Figure 3. CMTM6 is required for efficient endocytic recycling of PD-L1.

a. IFN-γ treated CMTM6 knockout or parental WM-852 Cas9 cells were pulse-labelled with ³⁵S-methionine/cysteine for 30 min, chased at 37°C and PD-L1 immunoprecipitated from detergent lysates at the indicated times. Eluates were split and incubated ± Endoglycosidase-H. Scans quantitated in ImageJ. Representative of 3 experiments. b-e. Cell surface PD-L1 is targeted for degradation in the absence of CMTM6. b&c. IFN-γ treated WM-852 cells were treated as described (b). Representative of six experiments (see Extended Data Fig. 7a & 9a). d&e. Cell surface IP (CS-IP). IFN-γ treated WM-852 cells were treated as described (d) and PD-L1 immunoprecipitates analysed by immunoblot. f. CMTM6 and PD-L1 are identified in recycling endosomes. MDA-MB-231 cells were fixed and immunostained for CMTM6 (large panel) or PD-L1 (right panel) plus markers of the ER (calnexin (CANX)), Golgi (GM130 and TGN46), early endosome (EEA1), late endosome/lysosome (LAMP1) or recycling endosome (RAB11 and transferrin receptor (TFRC)). Scale bar: 10μm. g&h. PD-L1 recycling is impaired in the absence of CMTM6. g. IFN-γ treated WM-852 cells were labelled with an unconjugated PD-L1-specific antibody before incubation at 37°C ± primaquine (described in Extended Data Fig. 8a). Remaining antibody-labelled surface PD-L1 was detected with an AF647-conjugated anti-mouse antibody and analysed by flow cytometry. h. PD-L1 recycling in IFN-γ-treated WM-852 cells (assay described in Extended Data Fig. 8c). Graph shows the proportion of internalised antibody-labelled PD-L1 recycled at each time-point. g&h. Mean and s.e.m. from 3 experiments.