Abstract 82: Single-cell cytokine secretion kinetics probed by Time-lapse flow (TM) cytometry
Authors: Fahlberg Marissa , Forward Sarah , Assita Emane Rose , Brown Trevor , Chattopadhyay Pratip , Kwok Sheldon 
Published: March 2024

Abstract
Cytokine secretion from immune cells is a central element of the immune response and a major determinant of immunotherapy efficacy. Several methods, including bulk in-solution assays and flow cytometry, have been used to measure cytokine secretion. Population-level analyses have shown that different cytokines can have different rates of secretion, depending upon their differentiation state or the stimulus itself. However, no current method can interrogate cytokine secretion from individual cells over time.
Here, we describe a novel flow cytometry-based assay for measuring single-cell cytokine secretion kinetics. Our approach, called “Time-lapse flowTM” uses semiconductor probes called laser particles (LPs) to optically barcode individual cells, providing a means to track single cells across a series of measurements over time. We applied this approach to measure T-cell cytokine secretion kinetics using stimulated primary human T cells.
Isolated T cells from cryopreserved human PBMCs were barcoded with LPs and stained with a bispecific antibody that binds to the cell surface on one end and “catches” secreted cytokines (IFNy and IL-2) on the other end. This permits analysis of cytokines from live cells. Next, T cells underwent three sequential stimulations over the course of 3 hours with PMA/Ionomycin. After the first stimulation, cells were stained with a surface marker panel that defined T cell memory phenotypes (CD3, CD4, CD8, CD45RA, CCR7, CD57, CD27, and CD95) and “detection” antibodies that recognized IFNy and IL-2 bound to the cell surface. The detection antibodies were re-added after each stimulation to quantify the accumulating cytokine on the cell surface. In total, kinetic profiles from over 100,000 CD3+ cells were measured.
We discovered massive heterogeneity of responding cells, identifying at least three distinct groups of cells defined by their initial time of cytokine secretion (early, middle, and late responders) and varying intensities of cytokine accumulation over time. Cells peaking in cytokine expression at the middle time points were mainly memory types with higher intensity, while later responders skewered towards naïve cells with less cytokine. However, IFNy variation wasn’t solely due to these markers, as diverse kinetics occurred across all subsets. For IL-2, we found that most responses initiated later than those of IFNy, and there was a reduced heterogeneity of both intensity and kinetics at these time points. These results were consistent across multiple donors and replicates (n=3).
Laser particle barcoding enables measurement of single-cell function over time. This novel time-lapse data allows interrogation of functional heterogeneity of immune cells at scale, enabling discovery of new relationships between T cell responses, diseases, and immunotherapies.
Citation Format: Marissa Fahlberg, Sarah Forward, Emane Rose Assita, Trevor Brown, Pratip Chattopadhyay, Sheldon Kwok. Single-cell cytokine secretion kinetics probed by Time-lapse flow (TM) cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 82.