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A New Approach Method for T Cell Immunotoxicity Using CD3/CD28 Co-stimulation and IL-2 NanoLuc Luciferase Reporter
Citation:
Martos, S., S. Simmons, AND K. Slentz-Kesler. A New Approach Method for T Cell Immunotoxicity Using CD3/CD28 Co-stimulation and IL-2 NanoLuc Luciferase Reporter. Presented at SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25533373
Impact/Purpose:
Presentation to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity
Description:
Background and Purpose: T cell receptor (TCR)-dependent activation of T cells dictates immune specificity and establishes immunological memory, key features of adaptive immunity. Canonical activation of T cells occurs through engagement of TCRs by interaction with the major histocompatibility complexes of antigen presenting cells and presented antigens along with co-engagement of costimulatory receptors (e.g., CD28). The activated signaling pathways control transcription of genes necessary for proliferation and differentiation, including IL-2, a critical cytokine for primary and secondary T-cell responses. Current in vitro immunotoxicity approaches for assessing altered T cell activation rely on polyclonal activation through chemical stimulation with Phorbol 12-myristate 13-acetate (PMA) and Ionomycin (Io), which bypasses TCR/CD3 complex and CD28 signaling. In contrast, the approach reported here incorporates a more biologically relevant activation through co-stimulation of CD3 and CD28. To develop an approach compatible with quantitative, high-throughput systems, we generated a Jurkat-derived cell line with inducible expression of NanoLuc luciferase regulated by a human IL-2 promoter sequence. The objective of this study is to evaluate the IL-2 NanoLuc reporter cell line in response to: 1) activation by PMA/Io co-exposure or CD3/CD28 co-stimulation and 2) suppression of T cell activation by a well-known inhibitor, Cyclosporin A. Methods: To assess the chemical activation response, we exposed IL-2 NanoLuc cells and cells from a commercially available, firefly luciferase IL-2 reporter system (T Cell Activation Bioassay, “IL-2 Promega”) to a mixture of PMA/Io at seven concentrations (3.125/0.125 to 125/5 nM PMA/uM Io) in triplicate and measured luminescence at 6 and 24 hours. To assess activation in response to CD3/CD28 co-stimulation, we stimulated both cell lines with seven concentrations (100 to 6400 beads/ul) of DynabeadsTM Human T-Activator CD3/CD28 (Gibco) in triplicate and measured luminescence at 6 and 24 hours. In these experiments, negative controls included vehicle-only (non-activated), no cells, and lysed cells. To assess suppression of activation, we incubated IL-2 NanoLuc and IL-2 Promega cells with cyclosporin A at seven concentrations (1.00 to 1000 nM) for one hour, added CD3/CD28 beads (1600 beads/ul) to activate, and measured luminescence after 24 hours of activation. Controls included vehicle (activated), vehicle (non-activated), and no cells. Results: For PMA/Io activation, the luminescent signal intensity at max stimulation (125 nM PMA/5 uM Io) was an order of magnitude higher in the IL-2 NanoLuc cells compared to the IL-2 Promega cells at both 6 and 24 hours. For CD3/CD28 co-stimulation, the luminescent signal intensity in the IL-2 NanoLuc cells at max stimulation (1600 beads/ul) was one order of magnitude higher at 6 hours and two orders of magnitude higher at 24 hours. Although the max luminescent signal intensity was higher for the IL-2 NanoLuc cells, there was a strong correlation between NanoLuc and Promega IL-2 reporter responses to activation by PMA/Io at 6 hours (R=0.98, p<2.2x10-16) and 24 hours (R=0.97, p<2.2x10-16) and by co-stimulation with CD3/CD28 beads at 6 hours (R=0.98, p<2.2x10-16) and 24 hours (R=0.95, p=9x10-9). For suppression of activation, the luminescent signal in activated vehicle controls was two orders of magnitude higher in IL-2 NanoLuc cells, compared to IL-2 Promega cells. There was strong correlation between NanoLuc and Promega IL-2 reporter responses to treatment of CD3/CD28-stimulated cells with cyclosporin A after 24 hours of activation (R=0.84, p=7.5x10-9). The half-maximal inhibitory concentration (IC50) for cyclosporin A at 24 hours post-activation was 1.09 nM for IL-2 NanoLuc cells and 6.95 nM for Promega cells. Conclusions: We developed a novel in vitro model to assess T cell activation using a more biologically relevant activation system than is currently . . .
URLs/Downloads:
DOI: A New Approach Method for T Cell Immunotoxicity Using CD3/CD28 Co-stimulation and IL-2 NanoLuc Luciferase Reporter
EPA_MARTOS_SOT2024_V4_3APR24.PDF (PDF, NA pp, 8196.001 KB, about PDF)