Abstract 550: Evaluating changes in T cell activation by measuring expression of IL-2, CTLA-4, NFAT, and NF-kB in Jurkat cells

Abstract Insufficient stimulation of T cells due to activated checkpoint pathways can lead to ineffective clearance of tumor cells and thus, tumor growth and cancer. Developing methods to measure changes in the activation of such pathways are needed to evaluate the efficacy of immunotherapy approaches to treating cancer by altering T cell activation. Current methods that use measurments of IL-2 expression are limited by the fact that IL-2 is not specific to T cell activation and that it provides little insight into the mechanisms by which activation is affected. This project aims to address such limitations by determining whether changes in T cell activation can be effectively evaluated by measuring expression of CTLA-4, NFAT, and NF-kB, in addition to IL-2. Jurkat cells were cultured and activated with PMA (100 ng/mL) and ionomycin (500 ng/mL) for five hours. Staining for IL-2 and CTLA-4 with antibodies and transfection of the cells with genetic constructs that link NFAT and NF-kB to fluorescent proteins provided the means to link expression of fluorescence to expression of the molecules of interest. FACS analysis was then used to measure changes in the expression of the four signaling molecules in response to the induced changes in activation. Activation of the cells resulted in increased expression of all four molecules. Following activation, fluorescence due to CTLA-4, NFAT, and NF-kB expression changed by average fold increases of 2.58, 1.10 and 1.13, respectively. By measuring these key elements of T cell signaling pathways in addition to IL-2, these methods provide a potentially more effective alternative to current methods for evaluating the efficacy of immunotherapeutic treatments for cancer. Citation Format: Jacquelyn M. Bement, Yuko J. Miyamoto. Evaluating changes in T cell activation by measuring expression of IL-2, CTLA-4, NFAT, and NF-kB in Jurkat cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 550.