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.

The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes. Although T cells are the predominant TIL population, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8 T cells with features of tissue-resident memory T (T) cell differentiation and that these CD8 T cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8 T gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8 T cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of T cells will be crucial for successful immunotherapeutic development in BC.

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression.Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2 þ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach.Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8 þ T cells following antigen-specific stimulation with PD-L1 þ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2 þ tumors treated with anti-Her-2 T cells in combination with anti-PD-1antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1 þ CD11b þ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer.