A 61-Parameter CyTOF Panel for Comprehensive Profiling of Human PBMC to Characterize Activation, Differentiation, Checkpoints and Cytokines 2259198

Abstract Introduction High-parameter cytometric analysis enables discoveries of potential therapeutic targets and informs disease prognoses in translational and clinical research. CyTOF™ technology is a single-cell analysis platform that uses metal-tagged antibodies to resolve 50-plus markers in a single tube. Notably, antibody cocktails and stained samples can be frozen for later use and acquisition, or barcoded and pooled together, minimizing technical variation. Unlike fluorescence-based cytometry, spectral unmixing and single-stain controls are not required. Therefore, it is uniquely possible with CyTOF technology to rapidly design high-parameter panels and use intracellular markers to gain functional insights. The goal of this study was to design a 61-parameter CyTOF panel for in-depth functional immune profiling of human PBMC. Methods The panel contains lineage markers for major immune cell subsets and a diverse array of phenotyping T cell targets focused on activation, differentiation, checkpoint and cytokines and can be used to identify over 60 cell populations. Untreated and stimulated PBMC were barcoded, pooled and stained. Samples were frozen and acquired on a later day using a CyTOF XT PRO system. Results High-dimensional analysis of stimulated PBMC revealed striking cross-lineage immuno-functional diversity at the single-cell level. The expression of over 20 markers spanning the functional landscape of activation, checkpoints and cytokines was revealed across effector, memory, cytotoxic, regulatory and exhausted immune cell populations. Conclusion CyTOF systems enable the highest number of simultaneous measurements in a single panel, allowing for wide immune coverage and high resolution of intracellular targets to interrogate functional potential. Overall, studying functional immunology using CyTOF technology can elucidate the complex nature of immune responses to provide an understanding of how they relate to disease and treatments. For Research Use Only. Not for use in diagnostic procedures., Funding Source n/a Topic Categories Immune Mechanisms of Human Disease (HUM)