Abstract B90: Deep characterization of in vitro chronically stimulated T cells through single-cell multiomic analysis

A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function, and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) What types of cells are generated during chronic stimulation? 2) How many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study—with unprecedented depth—how T cells change over the course of chronic stimulation. Human PBMCs from three healthy donors were continuously stimulated for 14 days in the presence of CD3/CD28 antibody-coated beads and recombinant human IL-2. This model system was developed to resemble culture conditions that may be used for CAR T cell expansion. Cells were collected at different time points (day 0, 3, 7, and 14) prior to downstream analysis. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naive to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naive-associated markers and upregulation of effector molecules, proliferation regulators, coinhibitory and costimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely upregulated upon 14 days of stimulation (CD39, ENTPD1, TNFSF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures in response to different activation protocols. Citation Format: Mirko Corselli, Margaret Nakamoto, Chip Lomas, Ian Taylor, Suraj Saksena, Tariq Arshad, Pratip K. Chattopadhyay. Deep characterization of in vitro chronically stimulated T cells through single-cell multiomic analysis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B90.