Abstract
The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (51Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate 51Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release 51Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.
Highlights
Cell-mediated killing is a pivotal mechanism in host immune responses to pathogens
The killing function of cytotoxic Natural Killer (NK) cells was determined by cytotoxicity assays using tumor target cells labelled with radioactive 51Cr, co-cultured at increasing ratios of NK effector cells at an effector-to-target (E:T) ratios ranging from 40:1 to 1:1 over a period of 4 h
The percentage of cytotoxicity was calculated by measuring the target cells’ viability, which in classic chromium release assays, is determined by the amount of radioactive chromium released in the supernatant
Summary
Cell-mediated killing is a pivotal mechanism in host immune responses to pathogens. NK cells are the main players of innate immunity and interact with target cells via inhibitory and activating receptors. CTLs are restricted by the major histocompatibility complex (MHC) molecules and utilize the T cell receptor to recognize antigenic peptides. Both cytotoxic cells kill the target by inducing cell lysis through the release of perforin and granzyme A/B and through the Fas–Fas ligand pathway [1]. For in vivo proof-of-concept and clinical evaluation of the efficacy of cell-mediated immunotherapy, it is mandatory to assess the effector functions of NK cells and CTLs against tumor target cells. A more accurate understanding of the kinetics of cell-to-cell killing might help to design more appropriate experimental protocols with well-timed data acquisition points
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