Differential T Cell Responsiveness to Proliferative Stimuli Assessed with a Novel, Homogeneous Ki-67 Immunoassay

Abstract Proliferative capacity is an important component of T cell fitness and a relevant parameter for characterization of CAR T cells and T cell activating agents. Ki-67 protein is a well-validated marker of cell proliferation. We used Lumit™ technology to develop a homogeneous, luminescent immunoassay for determination of intracellular Ki-67 levels. Purified CD8 +T cells from different human donors were plated in 96-well format, then treated with a panel of T cell activators +/− IL-2, including αCD3/αCD28, αCD3/αCD28/αCD2, phytohemagglutinin, concanavalin A, and a cocktail of PMA and ionomycin. An optimized lytic reagent was added to control and treated cells, followed by two anti-Ki-67 monoclonal antibodies labeled with SmBiT and LgBiT subunits, respectively. Upon coincident binding of these labeled antibodies to Ki-67 protein molecules, the resultant proximity of SmBiT and LgBiT reconstitutes NanoBiT luciferase. Addition of Lumit Detection Reagent produces light proportional to the level of Ki-67 expression, with a total assay time of 2 h. It was determined that stimulation of Ki-67 levels varied markedly between T cell activators tested, with the maximum responses observed exceeding 13-fold. Notably, T cell responses varied significantly between donors, including with respect to the degree of IL-2 dependence. This assay system was compatible with same-well fluorescent determination of cell death. In parallel treated wells, Lumit cytokine immunoassays were used to assess concomitant increases in cytokine levels. This simple, no-transfer, no-wash immunoassay for quantitative determination of Ki-67 levels provides an improved workflow for rapid and efficient comparative assessments of T cell proliferative responses.