Single-cell resolution of intracellular T cell Ca²⁺dynamics in response to frequency-based H₂O₂stimulation

Abstract

Adaptive immune cells, such as T cells, integrate information from their extracellular environment through complex signaling networks with exquisite sensitivity in order to direct decisions on proliferation, apoptosis, and cytokine production. These signaling networks are reliant on the interplay between finely tuned secondary messengers, such as Ca2+ and H2O2. Frequency response analysis, originally developed in control engineering, is a tool used for discerning complex networks. This analytical technique has been shown to be useful for understanding biological systems and facilitates identification of the dominant behaviour of the system. We probed intracellular Ca2+ dynamics in the frequency domain to investigate the complex relationship between two second messenger signaling molecules, H2O2 and Ca2+, during T cell activation with single cell resolution. Single-cell analysis provides a unique platform for interrogating and monitoring cellular processes of interest. We utilized a previously developed microfluidic device to monitor individual T cells through time while applying a dynamic input to reveal a natural frequency of the system at approximately 2.78 mHz stimulation. Although our network was much larger with more unknown connections than previous applications, we are able to derive features from our data, observe forced oscillations associated with specific amplitudes and frequencies of stimuli, and arrive at conclusions about potential transfer function fits as well as the underlying population dynamics.

Time-resolved microwell cell-pairing array reveals multiple T cell activation profiles

“…Similar patterns have been reported in the literature, 42 also in response to other stimuli, 79,80 but never at this scale of single cell analysis, with a throughput approximately 10fold higher than that offered by existing platforms. 12 Although the distinction between fast and slow responders could be prone to bias due to manual classification, the correlation between the abundance of stimulatory peptide : MHC present on APC and the fraction of responding T cells that show a "pulsatile" response profile suggests that in the presence of a low abundance of epitopes, cells are more likely to reach the activation threshold through multiple subthreshold spikes.…”

Oscillatory IL-2 stimulus reveals pertinent signaling timescales of T cell responsiveness

“…A custom GUI controlled pinch valves to enable precise delivery of stimulus and medium to the trapped cells. Prior studies with fluorescein labeled buffer have characterized that the pulsatile properties of this platform are well-matched to the desired input function [ 22 ]. Cells were exposed to either a constant flow of medium containing 10 pM or 100 pM IL-2 or to a pulsatile input of 100 pM IL-2, alternating with growth medium without IL-2.…”

“…Sensitivity of cellular response to quick oscillations of input is observed in other systems, such as intracellular T cell Ca 2+ dynamics in response to extracellular H 2 O 2 oscillations [ 22 ]. This raises the question of how such dynamics could affect cellular response to natural ligands that undergo binding and internalization such as cytokines.…”

Engineering Approaches to Study Cellular Decision Making

“…Of these, only imaging is able to track the same cell in time to monitor both signaling events and downstream effects. Numerous tools have been developed to monitor individual signaling pathways in real time, including fluorescent reporters to monitor protein localization, kinase activity (136, 137), hydrogen peroxide localization (138), and Ca 2+ (139). Importantly, many of these readouts can be multiplexed with single-cell endpoint analysis such as single-molecule fluorescent in situ hybridization for gene expression (140) or microwells for the analysis of secreted proteins (141).…”