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Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
Published on: February 2, 2013
A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells
Sumana Sharma1, Toby Whitehead2, Mateusz Kotowski2
1MRC Translational Immune Discovery Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK sumana.sharma@rdm.ox.ac.uk.
Abstract:
The recent success of immunotherapies relying on manipulation of T-cell activation highlights the value of characterising the mediators of immune checkpoint signaling. CRISPR/Cas9 is a popular approach for interrogating signaling pathways; however, the lack of appropriate assays for studying inhibitory signaling in T cells is limiting the use of large-scale perturbation-based approaches. Here, we adapted an existing Jurkat cell-based transcriptional reporter assay to study both activatory and inhibitory (PD-1-mediated) T-cell signaling using CRISPR-based genome screening in arrayed and pooled formats. We targeted 64 SH2 domain-containing proteins expressed by Jurkat T cells in an arrayed screen, in which individual targets could be assessed independently, showing that arrays can be used to study mediators of both activatory and inhibitory signaling. Pooled screens succeeded in simultaneously identifying many of the known mediators of proximal activating and inhibitory T-cell signaling, including SHP2 and PD-1, confirming the utility of the method. Altogether, the data suggested that SHP2 is the major PD-1-specific, SH2 family mediator of inhibitory signaling. These approaches should allow the systematic analysis of signaling pathways in T cells.
Insights
Researchers developed a new CRISPR-based screening method to study T-cell signaling pathways. This method identified SHP2 as a key mediator in PD-1 inhibitory signaling, advancing immunotherapy research.
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.
