Decoding T Cell Cytotoxicity via Single-Cell CRISPR Screens in Droplets

In the first part of the presentation, I will present a microfluidics-based platform for two-cell type CRISPR screens. Cytotoxic CD8+ T cells (CTLs) are pivotal effectors in tumor elimination, yet a comprehensive understanding of the genes regulating their cytotoxicity against cancer cells has been technically challenging. We developed a droplet-based microfluidics CRISPR screening platform that enables high-throughput, single-cell interrogation of CTL function. Individual CTLs with genetic perturbations are encapsulated alongside target cancer cells in picoliter droplets, where killing activity is assessed. Droplets are then sorted based on cytotoxic outcomes, followed by retrieval and analysis of sgRNAs. This approach faithfully recapitulated known mechanisms of T cell cytotoxicity and uncovered novel regulators.
In the second part of my presentation, I will briefly describe our efforts to use live bacteria to potentiate anti-tumor T cell responses. We found that oral administration of L-arginine increased the number of tumor-infiltrating T cells, acting synergistically with PD-L1 blockade to induce tumor regression. Due to practical limitations associated with high-dose L-arginine administration, we developed an engineered probiotic bacterium capable of converting ammonia—a common metabolic waste product in tumors—into L-arginine. This strategy demonstrated synergistic effects with PD-L1 blockade, leading to enhanced tumor clearance.