A simple, economic, time‐resolved killing assay (2014)

NK cells and CTLs are key elements of the cellular immune response and eliminate pathogen-infected and tumorigenic cells 1-3. Quantification of killer cell-mediated cytotoxicity is of great importance in immunotherapy 4, 5, to predict cancer risk 6 and to improve antibody-based treatments 7-9. A precise characterization of killer cell activity is also required to better understand the underlying molecular cytotoxic mechanisms 10-13. The time kinetic of the killing process reveals crucial information of killer cell activity. However, the standard endpoint cytotoxicity assays cannot obtain the time-resolved killing kinetics efficiently, especially when the killer cells are in short supply. We have developed a novel real-time killing assay that uses the fluorescent dye calcein-AM (acetoxymethyl) to directly monitor the loss of fluorescence during target cell death. In its acetoxymethyl ester form, calcein passes through the plasma membrane and remains in the cytosol after cleavage of the AM groups by endogenous esterases in living cells. Upon death of the target cells, calcein is released from the cytoplasm into the supernatant (Fig. 1A, and Video 1). Using the bottom-reading mode of a conventional plate-reader, we show that the fluorescence intensity correlates linearly with the number of calcein-loaded target cells, for both live and lysed cells, over four hours (Supporting Information Fig. 1A–C). This is a typical time frame for endpoint assays to analyze killing capacity. Based on this property, the fraction of killed cells can be calculated at each time point, as verified by the known numbers of an artificially lysed fraction of cells (Supporting Information Fig. 1D). Using this assay, we analyzed the killing kinetics of human NK cells (Fig. 1B). The obtained time course of killing shows an initially high killing rate that decays over time, which is in agreement with a granzyme B reporter-based analysis 14. To verify if the real-time killing assay with the plate reader correctly quantifies target cell killing, we directly monitored single-cell killing in parallel using an automated high-content microscopy system (BD Pathway BioImager 855). As expected, rapid lysis of target cells by NK cells was observed (Fig. 1C and Video 2). The fraction of lysed target cells at each time point from the BioImager was quantified (Fig. 1D) and was found to correlate well with the killing kinetics measured by the plate reader (R2 = 0.98; Fig. 1E). Moreover, the real-time killing assay performs well with high target number and at low effector to target cell ratios (Supporting Information Fig. 2 and Video 3). This shows that target lysis can be reliably quantified by the real-time killing assay. We next compared our assay against two standard cytotoxicity assays, the lactate dehydrogenase (LDH) release assay 15 and the propidium iodide (PI) uptake assay 16. The LDH release assay reported lower target cell lysis than the real-time killing assay at all time points (Fig. 2A), whereas results from the PI uptake assay and the real-time killing assay were very similar (Fig. 2B). We assume that a delayed release of LDH from cells compared to calcein release or PI uptake could result from differences in molecular weight. The molecular mass of LDH is 135–140 kD, which is about 200 times higher than that of calcein (666.5 D) or PI (668.4 D). Nevertheless, we cannot exclude that different target cell numbers, which were applied in the LDH release and the real-time killing assay due to the distinct optimal sensitivity ranges, may also contribute to this difference, even though identical E:T ratios were used. In addition, to obtain the same kinetics as the real-time killing assay, both LDH release and PI uptake assays require many more killer and target cells, depending on the number of time points to be determined. Next we characterized the killing kinetics of primary human (Fig. 2C, D) and mouse (Fig. 2E, F) CTLs using the new assay. We found clear changes in the maximum killing rates determined from the kinetics at day 3 and day 5, whereas only smaller differences were observed comparing the endpoints (240 min). The maximum killing rate thus serves as a very sensitive parameter for characterization of killing capacity, which can only be obtained with a kinetic assay. There are several possible reasons for the observed kinetic differences in human versus mouse CTLs, such as different time frames to gain effector functions (e.g. killing and/or migration behavior), the divergent functions of major human and mouse granzymes 17, or differential TCR-mediated signaling kinetics 18, 19. Finally, the quantification of this real-time killing assay is independent of a specific plate reader model as results were similar for two different plate readers tested (Supporting Information Fig. 3). CO2 gassing is not necessary for this assay, since the impact of CO2 on killing kinetics during the measurement is negligible (Supporting Information Fig. 4). In summary, the kinetics obtained with this new real-time killing assay provides an accurate tool for quantification of killing capacity. In particular, the different killing phases (for instance, faster and slower ones) that may be determined by this assay can help characterize the underlying killing mechanisms. Given its accuracy, simplicity in handling, low cost, and minimal requirement for the number of killer cells (especially from patients), the real-time killing assay can be applied to determine killer cell cytotoxicity on a routine basis for clinical purposes with widely available microplate readers. We thank the Institute for Clinical Hemostaseology and Transfusion Medicine for providing donor blood, Carmen Hässig and Cora Stephan for excellent technical help, Martina Sester and Jan Dirks for access to BD FACSCanto II, Varsha Pattu and Jens Rettig for mouse CTL. This project was funded by Sonderforschungsbereich 894 project A1 (to M.H.), by Sonderforschungsbereich 1027 project A2 (to B.Q.) and by Chinesisch-Deutsches Zentrum für Wissenschaftsförderung GZ 857 (to M.H.). F.Z. and B.B. are supported by Sonderforschungsbereich 894 project A17 and INST 256/271-1 FUGG. Research carried out for this study with humanmaterial has been authorized by the local ethics committee. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Figure S1. Fundamentals of the real-time killing assay. Figure S2. The real-time killing assay can be used over a broad dynamic range of target cell numbers and effector to target cell ratios. Figure S3. The real-time killing assay can be carried out reliably with different plate-readers. Figure S4. CO2 does not affect killing efficiency. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.