Mouse CD47 expression reduces the susceptibility of porcine cells to cytotoxicity by mouse macrophages. (A) Expression of murine CD47 (mCD47) on transfected LCL-13271 pig tumor cell lines. (Left) Flow cytometric analysis. Thin and bold histograms represent staining with isotype control and anti–mouse CD47 mAb (miap301), respectively. Neo transfectant LCL cells (LCL-neo), a representative clone (no. 1007) of mCD47 transfectant LCL cells (LCL-mCD47), and mouse CD47^+/+ A20 cells are shown. (Right) mCD47 RT-PCR. (Lane 1) LCL-mCD47 cells (clone no. 1007); (lane 2) LCL-neo cells; (lane 3) nontransfected LCL-13271 cells; (lane 4) CD47^+/+ mouse cell line A20. GAPDH was used as a DNA loading control. (B) LCL-mCD47 and LCL-neo cells were stained with different colors (CFSE or PKH-26), mixed at a 1:1 ratio, and cultured in culture plate (2.5 × 10⁴/well) with (○) or without (•) WT mouse intraperitoneal macrophages (5 × 10⁵/well) for 3 days. Shown are ratios of viable LCL-mCD47 to LCL-neo cells (left) and representative flow cytometric profiles (right; the percentages of LCL-mCD47 and LCL-neo cells are indicated) at the indicated time points. Combined results (mean ± SDs) from 3 independent experiments are presented. *P < .05; **P < .01; ***P < .001. (C) Numbers of LCL-mCD47 (▪/•) and LCL-neo (□/○) cells in the upper transwell chambers (inside the transwells) in cultures, in which the lower chambers (outside transwells) contained either both target cells (ie, a 1:1 mixture of LCL-mCD47 and LCL-neo cells) and mouse macrophages (T + M) or target cells only (T). Results (mean ± SDs) from a representative experiment of 3 are shown.