Article Text

Download PDFPDF

10 A multiparameter flow cytometry assay to monitor natural killer cell proliferation and activation in immuno-oncology clinical trials
  1. Jennifer Tsau1,
  2. Brittney Atzmiller1,
  3. David Quinn2,
  4. Tanya Mulvey2,
  5. Sema Kurtulus2,
  6. Jennifer Mataraza2,
  7. Shabnam Tangri1,
  8. Naveen Dakappagari1 and
  9. Ghanashyam Sarikonda1
  1. 1Navigate BioPharma Services, Inc., a Novartis subsidiary, Carlsbad, CA, USA
  2. 2Novartis Institutes for Biomedical Research, Cambridge, MA, USA


Background Natural Killer (NK) cells have garnered increasing interest as potential cellular therapies or as targets of biotherapeutic agents due to their ability to kill tumor cells in a non-antigen dependent manner. Hence, measurement of NK cell proliferation and/or activation following treatment can serve as a useful biomarker for assessing the efficacy of immunomodulatory therapies.

Methods We developed a novel 13-parameter flow cytometry panel incorporating cell differentiation (CD) markers important for identification of NK cell subsets (CD56, CD16), their proliferation (Ki-67), activation (CD25, CD335, NKG2D) and inhibition (CD159a) status. Additionally, CD markers that identify other cellular subsets known to be amenable to cytokine modulation (e.g., CD3 and CD14) were included for concurrent monitoring of T cell proliferation and monocyte activation. Method validation focused on analytical sensitivity, specificity and precision as key criteria of assay performance using peripheral blood mononuclear cells (PBMCs) stimulated with NK cell-activating cytokines and resting PBMCs from healthy donors.

Results The assay design allowed for robust quantitation of NK cell, T cell and monocyte functionalities. Lower limit of quantification (LLOQ) of target biomarker population was determined to be 1.0% of the parent population, based upon an analysis of 110 key target populations that displayed a co-efficient of variation (CV) of ≤25% and their frequencies ranged from 0.1% to 97.8% of the parent population. Additionally, ≤25% CV was observed in precision assessments, confirming the repeatability and reproducibility of the assay. Clinical trial utility of the assay was verified on cryopreserved PBMCs from patients with a variety of solid tumor malignancies. In these patients, the assay could clearly identify proliferating and activated NK cells, as well as proliferating T cells and activated monocytes, thus demonstrating its suitability for clinical trial applications.

Conclusions We developed and validated a novel multiparameter flow cytometry assay that allows for simultaneous measurement of proliferation, activation and inhibitory status of key immune cell subsets. Thus, this assay can help shed light on the mode of efficacy of novel therapeutic agents that modulate the immune system, aimed at treatment of cancer and autoimmune diseases.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See:

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.