Identification of Human Antigen-Specific T Cells Using Class II MHC Tetramer Staining and Enrichment

The development of peptide-major histocompatibility complex tetramers has enabled direct characterization and enumeration of antigen-specific T cells. However, the weaker interaction between class II tetramers and CD4⁺ T cells increases the challenge of using tetramers to analyze CD4⁺ T cell responses. Here, we provide an optimized class II tetramer staining protocol with a magnetic-bead enrichment strategy for the detection and functional analyses of human antigen-specific CD4⁺ T cells. This approach enables direct sampling of lymphocytes that recognize specific peptide-MHC complexes, including rare pathogen-specific CD4⁺ T cells from unexposed individuals.