI (blue) channels. The second, third and fourth panel column show the same cells viewed separately with the DAPI, green and Cy5 channels. Each represents a different phagocytic event.