PD-L1-deficient MC38 tumour cells were reconstituted with doxycycline inducible RFP (red circles), or PD-L1 (blue circle) using a lentiviral construct encoding constitutive GFP. PD-L1 reconstitution resulted in restored tumour outgrowth (a), while PD-L1-deficient tumours with doxycycline inducible RFP regressed. End-point analysis at day 24 showed that mixed cell (red/blue circles) inoculation led to tumours slightly smaller compared to tumours generated from PD-L1-enabled tumour cells only (b). While RFP+GFP+ tumour cells were detectable by flow cytometry at time of inoculation, this population was lost at endpoint, and solely PD-L1+GFP+ cells remained from the mixed inoculation (c). PD-L1 on target cells can inhibit antigen specific cytolytic T-cell activity in a dose dependent manner in vitro (d). Following complete regression of PD-L1-deficient CT-26 tumours, mice re-challenged with PD-L1-deficient (red) or wildtype (blue) tumours showed complete tumour regression (e), despite outgrowth of syngeneic EMT6 (black) tumours on the opposing flank (f). Following complete regression of MC38 PD-L1-deficient tumours, mice re-challenged with MC38 tumour cells over-expressing doxycycline induced PD-L1 (light blue) showed near complete tumor regression (g), despite over-expressed PD-L1 enabling slightly faster tumour progression in naive mice (h). Characterization of tumor subsets by flow cytometry in tumours was performed at day 19 post-inoculation. Data is representative of at least two individual study repeats, with 3 to 10 mice/group. Cytolytic in vitro assays were repeated four times with representative data shown. Dox, doxycycline; NA, naive; OVA, ovalbumin. Statistical significance was determined by Student’s t-test (*P<0.05; **P<0.01; ***P<0.001). Error bars depict s.d. from the mean. Mouse cartoon modified from ref. 15.