Abstract 2166: Deep characterization of in vitro chronically stimulated T cells via single-cell multiomic analysis

Abstract

Abstract A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for viable adoptive cell therapy, 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 using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system 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 throughout chronic stimulation. Human PBMCs from three healthy donors were stimulated for 14 days in the presence of CD3/CD28 antibody-coated beads and recombinant human IL-2. This model system resembles 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 naïve 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 naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory 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 up-regulated 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 heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. 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. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Mirko Corselli, Suraj Saksena, Margaret Nakamoto, Woodrow E. Lomas, Ian Taylor, Pratip K. Chattopadhyay. Deep characterization of in vitro chronically stimulated T cells via single-cell multiomic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2166.