Mass Cytometry Analysis of T-Helper Cells.

CD4 T cells or helper T cells play various roles in the immune response to pathogens, tumors, as well as in asthma, allergy, and autoimmunity. Consequently, there is great interest in the comprehensive investigation of different T helper cell subsets. Here, we use mass cytometry (CyTOF), which is similar to flow cytometry but uses metal ion-tagged antibodies, which are detected using time-of-flight mass spectrometry. CyTOF allows the simultaneous detection of over 40 different antibodies, allowing us to collect high-dimensional single-cell proteomic data on T helper subsets. We use an extensive staining panel with a large number of lineage markers, cytokines, and other functional markers to identify and characterize CD4 T cell subsets. In this method, human peripheral blood mononuclear cells are stimulated ex vivo with PMA and ionomycin, which activates T cells. The activated CD4 T cells can then be identified as Th1, Th2, or Th17 cells based on their production of IFNγ, IL-4, and IL-17, respectively. Tregs are identified as CD4CD25CD127. Once Th1, Th2, Th17, and Tregs have been identified, they can be characterized in more detail using the large number of phenotypic and functional markers included in the CyTOF staining panel. Finally, automated and unbiased high-dimensional data analysis tools can be employed to comprehensively characterize T helper cells and discover novel features.