Metabolic profiling of antigen-specific CD8⁺ T cells by spectral flow cytometry

Cytotoxic CD8⁺ T cells are essential mediators of immune responses against viral infections and tumors. Upon antigen encounter, antigen-specific CD8⁺ T cells undergo clonal expansion and produce effector cytokines, processes that require dynamic metabolic adaptation. However, profiling antigen-specific T cells at single-cell resolution remains technically challenging. We present a spectral flow cytometry-based workflow enabling metabolic profiling of antigen-specific CD8⁺ T cells identified via major histocompatibility complex (MHC) class I tetramers or CD137 upregulation. The approach integrates the analysis of metabolic protein expression to infer pathway activity, uptake of fluorescent probes to measure functional metabolism and metabolite utilization, and assays evaluating cellular energy metabolism. Applied to human and mouse samples, this method defined the metabolic profiles of cytomegalovirus-, SARS-CoV-2-, and tumor-specific CD8⁺ T cells across distinct activation states and tissues. By detailing each component of the workflow, we provide practical guidance for applying metabolic spectral flow cytometry to dissect disease mechanisms and therapeutic responses.