A Synthetic Cytotoxic T cell Platform for Rapidly Prototyping TCR Function

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented and validated a granzyme-activatable sensor of T cell cytotoxicity in a novel universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered using contemporary gene-editing techniques to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs, with significant responses (p < 0.05 and Cohen's d >1.9) in all cases. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24-hour exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate new knowledge regarding the patterns of T cell receptor recognition, and optimize novel therapeutic T cell receptors for improved cytotoxic potential and reduced cross-reactivity to undesired antigenic targets.