In-depth monitoring of the immune response to cancer and infection is vital to ascertain disease status and to assess immunotherapeutic options. Time-of-flight technology, the basis of CyTOF® mass cytometry, enables multiplex proteomic cellular phenotyping with 50 or more markers, making it ideal for comprehensive immune profiling. Unlike fluorescence-dependent approaches, which require signal compensation that makes the development of larger panels more challenging, CyTOF utilizes monoisotopic metal-tagged antibodies that exhibit minimal background signal, enabling the highest-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated dried-down 30-antibody cocktail preparation for immune profiling of human whole blood or PBMC. This assay is used with Maxpar Pathsetter™ software, which resolves whole blood into 37 immune populations comprising major lineage populations and their subsets, such as CD4 Th subsets and B and T cell memory cells, as well as stratifications of myeloid populations. The resulting system is a simple sample-to-answer solution for immune monitoring studies. Here, we expanded the 30-marker assay with 14 additional antibodies comprising pertinent targets of immunotherapy, including the exhaustion markers PD-1, PD-L1, Tim-3 and CTLA-4, and co-stimulation markers 4-1BB and ICOS. We also demonstrated the compatibility of the assay with downstream intracellular staining for cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B for assessment of cellular function in 4h PMA/ionomycin-stimulated whole blood cultures. Next, we modified the existing Pathsetter model to automate the analysis of whole blood stained with the expanded panel to generate reports on key immune cell populations, percentages of exhausted cells and cell subsets producing cytokines. Last, to demonstrate the ability of this expanded panel to identify antigen-specific T cell subsets accompanied by their in-depth phenotypic assessment, we tested this panel against CMV peptide-stimulated whole blood samples. We demonstrated the flexibility of the Maxpar Direct Immune Profiling Assay in panel customization and a streamlined workflow for automated analysis to enable comprehensive immune profiling of human whole blood. For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Shariq Mujib, Stephen K. Li, Nick Zabinyakov, Christina Loh. A comprehensive whole blood CyTOF immune monitoring panel with expanded surface and intracellular markers using the Maxpar Direct Immune Profiling Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1676.