As expected, Pdcd1 expression was > 100-fold increased in PD-1 OE and > 4-fold in CD3/CD28-activated T-cells compared to WT B16-F10 melanoma cells, but not detected in negative control, PD-1 KO B16-F10 or activated T-cells (Fig. 1a), thus confirming specificity of both primer sets for Pdcd1. Immunoblotting corroborated PD-1 protein expression by B16-F10 WT and PD-1 OE, but not PD-1 KO melanoma cells, and by unactivated and activated WT, but not PD-1 KO T-cells at an expected molecular weight of ~ 37–50 kDa (Fig. 1b), consistent with previous studies5,15. Here, CD28 is linked to melanoma.