Directly test individual T cell function with fewer cells on the berkeley lights lightning™ platform

Abstract

Background & Aim T cell-based therapies have shown great promise. However, developing these therapies is challenging because T cell-mediated tumor death relies on complicated cell-cell interactions and several complex mechanisms. To minimize side effects, the dose of T cell therapies would be tailored to mediate the rapid destruction of tumor cells while avoiding high levels of cytokine release, an effect that would be facilitated by relatively low numbers of T cells quickly killing multiple target cells. Current technologies rely on correlating phenotype, function, and gene expression based on experiments performed on different populations of T cells because no one platform is able to assess cell surface marker expression, cytokine secretion, and tumor cell killing activity of the same T cell and recover this cell for downstream genomic analysis. Here we demonstrate how the Lightning optofluidic platform can be used to directly link T cell phenotype and function (IFNγ secretion and tumor cell killing) to genotype (TCR sequence recovery) at a single-cell level and on the same T cell, enabling deeper and more thorough characterization of how T cells mediate tumor cell death and potentially the development of more efficacious therapies. Methods, Results & Conclusion The T Cell Analysis Suite on the Lightning platform enables phenotypic and functional screening of 1000s of individual T cells in a single experiment, followed by live cell export for downstream analysis of T cells of interest. Individual T cell-target cell interactions are precisely assembled in thousands of nanoliter sized NanoPen chambers across an OptoSelect microfluidic chip using light in a process called opto-electropositioning (OEP). T cells are selectively penned based on cell surface phenotype and co-cultured with target cells and IFNγ capture beads. Timelapse imaging is then used to assess T cell function and killing activity. Individual T cells of interest are exported for downstream analysis such as TCR sequencing. Using a CD19 CAR-T model, we used this workflow to phenotype single T cells based on their kinetics of target cell killing and correlate this to IFNγ secretion. We also extended these experiments to an endogenous tumor-specific T cell system, analyzing the kinetics of single T cell killing, cytokine secretion and performed TCR sequencing on recovered cells. We demonstrate how this platform can be used for CAR-T cell phenotypic and functional screening as well as the discovery of TCRs associated with T cell behaviors.