Superior Intracellular Detection of Cytokines, Transcription Factors, and Phosphoproteins by CyTOF Compared With Fluorescent Cytometry

Unraveling biological complexity, such as immune subset distribution in infectious disease(s), autoimmunity, or tumor heterogeneity, requires technologies capable of single-cell proteomic analysis such as flow cytometry. Surface phenotyping alone is often insufficient, as interrogating functional capacity is required to determine cellular mechanisms and effectively inform diagnostic biomarker discovery as well as development of novel therapeutics and vaccines. However, large flow panels with intracellular markers are subject to numerous challenges, including spectral overlap and background cellular autofluorescence, reducing resolving power for rare subsets or populations defined by low-abundance expression. We posited that use of mass cytometry may overcome such limitations; to address this, three small (10-12-plex) clone-matched antibody panels were evaluated by spectral flow and mass cytometry. Panels were comprised of surface and intracellular targets (phospho-epitopes, transcription factors or cytokines) and designed to minimize fluorescence spectral overlap. Overall, CyTOF technology demonstrated superior signal resolution compared with fluorescent counterparts for all three types of the intracellular targets that were compared. There was clear stimulation-specific resolution of IL-10 and IL-13 cytokine-producing cells from using mass cytometry that was not seen in the fluorescent panel and is not routinely detectable using that platform. Thus, accurate detection of immune cells with distinct functional signatures was enabled, permitting more unique and understudied populations to be quantified, including T regulatory 1 (Tr1) and cytotoxic type 2 (Tc2) T cell subsets. These results indicate that CyTOF technology has a unique ability to provide a high-resolution, comprehensive picture of the immuno-diversity present in a sample. Therefore, we posit that a new focus on use of mass cytometry for intracellular readouts could catalyze seminal discoveries in functional immune profiling, driving therapeutic design and diagnostics.