Abstract A74: T cell activation standardization for therapeutic assay development

Abstract

Introduction: Several cancer immunotherapy strategies rely on T-cell activation (1). While T-cell activation mechanisms are well established, there is a dearth of quantified standardization of these activation pathways. Standardization with quantified end-points [induced by known activators and pathways] allows the development of useful assays to evaluate emerging immunotherapies. Our initial work has focused on T-cell activation. Materials and Methods: Activation of the Jurkat T-cell line was induced by the combined action of ionomycin and PMA (phorbol 12-myristate 13-acetate) (2). Jurkat cells were cultured in 24 well plates in RPMI 1640 media (with 10% FBS and 1% Penicillin/Streptomycin) at a density of 106 cells/ml. We looked at a wide concentration range for both activators (500 - 1500 ng/ml for ionomycin and 10 - 100 ng/ml for PMA), in order to identify their most synergistic combination for T-cell activation as quantified by biomarker expression (i.e. IL-2 ELISA). The negative controls were 1% DMSO (used for solubilizing the activators), and PBS (phosphate buffered saline). For a specific combination of 1000 ng/ml ionomycin and 100 ng/ml PMA, the designed time points were 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. The cells and supernatant were preserved at the end of these time-points. The viability of the cells was tested using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and the IL-2 production was detected using ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: In addition to identifying the optimal activating concentrations of ionomycin and PMA, the obtained results characterized the time-course of maximal Jurkat cell activation over a 24 h period. During this 24 h period, the IL-2 levels and the cell proliferation rates progressively increased before starting to plateau. The production of IL-2 started increasing at 4 h from 157.6 pg/ml and continued to go up until 24 h to 687.6 pg/ml. Cell proliferation decreased a little during the first 2 h and increased from 57% to 114% (of the PBS control) between 8 to 24 h. Conclusions and Discussion: Our work establishes a standardized basis to determine the extent and the time-course of maximal T-cell activation through a given mechanism - in this case through the mechanism of calcium ionophore induced (ionomycin) and phorbol ester induced (PMA) T-cell activation (3). Such quantified standardization and maximization of T-cell activation in turn presents a cell-culture model (or assay) to evaluate candidate therapeutics designed to work through the same activation pathway. In addition to T-cells, we are working on the quantified standardization of the activation of NK-cells (4) and dendritic cells (5), with activation measured by the end-points of biomarker expression profiling (ELISA/Luminex), proliferation (MTT assay), microscopy and flow cytometry (CD receptor expression profiling). References: 1. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: Moving beyond current vaccines. Nature Med 2004;10(9): 909. 2. Manger B, Weiss A, Weyand C, Goronzy J, Stobo JD. T cell activation: Differences in the signals required for IL 2 production by nonactivated and activated T cells. J Immunol 1985;135(6):3669-73. 3. Hashimoto S, Takahashi Y, Tomita Y, et al. Mechanism of calcium ionophore and phorbol ester-induced T-cell activation. Scand J Immunol 1991;33(4):393-403. 4. Ljunggren HG, Malmberg KJ. (2007). Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol 2007:7(5):329. 5. Melief CJ. Cancer immunotherapy by dendritic cells. Immunity 2008;29(3):372-83. Citation Format: Shilan Dong, Jason Cahoon, Tasnim Khalifa, Rachit Ohri. T cell activation standardization for therapeutic assay development [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A74.