Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell mediated immunosurveillance1,2. The success of therapies that disrupt PD-L1 mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression1. Using a genome-wide CRISPR/Cas9 screen we identified the uncharacterized protein CMTM6 to be a critical regulator of PD-L1 in a broad range of cancer cells. CMTM6 is 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 remarkable 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 in vitro and in vivo. These findings provide novel insights into the biology of PD-L1 regulation, identify a previously unrecognised master regulator of this critical immune checkpoint and highlight a new potential therapeutic target to overcome immune evasion by tumour cells.

Purpose: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. Experimental Design: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoStringbased analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses. Results: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8 þ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8 þ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/ 10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB. Conclusions: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

We have therefore revealed a potentially novel biomarker for the efficacy of anti-PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer.