Survival of mice transplanted with CMTM6-depleted cells was significantly increased compared to control shRNA-expressing cells (Physique 4h). malignancy cells. CMTM6 is usually a ubiquitously expressed, protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome we find that CMTM6 displays amazing specificity for PD-L1. Importantly, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC Class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour specific T-cell activity and was the only recognized regulator of PD-L1 expression (Physique 1c). Depletion of CMTM6 using specific sgRNAs or short hairpin RNAs (shRNAs) led to a dramatic reduction in total cellular levels of PD-L1 (Physique 1d and Extended Data Fig. 1c/d). These findings have broad relevance as CMTM6 is usually a major regulator of PD-L1 expression in cell lines representative of melanoma, breast and lung malignancy (Physique 1e and Extended Data Fig. 2&3), diseases that respond to immune checkpoint blockade1,2. Importantly, CMTM6 depletion reduces both constitutive and IFN- induced PD-L1 expression without compromising antigen presentation by reducing cell surface MHC class I levels (Extended Data Fig. 4). Exogenous expression of CMTM6 in CMTM6 knockout cells regulates PD-L1 in a dose dependent manner and Aligeron restores both total and cell surface PD-L1 levels (Physique 1f and Extended Data Fig. 5a). In myeloid lineage cells, CMTM6 depletion specifically downregulates cell surface expression of PD-L1 but not PD-L2 (Extended Data Fig. 6a/b). Interestingly, CMTM6 levels are not influenced by IFN- activation (Extended Data Fig. 1c, ?,4b4b and ?and5b)5b) and, in contrast to other recently described regulators of PD-L1 expression1,2, CMTM6 does not function as a transcriptional regulator of PD-L1 either in the presence or absence of IFN- (Physique 1g). Open in a separate window Physique 1 CMTM6 is usually a principal regulator of PD-L1 expression in multiple tumour typesa. A genome-wide CRISPR/Cas9 screen identifies genes essential for cell surface PD-L1 expression. BxPC-3 pancreatic malignancy cells expressing Cas9 were mutagenised with a pooled lentiviral sgRNA library and PD-L1 low cells enriched by FACs sorting b&c. Significant hits from screens in cells pre-treated with IFN- before sorting (B) and non-IFN- treated cells (C). Dotted collection indicates Bonferroni-corrected significance threshold. d. Immunoblot in MDA-MB-231 cells expressing Cas9 and sgRNAs targeting either CMTM6 or PD-L1. e. Surface PD-L1 in IFN–treated cells transduced with CMTM6-specific sgRNAs versus parental Cas9 expressing cells. Observe Extended Data Fig. 3 for full dataset. f. PD-L1 expression in CMTM6 knockout MDA-MB-231 cells CMTM6 cDNA analysed by circulation cytometry and immunoblot. Representative of 3 experiments. g. qRT-PCR analysis in control and CMTM6-depleted cells treated 500IU/ml IFN- for 48h. 2 biological replicates (mean, s.e.m.). CMTM6 belongs to a family of proteins, primarily encoded by two unique gene clusters, on chromosome 16 (CMTM1-4) and chromosome 3 (CMTM6-8)6. Whilst largely uncharacterised, CMTM family members contain a MARVEL domain name comprising at least three transmembrane helices7. Interestingly, MARVEL domain name proteins have been implicated in regulating trafficking of transmembrane and secretory proteins7. To determine whether CMTM6 interacts with PD-L1, we performed reciprocal co-immunoprecipitation experiments using detergent conditions that solubilise the membrane to a variable degree. CMTM6 was readily detected in association with PD-L1; Aligeron however, this conversation is maintained only under Aligeron conditions that preserve the integrity of a membrane-associated complex (Physique 2a/b). In agreement with this, CMTM6 co-localises with Aligeron PD-L1 at the cell surface both in the presence and absence of IFN- activation (Physique 2c and Extended Data Fig. 5c/d). Open INCENP in a separate window Physique 2 CMTM6 shows functional specificity for Aligeron PD-L1a. PD-L1 is usually readily detected in association with CMTM6. Immunoprecipitation of CMTM6 (left panel) or PD-L1 (right panel) from digitonin lysates of IFN- treated MDA-MB-231 cells. Analysis by immunoblot. Lysate = 5% of input. Experiments performed twice. b. Conversation of CMTM6 with PD-L1 is usually detergent-sensitive. Cells were lysed in 1% digitonin (Dig) and adjusted to the indicated detergent concentrations prior to immunoprecipitation of PD-L1. Experiment performed twice. c. PD-L1 and CMTM6 co-localise at the plasma membrane. PD-L1 knockout, CMTM6.