TY - JOUR T1 - Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens JF - bioRxiv DO - 10.1101/2020.06.28.175596 SP - 2020.06.28.175596 AU - Efthymia Papalexi AU - Eleni Mimitou AU - Andrew W. Butler AU - Samantha Foster AU - Bernadette Bracken AU - William M. Mauck III AU - Hans-Hermann Wessels AU - Bertrand Z. Yeung AU - Peter Smibert AU - Rahul Satija Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/06/28/2020.06.28.175596.abstract N2 - The expression of inhibitory immune checkpoint molecules such as PD-L1 is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here we apply ECCITE-seq, a technology which combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1, and leverage our multi-modal data to identify both transcriptional and post-transcriptional modes of regulation. In particular, we discover that the kelch-like protein KEAP1 and the transcriptional activator NRF2, mediate levels of PD-L1 upregulation after IFNγ stimulation. Our results identify a novel mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multi-modal single-cell perturbation screens.Competing Interest StatementIn the past three years, RS has worked as a consultant for Bristol-Myers Squibb, Regeneron, and Kallyope, and served as an SAB member for ImmunAI and Apollo Life Sciences GmbH. PS is a co-inventor of a patent related to this work. BZY is an employee at BioLegend Inc., which is the exclusive licensee of the New York Genome Center patent application related to this work. ER -