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958 Simplifying high-parameter phenotypic and functional characterization of cancer immune cells
  1. Deeqa Mahamed,
  2. Geneve Awong,
  3. Leslie Fung and
  4. Christina Loh
  1. Standard BiTools, Markham, ON, Canada
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Interrogating immune cell composition and function in patients with cancer is critical for making disease prognoses, monitoring clinical efficacy of tumor immunotherapies, identifying novel therapeutic targets, and discovering predictive biomarkers of disease. Both the adaptive and innate arms of the immune system play important roles in generating pro- or anti-tumor milieus. Since immune cells can also indirectly impact CAR T cell or antibody-based immunotherapies, characterizing these cells using optimized and reproducible assays is critical.

Unlike traditional fluorescence-based flow cytometry, CyTOF® technology uses metal-isotope-tagged antibodies, which eliminates autofluorescence issues and minimizes signal spillover, allowing for the rapid design and application of 40-plus-marker panels. To enhance the utility of the 30-marker Maxpar® Direct™ Immune Profiling Assay™ (Maxpar Direct Assay), we developed nine add-on Expansion Panels for deeper phenotyping of immune cells. Here we demonstrate combining the Maxpar Direct Assay with Expansion Panels to create a 42-marker panel to characterize multiple myeloma PBMC.

Methods PBMC from healthy donors and donors with multiple myeloma were stimulated in vitro, then stained in the 30-antibody Maxpar Direct Assay tube with the NK Cell Expansion Panel 1 (CD181, NKp30, NKp46, PD-1, NKG2A, ICOS, and TIGIT) or the T Cell Expansion Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4–1BB) as drop-in antibodies. Surface staining was followed by intracellular staining with the Basic Activation Expansion Panel antibodies (IL-2, TNFα, IFNγ, perforin, granzyme B) after cell fixation and permeabilization. Anti-CD107a was added during stimulation to measure degranulation. Samples were acquired on a CyTOF XT™ instrument in automated batch acquisition mode. Automated analysis was performed with Maxpar Pathsetter™ software to enumerate immune cell types and quantify marker expression.

Results In addition to the 37 populations identified by Pathsetter with the base Maxpar Direct Assay, the new Expansion Panels allowed deeper profiling of NK and T cells, while the Basic Activation Panel revealed their cytokine responsiveness and cytotoxic potential. After activation, IFNγ, TNFα, and PD-1 were upregulated in early and late NK cells, as well as in effector, effector memory and central memory CD4 and CD8 T cells.

Conclusions The Maxpar Direct Assay Expansion Panels enabled deeper phenotyping of specific cell types and activation states, including ex vivo and activated myeloid cells, T cells, and NK cells. Thus, the optimized single-tube Maxpar Direct Assay is a powerful tool that can be further expanded and customized with predefined panels or antibodies to comprehensively study immune cells in health and disease.

Ethics Approval The samples obtained for this study were sourced from an accredited commercial provider.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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