Single-cell signaling network profiling during redox stress reveals dynamic redox regulation in immune cells

In eukaryotic cells, reactive oxygen species (ROS) serve as crucial signaling components. ROS are potentially toxic, so constant adjustments are needed to maintain cellular health. Here we describe a single-cell, mass cytometry-based method that we call signaling network under redox stress profiling (SN-ROP) to monitor dynamic changes in redox-related pathways during redox stress. SN-ROP quantifies ROS transporters, enzymes, oxidative stress products and associated signaling pathways to provide information on cellular redox regulation. Applied to diverse cell types and conditions, SN-ROP reveals unique redox patterns and dynamics including coordinated shifts in CD8⁺ T cells upon antigen stimulation as well as variations in CAR-T cell persistence. Furthermore, SN-ROP analysis uncovers environmental factors such as hypoxia and T cell exhaustion for influencing redox balance, and also reveals distinct features in patients on hemodialysis. Our findings thus support the use of SN-ROP to elucidate intricate redox networks and their implications in immune cell function and disease.