Product(s) used in this publication: PepMix™ Peptide Pools
Ag-specific CD4(+) T cells orchestrating adaptive immune responses are crucial for the development of protective immunity, but also mediate immunopathologies. To date, technical limitations often prevented their direct analysis. In this study, we report a sensitive flow cytometric assay based on magnetic pre-enrichment of CD154(+) T cells to visualize rare Ag-reactive naive and memory Th cells directly from human peripheral blood. The detection limit of ≈ 1 cell within 10(5)-10(6) permitted the direct enumeration and characterization of auto-, tumor-, or neo-Ag-reactive T cells within the naive and even memory CD4(+) T cell repertoire of healthy donors. Furthermore, the analysis of high target cell numbers after pre-enrichment of rare Ag-specific T cells from large blood samples dramatically improved the identification of small subpopulations. As exemplified in this work, the dissection of the Ag-specific memory responses into small cytokine-producing subsets revealed great heterogeneity between pathogens, but also pathogen-related microsignatures refining Th cell subset classification. The possibility to directly analyze CD4(+) T cells reactive against basically any Ag of interest at high resolution within the naive and memory repertoire will open up new avenues to investigate CD4(+) T cell-mediated immune reactions and their use for clinical diagnostics.