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Updated: Nov 15, 2025
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens
Efthymia Papalexi1,2, Eleni P Mimitou3, Andrew W Butler1,2
1Center for Genomics and Systems Biology, New York University, New York, NY, USA.
Abstract:
The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that 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 multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.
Insights
Researchers uncovered new regulators of programmed death-ligand (PD-L)1, a key immune checkpoint in cancer. They identified KEAP1 and NRF2 as crucial for PD-L1 upregulation, offering novel therapeutic targets for cancer immunotherapy.
Area of Science:
- Immunology
- Cancer Biology
- Genomics
Background:
- Immune checkpoint molecules like programmed death-ligand (PD-L)1 are often expressed in cancers, suppressing anti-tumor T cell responses.
- Understanding the regulation of PD-L1 is critical for developing effective cancer immunotherapies.
Purpose of the Study:
- To explore the molecular networks regulating PD-L1 expression using advanced single-cell technologies.
- To identify novel regulators of PD-L1 and elucidate their mechanisms of action.
Main Methods:
- Utilized expanded CRISPR-compatible (EC)CITE-seq for pooled CRISPR screens combined with single-cell mRNA and protein measurements.
- Developed and applied the computational framework 'mixscape' to enhance signal-to-noise in single-cell perturbation screens.
- Integrated multimodal data to identify transcriptional and post-transcriptional regulatory mechanisms.
Main Results:
- Identified and validated key regulators of PD-L1 expression.
- Discovered that KEAP1 and NRF2 mediate PD-L1 upregulation following interferon-gamma (IFN-γ) stimulation.
- Uncovered both transcriptional and post-transcriptional modes of PD-L1 regulation.
Conclusions:
- Identified a novel mechanism regulating immune checkpoints, specifically PD-L1.
- Presented a powerful analytical framework for multimodal single-cell perturbation screens.
- Findings offer potential new therapeutic targets for modulating anti-tumor immunity.
