Background The key challenges to developing T cell-based therapies center on the fact that T cell-mediated tumor death relies on complicated cell-cell interactions and several complex mechanisms. These therapies have also been associated with significant side effects related to cytokine release syndrome (CRS) and neurotoxicity, placing importance on understanding T cell anti-tumor functions like cytokine release and killing kinetics. Ideally, T cell therapies would be tailored to mediate the rapid destruction of multiple tumor cells while reducing these side effects.

Methods The Berkeley Lights Opto™ Cell Therapy Development Workflow is a collection of software capabilities, reagents, and protocols that allow scientists to selectively measure cytokine secretion, visualize killing behavior, and sequence TCRs from individual cells in parallel. Here, we demonstrate its use for CAR-T cell phenotypic and functional screening as well as the discovery of TCRs associated with specific T cell behaviors.

Results The cumulative percentage of pens with tumor cell caspase-3 activity increased over time in pens loaded with CD19+ tumors, peaking at 50% tumor cell death after 16 hours of incubation. This is in contrast to only 10% of pens displaying tumor cell death in control pens loaded with CD19- tumor cells; control pens also exhibited slower killing kinetics. The single-cell resolution of the OptoSelect™ microfluidic chip enabled us to analyze each significant T cell-tumor cell interaction. We were able to directly compare differences in killing kinetics of individual T cells and link this tumor killing behavior to IFNγ secretion. We identified fast-killing and slow-killing CAR-T cells in a single-day experiment, which could then be exported for genomic analysis. We highlight an example where TCR alpha and beta sequences are recovered from single T cells after export.

Conclusions The Opto™ Cell Therapy Development Workflow on Berkeley Lights systems enables researchers to correlate cytokine secretion to target cell killing behavior in CAR-mediated antigen recognition, discriminate CAR-T cell subsets based on kinetics of target cell killing, and link cytokine secretion and target cell killing behavior to TCR sequence in TCR-mediated antigen recognition.