A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• T-cell activation is crucial for immunotherapies, necessitating detailed characterization of immune checkpoint signaling mediators.
• Current methods for studying inhibitory T-cell signaling, particularly using large-scale CRISPR screens, are limited by a lack of suitable assays.

Purpose of the Study:

• To adapt and validate a CRISPR-based genome screening assay for studying both activatory and inhibitory T-cell signaling pathways.
• To identify key mediators of Programmed Death-1 (PD-1) mediated inhibitory signaling in T cells.

Main Methods:

• Adaptation of a Jurkat cell-based transcriptional reporter assay for CRISPR screening in arrayed and pooled formats.
• Targeted screening of 64 SH2 domain-containing proteins in Jurkat T cells using arrayed CRISPR screens.
• Pooled CRISPR screens to identify mediators of proximal activating and inhibitory T-cell signaling.

Main Results:

• Arrayed screens demonstrated the utility of the assay for studying mediators of both activatory and inhibitory signaling.
• Pooled screens successfully identified known mediators of T-cell signaling, including SHP2 and PD-1.
• Data strongly suggest SHP2 is the primary SH2 family mediator of PD-1-specific inhibitory signaling.

Conclusions:

• The developed CRISPR-based assay is effective for large-scale analysis of T-cell signaling pathways, including inhibitory checkpoints.
• SHP2 plays a significant role as a mediator in PD-1-mediated T-cell inhibition.
• This methodology enables systematic interrogation of signaling pathways critical for T-cell function and immunotherapy.