PROJECT SUMMARY Cytokines represent a broad range of small signaling proteins utilized by immune cells in cell-to-cell communications. Dysregulated cytokine release contributes to acute and long-term conditions, from life- threatening cytokine release syndrome to delayed wound healing. Targeting the cytokine release is an attractive strategy because it can regulate multiple cytokines involved in complex diseases. However, no FDA- approved drugs target this process, and related clinical trials remain scarce. The lack of clinical translation represents an urgent need to develop advanced technologies to better understand the cytokine release process at the molecular level. To date, existing assays for cytokine quantification, such as ELISA and flow cytometry, provide a low resolution that masks detailed mechanistic information in space and time. By capitalizing on the PI’s unique expertise in immune imaging, the R35 proposal will address this need by developing ultrasensitive cytokine quantification techniques using T cells and interleukin-2 as a model system. Cytokine quantification will be achieved in three specific areas: 1) at the resolution of single-vesicle fusion events with the plasma membrane, 2) at the nanoscale membrane release sites, and 3) in the membrane- enclosed form of extracellular vesicles (EVs). Enhanced mechanistic understanding will be obtained at the single-cell level regarding the temporal and directional profiles of cytokine release, proximity-based regulation by membrane calcium channels, and the dynamic distribution between soluble and EV-associated cytokines during T cell activation. Each of these areas will potentially enable compound screening targeting specific spatial and temporal phenotypes, investigations of membrane channel inhibitors, and targeting EVs for cytokine modulation. Future studies will be expanded to other essential cytokines from adaptive and innate immune cells. Ultrasensitive quantification of cytokines will enhance mechanistic understanding of the cytokine release in search of novel membrane targets to modulate the process.