Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry


  • Rachel A. Cunningham Dana-Farber Cancer Institute, Center for Immuno-Oncology
  • Martha Holland Dana-Farber Cancer Institute, Center for Immuno-Oncology
  • Emily McWilliams Dana-Farber Cancer Institute, Center for Immuno-Oncology
  • Frank Stephen Hodi Dana-Farber Cancer Institute, Center for Immuno-Oncology
  • Mariano Severgnini Dana-Farber Cancer Institute, Center for Immuno-Oncology



checkpoint markers, flow cytometry, immuno-phenotype, immunotherapy


As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4+ and CD8+ T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.


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How to Cite

Cunningham RA, Holland M, McWilliams E, Hodi FS, Severgnini M. Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry. J Biol Methods [Internet]. 2019Jun.3 [cited 2022Oct.6];6(2):e114. Available from:




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