How is natural killer (NK) cell function measured?

How NK Cell Function is Measured

NK cell function is measured primarily through flow cytometry-based assays that assess cytotoxicity, cytokine production (especially IFN-γ), and activation marker expression (CD69, CD25), with functional assays being superior to simple cell counts for clinical decision-making. 1, 2

Primary Measurement Methods

Flow Cytometry-Based Cytotoxicity Assays

• Flow cytometry has replaced traditional 51Cr-release assays as the preferred method for measuring NK cell responses because it allows simultaneous assessment of both effector cell function and target cell death 1, 3

• NK cells are identified as CD3-negative, CD56-positive, and/or CD16-positive populations within the viable lymphocyte gate defined by forward scatter (FSC) and side scatter (SSC) parameters 4, 3

• Target-induced NK cell loss (TINKL) measures NK cell responses by quantifying the disappearance of NK cells from the lymphocyte gate after interaction with target cells, capturing both NK cells that remain bound to targets and those undergoing target-induced apoptosis 3

• CD107a surface expression (a degranulation marker) combined with Annexin V binding to target cells provides a sensitive measure of NK cytotoxic activity 1

• The assay can measure natural killing, antibody-dependent cellular cytotoxicity (ADCC), and NK cell alloreactivity depending on the target cells used 3

Cytokine Production Assessment

• IFN-γ production is the gold standard functional readout, measured either by:

  • Intracellular flow cytometry staining with specific antibodies 1, 2
  • ELISPOT assays for extracellular IFN-γ quantification 1
  • Luminex-based multiplex cytokine detection 5

• Granzyme B ELISPOT correlates better with standard cytotoxicity assays than MHC tetramer or IFN-γ ELISPOT in clinical settings 1

Activation Marker Expression

• CD69 and CD25 expression on NK cells indicates activation status and correlates inversely with tumor burden 1, 2

• Intracellular perforin, granzyme B, and granulysin levels measured by flow cytometry serve as indicators of cytotoxic capacity 6, 7

Clinical Application Algorithm

When to Use Functional Assays vs. Cell Counts

• Always prioritize functional assays (cytotoxicity, cytokine production) over absolute NK cell counts when monitoring patients with malignancies or EBV-associated diseases 2, 8

• Functional assays provide prognostic information: lower NK activity correlates with higher cancer incidence and faster progression, while preserved function predicts better outcomes 8

Standardization Requirements

• A minimum three-color panel (CD3/CD16/CD56 or CD3/CD19/CD16+CD56) is required for basic NK cell identification 4

• A four-color panel (CD45/CD3/CD19/CD16+CD56) is recommended for comprehensive lymphocyte analysis 4

• CD45/side scatter gates must contain >95% lymphocytes to avoid contamination with monocytes or basophils 4

• Include positive controls (mitogens), negative controls, and internal quality standards for each assay to ensure consistency between sites 1

Important Caveats and Pitfalls

Technical Considerations

• NK cells have bright CD45 fluorescence but slightly higher side scatter than other lymphocytes, requiring careful gating to ensure complete inclusion of all NK subsets 4

• Monoclonal antibodies for T-cells (CD3), B-cells (CD19/CD20), and NK-cells (CD16/CD56) should account for all lymphocytes in the specimen as a quality control measure 4

• Standardized processing, cryopreservation, storage, and thawing protocols are essential—consider the protocols tested by immunologic monitoring consortia 1

Clinical Interpretation Pitfalls

• Tumor burden progressively impairs NK function: increasing malignant cell load correlates with decreased cytotoxicity, reduced IFN-γ production, and lower CD69/CD25 expression 1, 8

• Volatile anesthetics (isoflurane, sevoflurane) suppress NK cytotoxicity and cytokine production for several days postoperatively, which must be considered when timing functional assessments 1

• Tumor-derived cytokines (IL-1, IL-6, TNF-α) and immunosuppressive factors (IL-10, TGF-β) create a hostile microenvironment that diminishes NK activation independently of cell numbers 1, 8

Alternative Methods for Specific Contexts

• 7-AAD staining can measure apoptosis and death of target cells following NK cell incubation as an alternative to chromium release 9

• Multicolor polyfunctional flow cytometry allows simultaneous assessment of multiple cytokines (IFN-γ, IL-2, TNF-α) and provides more comprehensive functional profiling 1

• For virus-infected target cells, both 51Cr-release and 7-AAD methods remain valid options depending on laboratory capabilities 9