Fig. 6. IFN-γ secreted by T cell modulates reciprocal expression of TRIM21 and CD73 through a feedback loop.

(A) MDA-MB468 cells were treated with IFN-γ (100 μg/ml), and cell lysates were collected at indicated time point. TRIM21 and CD73 protein levels were determined by immunoblotting. (B) Normalized TRIM21 and CD73 expression bar graph of (A). (C) MDA-MB468 cells were cocultured with or without human PBMCs and treated with IFN-γ–neutralizing antibody (Ab; 1 μg/ml), and TRIM21 and CD73 protein levels were determined by immunoblotting. Feedback regulation of CD73 by secreted IFN-γ from T cells. Activated T cell suppresses the CD73 expression level in tumor cells compared to control, which could be reversed by IFN-γ antibody (right). (D) MDA-MB468-CD73^WT and MDA-MB468-CD73^4KR cells were treated with IFN-γ and MG-132. Cell lysates were collected at indicated time points and followed by measuring CD73 protein expression (D). (E) Normalized CD73 expression bar graph of (D) suggesting replacement of four lysine residues on CD73 by arginine significantly blocks the IFN-γ–induced down-regulation of CD73 in MDA-MB468 cells. (F and G) Replacement of four lysine residues on CD73 by arginine significantly blocks the IFN-γ–induced down-regulation of CD73 in MDA-MB231 cells.

**P < 0.01, ***P < 0.001, ****P < 0.0001. Data (means ± SEM) are representative of at least three independent experiments.