W166 - Novel Functional Diversity of Human T Cells Is Revealed as a Result of the Unprecedented Resolution of Intracellular Cytokines, Transcription Factors and Phosphoproteins Using Mass Cytometry (CyTOF XT)

Abstract Text: Measuring functional signatures provides key insights into several facets of immunology, including T/B memory cell diversity following infection and/or vaccination and the panoply of immune activity in response to cell therapies, specifically the impact of inflammatory and immunosuppressive lineages on treatment efficacy. However, intracellular readouts using flow cytometry have been historically difficult to detect due to complications with spectral overlap and autofluorescence. We posited that mass cytometry may enable superior signal resolution within the cell compared with fluorescence cytometry. We evaluated three small (11–12-plex) clone-matched antibody panels using a spectral flow and mass cytometer. Each panel measured surface and intracellular analytes (cytokines and other markers of functional potential phosphorylation events and transcription factors); the fluorescent panels were designed to minimize spectral overlap. PBMC were stimulated, split, stained with fluorochrome- or metal-conjugated antibodies and acquired. Data was analyzed using PhenoGraph clustering and visualized with opt-SNE to determine cellular functional diversity. Most strikingly, stimulation-specific IL-10 and IL-13 positive cells were only detected using a CyTOF™ XT mass cytometer. Moreover, CyTOF technology provided cleaner signal-to-noise distinction for most phosphorylation events and transcription factors measured. Superior resolution translated to more accurate population clustering and a higher number of unique immune cell subset signatures, including clear detection of Tr1 and Tc2 populations. Overall, CyTOF technology uniquely provided a comprehensive picture of the immuno-diversity present in a sample; this platform will help illuminate swaths of currently “invisible” non-inflammatory immune responses, revealing new, broader classes of therapeutic targets and approaches to treat chronic inflammatory diseases.