Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Assessing the cytotoxic potential of T cell-based therapies like CAR T cells is crucial for clinical application.
• Traditional bulk assays lack the resolution to capture functional heterogeneity within CAR T cell populations.

Purpose of the Study:

• To develop a high-throughput, single-cell method for assessing CAR T cell cytotoxicity.
• To enable dual quantification of effector molecule release and target cell death.

Main Methods:

• A double-emulsion droplet-based microfluidic system for co-encapsulating single effector CAR T cells with single target cells (CD19+ JeKo-1).
• Utilized reagents for visualizing Granzyme B secretion and propidium iodide (PI) staining for cell death.
• Employed standard flow cytometry for analyzing functional diversity within droplets.

Main Results:

• Demonstrated successful generation of droplets containing single CAR T and target cells.
• Showcased the ability to assay functional heterogeneity and temporal kinetics of CD19-specific CAR T cell killing.
• Validated the dual quantification of effector molecules and target cell death at the single-cell level.

Conclusions:

• The developed droplet-based assay provides detailed insights into CAR T cell functional heterogeneity and cytotoxic potential.
• This method is adaptable for various T cell types, cytotoxic immune cells, and other effector functions like cytokine secretion.
• Offers a valuable tool for the preclinical assessment and optimization of adoptive cell therapies.