Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens

Nat Genet. 2021 Mar;53(3):322-331. doi: 10.1038/s41588-021-00778-2. Epub 2021 Mar 1.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • B7-2 Antigen / metabolism
  • B7-H1 Antigen / genetics*
  • B7-H1 Antigen / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism
  • Humans
  • Immune Checkpoint Proteins / physiology*
  • Kelch-Like ECH-Associated Protein 1 / genetics
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism
  • Programmed Cell Death 1 Ligand 2 Protein / metabolism
  • Receptors, Interferon / genetics
  • Reproducibility of Results
  • Signal-To-Noise Ratio
  • Single-Cell Analysis / methods*
  • THP-1 Cells
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • B7-2 Antigen
  • B7-H1 Antigen
  • BRD4 protein, human
  • CD274 protein, human
  • CUL3 protein, human
  • Cell Cycle Proteins
  • Cullin Proteins
  • IFNGR2 protein, human
  • Immune Checkpoint Proteins
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • PDCD1LG2 protein, human
  • Programmed Cell Death 1 Ligand 2 Protein
  • Receptors, Interferon
  • Transcription Factors