Development of single-cell ROS regulome profiles of CD8+ T cells 

Cells under oxidative stress regulate their functions, but current methods for detecting this stress mostly use mass spectrometry or fluorescent probes sensitive to reactive oxygen species (ROS). While valuable, these methods can't simultaneously characterize specific ROS-mediated pathways at the single-cell level. Thus, we developed an approach to characterize the ROS regulome of single-cells together with their phenotypic identity, termed single-cell ROS regulome profiling (scROP). To accomplish this, we first assessed over 100 commercially available antibodies by fluorescent cell barcoding screening and then selected a subset of ROS antibodies (n = 25). We combine our method with in vitro-activated OTI-CD8⁺ T cells to quantify the proteins that regulate the activity of the ROS pathway, it provides a more detailed sequential progression of how ROS regulome response through oxidative and redox pathways in different CD8⁺ T cell states that lead to exhaustion. Moreover, our results show that T cell exhaustion rates differ under hypoxic conditions or in the presence of antioxidants. We confirmed our findings using clinical samples, such as blood from hemodialysis patients, CD8+ T cells from CAR-T patients, and various tumor sections from hepatic carcinoma patients. Overall, our novel single-cell ROS regulome profiling approach revealed a comprehensive molecular roadmap of CD8⁺ T cells in response to sustained stimulations.