Protocol for deep phenotyping of human antigen-specific T cells across conventional and unconventional subsets by 22-color spectral flow cytometry

Analyzing antigen-specific T cells at the subset level is crucial for studying antiviral immunity and vaccine efficacy. We present a 22-color spectral flow cytometry protocol to characterize these cells from human PBMCs, covering conventional αβT and unconventional (MAIT, γδ T) subsets. We describe steps for PBMC thawing and plate setup, peptide stimulation, three-step surface staining, and intracellular staining. We then detail procedures for sample acquisition and Flowjo data analysis. This approach enables precise immunophenotyping with limited samples.