Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens.

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 IFN? 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. Overall design: Identification of novel immune checkpoint regulators using ECCITE-seq. Please note that the ECCITE_ADT_Barcodes.csv and ECCITE_Arrayed_ADT_Barcodes.csv files are identical. To avoid any confusion, duplicated files were generated to link to each experiment type for users who may try to analyze a specific experiment (ECCITE or ECCITE_Arrayed).