Strategies for Accurately Measuring Cell‑Mediated Cytotoxicity
A Review of the Types of Cell-Mediated Cytotoxicity and Appropriate Measurement Methods

Introduction

Cell-mediated cytotoxicity is a cornerstone of the adaptive immune system, allowing our bodies to effectively identify, target, and lyse cells to help contain pathogens.

Scientists’ ability to harness this targeted killing power, first described in reports in the 1960s, is the reason the immune system has been studied extensively to fight bacteria, viruses, and infections — including cancer.

T cells and natural killer (NK) cells have evolved to possess the unique ability to circulate as effector cells, target cells infected by viruses or other intracellular parasites, and kill infected cells — all while causing minimal damage to surrounding tissue.

Because cell-mediated cytotoxicity is so critical to enhancing the immune system and developing targeted immunotherapies, scientists need to be able to measure this function accurately in vitro.

There are multiple forms of cell-mediated cytotoxicity and several methods for measuring it, which we will discuss below.

3 Forms of Cell-Mediated Cytotoxicity

cell-mediated cytotoxicity
Figure 1. A CD8+ T cell attaching to a target cell to initiate cell-mediated cytotoxicity.

1) Antigen-Specific T Cell-Mediated Cytotoxicity

Perhaps the most common form of cell-mediated cytotoxicity used in cancer immunotherapies, this is employed by drugs targeting immune checkpoints, like Keytruda® and Opdivo®.

In this mechanism, antigen-specific T cell receptors expressing the CD8 glycoprotein bind tightly to Major Histocompatibility Complex (MHC) class I molecules expressed by cancer cells. This binding triggers the CD8+ T cell to release perforin and granzymes, initiating lysis of the cancer cells.

T cell receptors expressing CD4 glycoproteins become helper T cells, which signal the presence of cancer cells to CD8+ T cells, which can, in turn, perform cell lysis.

fc receptors
Figure 2. Fc receptors on the NK cell recognize and bind to antibodies on the surface of the target cell.

2) Antibody-Dependent Cellular Cytotoxicity

ADCC relies upon effector cells — typically NK cells — to bind to antibodies on the surface of target cells and initiate cell lysis. While NK cell-mediated ADCC is the most common example, macrophages, neutrophils, and eosinophils have also been shown to mediate ADCC.

This mechanism starts with antibodies that are bound to the surface of an infected target cell. The Fc receptors on NK cells recognize and bind to the antibody, initiating the release of cytotoxic granzymes and ultimately cell lysis.

Certain antibody-based drugs, such as Herceptin® and Rituxan®, utilize the ADCC mechanism.

nk cell receptors
Figure 3. A variety of NK cell receptors detect target cell alterations and can trigger the release of cytokines.

3) Natural Killer Cell-Mediated Cytotoxicity

Unlike the previously described antibody-dependent adaptive immunity and antigen-specific cytotoxicity mechanisms, this cell killing method of the innate immune system requires neither antibody expression nor antigen expression.

NK cells can directly recognize tumor cells by employing a variety of receptors to detect cell alterations caused by infections and stress. Either a lack of signal through inhibitory receptors or too much signal from other receptors can initiate the cytotoxic process through the natural release of cytokines.

Most important and relevant to fighting cancer are the surface MHC molecules, which play a critical role both in controlling the NK cell response and maintaining NK cell responsiveness for secondary responses to known pathogens. Loss of surface MHC molecules leads to a lack of inhibitory signal, which can result in NK cell killing.

This mechanism is less commonly used in immunotherapy development. Chimeric antigen receptors (CARs) are being engineered into NK cells to take advantage of their cytotoxic capabilities and serve as an alternative to T cells.

Methods for Measuring Cell‑Mediated Cytotoxicity

Chromium-51 (51Cr) Release

Considered the gold standard since its creation in 1968, this method is not used as often today due to the required hazardous materials handling. In a Chromium-51 release assay, target cells are labeled with 51Cr. When introduced to the effector cells, the target cells release 51Cr through cell lysis. The amount of 51Cr in the supernatant of the centrifuged sample can then be measured.

Pros

  • Sensitive — the release of 51Cr is easy to detect
  • Measures death of target cells, not death of killer cells

Cons

  • Some leakage of the label, higher background
  • Requires hazardous materials

Materials Needed

  1. Target cells
  2. Effector cells: NK cells, PBMC containing Nk cells, T cells, macrophages, neutrophils, or eosinophils
  3. Chromium-51
  4. Culture medium
  5. Buffer (optional)
  6. Detergent
  7. 96-well U-bottom microplate
  8. Gamma counter or liquid scintillation counter

Lactate Dehydrogenase (LDH) Release

The LDH release assay measures the amount of soluble cytosolic enzyme (LDH) released during cell death using a colorimetric readout. LDH is present in most living cells, making it a convenient and reliable marker of cell death.

LDH release reduces NAD+ to NADH and H+ and oxidizes in the cell culture. The diaphorase then reduces tetrazolium salt to a red formazan. The amount of red color measured is representative of the amount of damaged or dead cells in the culture.

There is also a fluorometric version of the LDH release assay that is equally simple and effective.

Pros

  • LDH is more stable than other enzymes
  • No label required

Cons

  • Release of LDH is not limited to the target cells
  • High background

Materials Needed

  1. Target cells
  2. Effector cells: NK cells, PBMC containing NK cells, T cells, macrophages, neutrophils, or eosinophils
  3. Culture medium
  4. Substrate mix
  5. Buffer (optional)
  6. 96-well flat-bottom microplate
  7. Microplate reader

Calcein Release

The calcein release assay involves labeling target cells with a non-toxic, non-fluorescent compound, Calcein AM (acetoxymethyl). Calcein AM can easily penetrate live cells where it then produces Calcein, a highly fluorescent compound.

Upon introduction to effector cells, the live cells that are damaged or killed release Calcein into the culture. The fluorescence intensity in the culture can easily and quickly be measured using a microplate reader.

Pros

  • Can be detected using fluorescent plate reader
  • Label is specific to target cells

Cons

  • High background

Materials Needed

  1. Target cells
  2. Effector cells: NK cells, PBMC containing NK cells, T cells, macrophages, neutrophils, or eosinophils
  3. Calcein AM
  4. Culture medium
  5. Buffer (optional)
  6. 96-well flat-bottom microplate
  7. Microplate reader

Staining to Detect Degranulation

Another popular technique is staining of CD107a as a means of detecting degranulation of cytotoxic T cells or NK cells. CD107a is found on the granules in CTL and NK cells, which contain granzyme and perforin, two components involved in the lysis of target cells by these two effector cells.

Expression of CD107a is transient, but adding directly labeled anti-CD107a to T cells or NK cells prior to exposure to target cells allows for detection of this antigen during degranulation. You can also add other cell surface markers to identify subpopulations of cells that are degranulating.

Pros

  • Only requires one label
  • Low background

Cons

  • May miss degranulation
  • Flow cytometer needed

Materials Needed

  1. Target cells
  2. Effector cells: NK cells, PBMC containing NK cells, T cells, macrophages, neutrophils, or eosinophils
  3. Primary antibody
  4. Paraformaldehyde
  5. Methanol
  6. Buffer (optional)
  7. Detergent
  8. 96-well U-bottom microplate
  9. Fluorescence microscope
  10. Flow cytometer

Flow Cytometry

For a more advanced and real-time method of detecting cell-mediated cytotoxicity, consider flow cytometry-based assays. To use flow cytometry, first label the target cells with a fluorescent viability dye to differentiate them from the effector cells.

Pros

  • Deeper analysis
  • High sensitivity

Cons

  • Not as easily read in larger assays

Materials Needed

  1. Target cells
  2. Effector cells: NK cells, PBMC containing NK cells, T cells, macrophages, neutrophils, or eosinophils
  3. Primary antibody
  4. Culture medium
  5. 96-well U-bottom microplate
  6. Centrifuge
  7. Flow cytometer
Figure 4. K562, a cell line that is readily killed by NK cells, was labeled with CFSE and combined with increasing numbers of NK cells. Following overnight incubation, the dead cells were labelled using 7-amino-actinomycin D (7-AAD). Flow cytometric analysis was used to determine the percentage of dead cells (7-AAD-stained) among the target cells (CFSE-stained) while excluding the NK cells.

Conclusion

The method you choose for measuring cell‑mediated cytotoxicity should factor in your research goals, target cells used, equipment availability, and measurement and analysis expertise. While some methods can easily generate data from 96 wells (luminescent assays using a plate reader), other methods are better suited to a smaller number of samples that provide more extensive data that may provide needed insights.

Always account for spontaneous death or release of any label used. Calculation of the percent cytotoxicity uses spontaneous release to set a baseline. Samples that are killed by a toxic compound or detergent are used to set the maximum. The classic calculation is:

equation

Be sure to run your experiments in triplicate to control for experimental errors and bias. Studies that aim to maximize this important T cell function may lead to improved therapies.

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Image Sources

1 Adapted from original illustration by Dananguyen via Creative Commons
2 Adapted from original illustration by Satchmo2000 via Creative Commons
3 Adapted from Kristy C. Newman & Eleanor M. Riley (April 2007). Whatever turns you on: accessory-cell-dependent activation of NK cells by pathogens. Nature Reviews Immunology 7, 279-291. Accessed from www.nexcelom.com

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