Distinguishing perforin-mediated lysis and granzyme-dependent apoptosis

CHAPTER SIXTEEN

Distinguishing perforin-mediated lysis and granzyme-dependent apoptosis Jesse A. Rudd-Schmidta, Joseph A. Trapanib,c,*,†, Ilia Voskoboinika,b,*,† a

Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia c Rosie Lew Program in Immunotherapy and Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia *Corresponding authors: e-mail address: [email protected]; [email protected] b

Contents 1. Introduction 2. Methods to assess perforin activity using purified protein 2.1 Sheep red blood cell (SRBC) lysis using recombinant perforin 2.2 Death of nucleated cells using recombinant perforin 2.3 Live cell imaging of effector lymphocyte-target interaction 3. Conclusion: Choice of assay References

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Abstract Perforin is an indispensable effector protein of primary cytotoxic lymphocytes (CTL or NK cells) that typically defend the host against virus infection, or gene-modified (chimeric antigen receptor—CAR) anticancer T cells. Perforin’s pore-forming activity is necessary for the delivery of proapoptotic serine proteases, granzymes, into the cytosol of infected or cancerous target cells. The complete loss of perforin function is detrimental for the function of cytotoxic lymphocytes, and leads to fatal immune dysregulation in infants and predisposes the carriers of hypomorphic perforin mutations to various chronic inflammatory sequelae and blood cancers. Here, we describe several optimized and validated functional assays using purified effector proteins and cytotoxic lymphocytes that enable detailed analysis of perforin-mediated target cell death pathways.

†

These authors contributed equally to the paper.

Methods in Enzymology, Volume 629 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.07.034

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2019 Elsevier Inc. All rights reserved.

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1. Introduction Cytotoxic lymphocytes (CLs), represented by CD8 + T cells and natural killer (NK) cells, kill their cognate infected or transformed targets through the cytotoxic granule cell death pathway (de Saint Basile, Menasche, & Fischer, 2010; Voskoboinik, Whisstock, & Trapani, 2015). Cytotoxic secretory vesicles (CSVs, frequently referred to as “granules”) are lysosome-like organelles found in various cell types, most notably hematopoietic cells and melanocytes. In CLs, the CSVs contain an electron dense core in which are stored proapoptotic serine proteases (granzymes, with granzyme B being most cytotoxic) and a nonredundant pore-forming protein, perforin (Sutton et al., 2016). The recognition of a target by the CL is followed by a cascade of signaling events leading to “granule exocytosis”; the CSVs migrate to the site of cell–cell contact, fuse with the presynaptic membrane and release their cytotoxic cargo into the synaptic cleft (de la Roche, Asano, & Griffiths, 2016). There, by forming oligomeric transmembrane pores (Law et al., 2010; Leung et al., 2017; Metkar et al., 2015; Praper et al., 2011), perforin transiently disrupts the target cell plasma membrane, allowing diffusion of granzyme B into the cytosol (Lopez, Jenkins, et al., 2013; Lopez, Susanto et al., 2013), where it cleaves proapoptotic intermediaries such as Bid and procaspases to rapidly initiate apoptosis (Kaiserman et al., 2006; Sutton et al., 2000). The congenital loss of perforin expression or function completely inactivates cell death signaling, leading to failure to clear key pathogens and to a severe immunoregulatory disorder—familial hemophagocytic lymphohistiocytosis (de Saint Basile et al., 2010; Janka, 2012; Jenkins et al., 2015; Meeths et al., 2014). Due to its key pathogenic role, we refer to the spectrum of infective, immunoregulatory and neoplastic consequences of perforin-deficiency as “perforinopathies” (Voskoboinik & Trapani, 2013). Various methodologies for measuring the activity of CLs or their cytotoxins have been developed over the past several decades (Hagn, Sutton, & Trapani, 2014; Sutton et al., 2008; Waterhouse & Trapani, 2003). Cell population-based assays typically involve short-term co-incubation of target cells with purified perforin and granzyme B (or other granzymes, if required), or with CD8 + T or NK cells. The readout/s allow an overall assessment of effector protein or CL function, but shed no light on the mechanism of target cell death. In our experience, by far the most quantifiable, reliable and reproducible cytotoxicity assay is the 51Cr release assay,

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in which the cytosol of target cells is first loaded with Na51 2 CrO4, and the 51 release of Cr into the media by dying cells following coincubation with purified perforin/granzyme B or CLs indicates the level of cell death. Chromium label is released by dying cells due to loss of plasma membrane integrity (secondary necrosis), a late feature of apoptosis. In order to simultaneously measure DNA fragmentation, 125I-UdR may also be incorporated into the target cell DNA during the labelling phase, and the release of radioactive DNA fragments into the media monitored over time. While the 51Cr release assay is still commonly used due to its high sensitivity and versatility, 125 I release has largely been replaced by TUNEL or FACS-based assays that measure activated caspases, which are cheaper, safer and more convenient. Providing the FACS gating strategies can reliably distinguish target cells in a mixed population that also includes CLs, such assays can estimate a range of cell death-associated parameters such as plasma membrane permeabilization, DNA fragmentation (using DNA dyes, e.g., PI and 7-AAD), caspase activation (using fluorogenic substrates) or phosphatidylserine exposure using fluorescently labeled Annexin V (AV) When assessing perforin/granzymemediated apoptosis in response to purified or recombinant proteins, it is essential to concurrently assess markers of both apoptotic and necrotic cell death and to select appropriate protein doses and endpoint/s for analysis. A common and recurrent problem in many published papers is the assumption that double-positive (PI+/AV+) cells reflect apoptosis (Waterhouse, Sedelies, et al., 2006; Waterhouse, Sutton, et al., 2006). However, this may not be so, as membrane disruption due to perforin-induced necrosis (cell lysis) can allow AV access to the inner leaflet of the plasma membrane that is enriched for phosphatidyl serine, thus providing a false-positive readout. When using recombinant perforin to deliver granzyme/s in vitro, a critical preliminary step is to identify the “sub-lytic” concentration of perforin sufficient to cause transient membrane damage, so presenting a “window of opportunity” for granzyme entry, but insufficient to cause granzymeindependent cell death through osmotic lysis (discussed below). In the longer term, assaying clonogenic target cell survival following exposure to perforin/granzyme B or intact CLs is an excellent alternative to the short-term (4–16 h) assays described above, as it unequivocally measures target cells that survive and resume proliferating (Crowley, Christensen, & Waterhouse, 2016). However, this assay does not discriminate between different modes of cell death. Similarly, quantitative assays of in vivo CL-mediated killing are based on the injection of a predetermined number of differentially tagged target and control nontarget cells into the

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blood stream of experimental mice, and measuring the ratio of the two cell types remaining in blood or spleen 4–24 h later (Baran et al., 2006; Coles, Mueller, Heath, Carbone, & Brooks, 2002). Single cell microscopy examining fixed cells, or live cells in real-time, provides the most informative and detailed assessment of the biochemical and cellular pathways of perforin/granzyme mediated cell death. However, these assays are time consuming and live cell imaging requires specialized equipment to ensure adequate cell viability is maintained during the imaging process. Importantly, experiments involving single cell analyses ought to be read in a blinded fashion and estimate large numbers of synapse events to eliminate unconscious bias and minimize experimental error. A range of reporter constructs such as fluorogenic substrates of granzyme B or caspases can be introduced into the target cells to monitor various aspects of cell death, e.g., (Choi & Mitchison, 2013) but these should be used with care as they may inadvertently influence the course of cell death. The method described in Section 2 is a notable exception as it does not require any endogenous reporter. In addition to reducing potential artifacts, this also allows experiments to be performed using a small number of cells, which is particularly useful when working with samples such as primary tumor cells.

2. Methods to assess perforin activity using purified protein 2.1 Sheep red blood cell (SRBC) lysis using recombinant perforin Any cell exposed to sufficient concentrations of purified perforin in the presence of >1 mM free Ca2+ will undergo osmotic lysis. Simple assays that measure the release of intracellular proteins (such as hemoglobin from SRBCs) can therefore be used to detect perforin activity, for example, in fractionated lysates made from purified CLs, or when expressing recombinant perforin in baculovirus-infected insect cells. Sheep RBCs are commonly used as they are available commercially, but erythrocytes from other species may be used in their place. The assay can be scaled up or down but is typically carried out in a 96-well plate format. 1. Wash required number of SRBC three times in Buffer A containing 20 mM HEPES (pH 7.4) and 150 mM NaCl, and then prepare a suspension of 5  108 cells/mL. 2. Mix 20 μL SRBC suspension, 140 μL buffer A and 20 μL of 10 mM CaCl2 (also diluted in buffer A) at room temperature.

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3. Add perforin diluted in up to 20 μL buffer A, and transfer the plate to 37°C for 20 min. 4. As a negative control measuring spontaneous release of Hb, add 20 μL of buffer A instead of the CaCl2 solution. 5. As a positive control measuring total Hb release, resuspend 20 μL cells in 180 μL distilled water. 6. Following incubation, centrifuge the plate at 500  g for 4 min, transfer the supernatant into fresh wells, and measure Hb absorbance at 450 nm. 7. The following formula is used to calculate % specific SRBC lysis: [(Hb release with perforin) (spontaneous Hb release)]/[total Hb release) (spontaneous Hb release)]  100.

2.2 Death of nucleated cells using recombinant perforin Nucleated cells are required in cell death assays that read out the effects of perforin-granzyme synergy, or that assess killing by intact CLs (CD8+ T cells or NK cells). In each case, the first step is to label target cells with Na251 CrO4 —subsequent steps measure the amount of label release following cell death. 2.2.1 Perforin-mediated lysis of 51Cr-labeled nucleated cells This assay is carried out similarly to the SRBC lysis assay except that the readout is 51Cr release rather than Hb. The assay is robust, reproducible, quantifiable and relatively cheap, and is strongly recommended for laboratories that carry out cytotoxicity assays on a repetitive or regular basis. Its major disadvantage is that appropriate safeguards such as adequate training in the safe handling of 51Cr need to be implemented, so laboratories that may have only a passing interest in CTL/NK cytotoxicity may find it more convenient to use an alternative technology (see Section 2.2.3.). 2.2.1.1 Labelling cells with Na251 CrO4

1. Given the high sensitivity of the assay, the number of target cells labeled with Na251 CrO4 can be quite small. However, since the labelling reaction is carried out in a small volume (up to 300 μL) for 1-2 h, cell density should be limited to 107 cells per mL to maintain cell viability. The minimum number of cells that can be labeled is limited by the efficiency of Na251 CrO4 uptake, which varies widely between different cell types, and can only be deduced empirically. Typically, we combine 2  106 target cells with 50–250 μCi Na251 CrO4 in 200 μL medium at 37°C for 1–2 h.

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In order to minimize the volume of radio-label stock solution added, be sure to purchase Na251 CrO4 of high specific activity. 2. After the incubation, 51Cr-labeled cells should be washed three times in the same medium, and finally resuspended at the desired cell density at RT. For most subsequent assays, we utilize 20,000 target cells per well, and resuspending the labeled cells at 106/mL means the desired cell number can be delivered in 20 μL. The labeled cells can then be used in various ways: to test for (i) perforinmediated cell lysis in a manner similar to SRBC; (ii) exposed to a low dose of perforin along with purified granzyme B that induces apoptosis, or (iii) cell death induced by intact CD8+ CTL or NK cells (Sutton et al., 2008). Since purified perforin is rapidly neutralized by lipids in fetal calf serum, serumfree culture medium (RPMI or DMEM, as appropriate) supplemented with 0.1% (w/v) lipid-free BSA (fraction V) should be used in these experiments. 2.2.1.2 Perforin-mediated lysis of nucleated cells

3. First, 160 μL of medium is placed into a well of a 96 well plate, then typically 10–20,000 51Cr labeled target cells in 20 μL medium, followed by perforin diluted in 20 μL of the same medium. The plate is then incubated at 37°C for 1 h. 4. Following the incubation, the plate is centrifuged at 500  g for 4 min, 150 μL of the supernatant is carefully collected without dislodging the tiny cell pellet, and the radioactivity is measured using a γ-counter. Spontaneous release is assessed in cells that were not exposed to perforin, and maximum labelling is assessed by incubating the same number of target cells with 20 μL 10% Triton-X or 1 M HCl. High concentrations of HCl are used as they cause depurination of DNA and reduced viscosity of the resultant lysate. 5. % Specific cell lysis/death is estimated using the formula: [(51Cr release with perforin) (spontaneous 51Cr release)]/[total 51 Cr release) (spontaneous 51Cr release)]  100. 2.2.1.3

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Cr release assay to assess perforin-granzyme B synergy

1. Cell labeling is carried out exactly as above. 2. Critical for this assay is to distinguish direct perforin lysis from granzyme-mediated apoptosis that is facilitated by perforin. Therefore, the following optimization experiments are necessary to determine a “sub-lytic” concentration of perforin that nevertheless causes sufficient

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plasma membrane disruption to allow granzyme B diffusion into the target cell. Perforin should be titrated as per A, to determine the amount of perforin required to kill approximately 10% of the cells. To validate that level of perforin as “sub-lytic” rather than “non-lytic,” the following is suggested: 2 Resuspend the required number of 51Cr labeled cells in duplicate in 200 μL of medium (as above) and pre-warm in a 1.5 mL Eppendorf (or 4 mL FACS) tube at 37°C for 5 min. Add perforin at the various concentrations to be tested, mix, and after 2 min further incubation add 1 mL of ice-cold medium to one tube and transfer it onto ice for 10 min. Incubate the remaining tube at 37°C for a further 30 min. Collect the supernatants and measure 51Cr release. “Sub-lytic” concentrations of perforin will cause minimum lysis at 37°C, but will result in over 50% lysis at 4°C. This is due to the fact that the plasma membrane repair response is severely inhibited at 4°C, and even a small “sub-lytic” number of pores on the plasma membrane cannot be repaired (Lopez, Susanto, et al., 2013). A similar experiment can be conducted using propidium iodide staining instead, if the laboratory setup precludes the use of 51Cr. These experiments are particularly important when assessing the effect of drugs on perforin/granzyme function, as a number of studies have led to erroneous conclusions due to inadvertent use of perforin inhibitors (Martinvalet, Zhu, & Lieberman, 2005), such as Tiron (Susanto et al., 2013). 3. Once the desired dosage of perforin has been determined, add desired amount/s of granzyme B to cell suspension, then mix in perforin, and incubate at 37°C for 4 h. It is critical to add perforin last, as the pores on the plasma membrane are short-lived (Lopez, Susanto, et al., 2013). Assess 51Cr release as described above. 2.2.2 FACS-based assays to assess perforin lysis and perforin-granzyme synergy One of the advantages of simple FACS-based assays described below is that they permit simultaneous evaluation of markers of apoptosis (externalized phosphatidylserine [PS] using fluorescently labeled AV) and necrosis (using PI or another cell-impermeable DNA dye, such as 7-AAD, as a prototypic marker/stain). It is essential to assess both parameters, since Annexin V-positive cells can in fact be necrotic, rather than apoptotic, due to Annexin V diffusing into a necrotic cell and binding PS on the inner leaflet of the plasma membrane. Conversely, apoptotic cells ultimately undergo

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secondary necrosis, and it is impossible to distinguish these cells from others that died purely through necrosis without including a marker of apoptosis (Fig. 1). Furthermore, it is important to select an appropriate time-point for the analysis: in Fig. 1, panels (B) and (C) denote early and late apoptotic cells respectively, and panel (D) will not discriminate between primary necrosis and late stages of apoptosis. An appropriate time-point cannot be determined a priori as the sensitivity to granzyme B varies considerably between different cell types; therefore, this should be established empirically. To set up a FACS-based assay as above, cells are coincubated with carefully titrated doses of perforin and granzyme B (as described above and in Lopez, Susanto, et al., 2013) and, at various time-points (typically, 1–4 h) transferred to ice, and 1 μM propidium iodide (or an alternative cell impermeable dye, e.g., 7-AAD) and fluorescent conjugate of annexin V (diluted as per manufacturer’s instruction) added for 10 min. The cells are then centrifuged for 4 min at 500  g, resuspended in FACS buffer (phosphatebuffered saline supplemented with 2% fetal calf serum), and analyzed by flow cytometry at appropriate wavelengths (depending on the type of dyes/ fluorophores used). An alternative method is based on the assessment of DNA fragmentation in the ethanol-fixed cells using propidium iodide, where sub-G1 (hypodiploid) population of nuclei indicates apoptotic cells (Riccardi & Nicoletti, 2006) (for example, see supplementary fig. 1H in Lopez, Susanto, et al., 2013). While this is a popular assay, its main disadvantages are the loss of cell morphology (due to lysis) and inability to discriminate between necrotic and healthy cells, which is an important factor when assessing the synergy between granzymes and cytolytic (necrotic) perforin.

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2.2.3 Other assays Over the years, a number of commercial flow cytometry and bioluminescence assays have been developed, and these can be used (by following manufacturer’s instructions) as an alternative to the assays described above. However, in our opinion, these are less informative or quantitative, more expensive and require optimization and validation.

2.3 Live cell imaging of effector lymphocyte-target interaction This method provides a temporal analysis of the CL/target cell interaction, commencing with the formation of a stable immune synapse (signified by calcium signaling within the CL), subsequent perforin pore formation on the target (PI influx from the medium), and the early stages of apoptosis (target cell rounding and blebbing). The protocol can be applied to any combination of effector and target cells, where the effector cell utilizes the perforin-mediated cell death pathway. This protocol will focus on the use of T cell receptor transgenic mouse OTI T cells and cognate peptide (SIINFEKL)-pulsed MC57 targets as an example; however, our group has previously applied the same method to primary mouse and human NK cells, clonal human T cells, and a variety of targets (Lopez, Jenkins, et al., 2013). Propidium Iodide (PI) emits strong fluorescence upon binding to either DNA or RNA, and as it is impermeable to the cell membrane it only binds nucleic acids once a cell has lost membrane integrity—here, due to membrane disruption by perforin released into the immune synapse. In 2013, it was discovered that when used at 10s–100s μM (as opposed to a traditional 0.5–2 μM used for assessing cell necrosis), PI influx and binding to cytosolic RNA at the point of synapse formation could be visualized, providing the first real-time marker of perforin pore formation (Lopez, Susanto, et al., 2013). a. Reagents and equipment 2 MC57, mouse fibrosarcoma cell line. 2 C57BL/6.OTI mice. 2 Antigenic peptide (if required). 2 Fluo-4, AM (Thermo Fisher, cat # F14201)—1 mM stock in DMSO. 2 Pluronic F-127, 20% solution in DMSO (Life Technologies # P3000MP) 2 Propidium Iodide—1 mg/mL (1.5 mM) stock solution prepared in culture media.

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2 Culture media with appropriate supplements and fetal calf serum. 2 Ibidi treat 8 well microscopy chambers (cat # 80826) or similar. 2 Confocal microscope with standard green (488 nm excitation) and red (561 nm excitation) channels, equipped with temperature and CO2 control (We have used a Leica TCS SP5 confocal microscope in our previous publications, and this is listed as an example in this protocol). b. Cell culture 2 For T cell activation, isolate splenocytes from a C57BL/6.OTI mouse and resuspend the CD8+ T cells in sterile medium containing 10 nM SIINFEKL and 100 U/mL IL-2. We typically use 100 mL RPMI-1640 for 50% of one spleen. After 72 h culture, wash cells three times with 50 mL PBS to deplete SIINFEKL peptide from the media. Resuspend in fresh CTL media supplemented with 100 U/mL IL-2, and split cells daily thereafter to 500,000 cells/mL using the same medium. 2 Maintain MC57 cells in complete SAFC DMEM, ensuring cells don’t exceed >80% confluency. 2 On the day prior to the experiment, recover adherent MC57 target cells with trypsin, wash and resuspend cells at 1  105/mL and plate 30,000 cells per well of an ibidi itreat 8 well chamber-slide (300 μL media). Note:*if using a fluorescence microscope without brightfield capabilities, pre label the MC57 cells with Cell Trace Violet to allow clear distinction from the CLs. c. Experimental set up 2 On the day of the experiment, replace media on MC57 cells with fresh medium containing 1 μM SIINFEKL peptide. After 1 h, wash the cells 3 times with complete SAFC DMEM media to remove unbound peptide. This is important as any residual free peptide may be taken up by the CLs themselves, resulting in “fratricidal” killing. 2 Place the chamber slides into the slide holder. Maintain both controlled temperature (37°C) and constant CO2 concentration (5%). On the Leica SP5 microscope, the slide holder is located within a temperature controlled chamber, and CO2 is provided by infusion using a gas incubation system (“The Brick”; ibidi). 2 Mix 1 μL of Pluronic (a mild detergent assisting with dispersion of Fluo-4, AM) and 1 μL of Fluo-4, AM in a 1.5 mL Eppendorf tube, then add 1 mL of activated OTI T cells at 1  106/mL

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(final concentration 1 μM Fluo-4, AM), incubate for 15 min at 37° C, then wash the cells twice and resuspend them in 1 mL of complete media (RPMI). 2 Prepare a working Propidium Iodide stock at 150 μM in complete RPMI media. Remove all media from the first well of MC57 and add 200 μL of the 150 μM PI stock. Then add 100 μL of the 1  106/mL Fluo 4-am labeled T cells. The SIINFEKL labeled MC57 cells and the Fluo-4-am labeled T cells are suspended in media containing 100 μM PI and are ready for imaging. d. Imaging 2 Adjust focus to a z-plane that contains the cytoplasm of the target cell; this will facilitate visualization of PI fluorescence (the “PI blush”) in the target cell cytoplasm subsequent to perforin pore formation. 2 Set up two channels for sequential imaging; the Fluo-4, AM channel providing 488 nm excitation and also containing the Brightfield/DIC collection, and the PI channel providing 561 nm excitation. We use a standard pinhole setting for the PI channel (1.38μm for the 40 [NA 0.85] air objective which we use), and a larger 5 μm pinhole setting for the Fluo-4, AM channel, to allow for any movement of the effector cells in the z-axis when making calcium flux measurements. 2 Begin sequential imaging of the channels using the time-lapse setting (x, y, t), acquiring approximately 6–7 frames/min. Make record of the time between frames to apply to later analysis. Alternatively, acquisition details can be recalled by opening the .lif file in Metamorph light. 2 Adjust gain/voltage to appropriate levels during the first movie, in particular ensuring that the green channel does not saturate during Ca2+ flux (to allow differentiation between multiple calcium fluxes if this analysis is required). 2 As gain needs to be adjusted at the beginning of this first acquisition, some initial events may be missed. However, once gain settings are optimized, they can be applied to the subsequent movies, allowing for imaging to commence immediately (Fig. 2). Image analysis can be performed using appropriate software such as Metamorph (Universal Imaging, Downingtown, PA). By selecting regions of interest around synapse events using the region tool, record the frame number corresponding to the following events: synapse initiation (initial cell–cell contact), calcium flux, PI blush, CL detachment, target cell rounding, target cell secondary necrosis. Also note any other features of interest

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Fig. 2 Expected result; six frame montage highlighting the key events of a T cell receptor transgenic mouse OTI T cell killing a SIINFEKL pulsed MC57 target. In this movie Calcium flux occurs at 3:25, PI blush 2 min later at 5:25. After PI blush the target begins to round (indicating caspase activation) and the effector cell detaches by 6:45. Reproduced from Voskoboinik, I., Whisstock, J. C., & Trapani, J. A. (2015). Perforin and granzymes: function, dysfunction and human pathology. Nature Reviews. Immunology, 15(6), 388-400. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25998963. doi:10.1038/nri3839 Time 0 sec

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Fig. 3 Quantitative analysis of Calcium flux within a Human NK cell and PI blush within its HeLa target cell. (A) Calcium flux is seen to occur after 30 s while PI blush in the target begins at 110 s. (B) Measurements of fluorescence intensity were made within Metamorph software and graphed as Fold increase in fluorescence vs Time for both Fluo-4, AM and PI. This method of analysis highlights the distinct rise in Fluo-4, AM fluorescence proceeding the PI blush in the target cell, and allows for a clear visualization of the temporal data. Here, arrows highlight the point of both Calcium Flux in the effector cell and PI blush in the target cell. Image reproduced from Lopez, J. A., Jenkins, M. R., RuddSchmidt, J. A., Brennan, A. J., Danne, J. C., Mannering, S. I., … Voskoboinik, I. (2013). Rapid and unidirectional perforin pore delivery at the cytotoxic immune synapse. Journal of Immunology, 191(5), 2328-2334. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/ 23885110. doi:10.4049/jimmunol.1301205.

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and reasons why some events may need to be excluded (i.e., cells drifting out of focus, too many cells in one area leading to an obscured image). Calculate the number of frames between key synapse events, and record in a table. These frame values can then be converted to time values (usually seconds) using the known acquisition details (seconds/frame). Further analysis should be conducted as per software instructions; an example of analysis parameters is shown in Fig. 3.

3. Conclusion: Choice of assay Many cell death assays have been developed and utilized extensively over the years, and their application varies widely depending on the research question, laboratory setup (e.g., availability of 51Cr or live cell microscopy) and the mix of skills in a particular lab. Every assay has its limitations: some do not discriminate between the modes of cell death or can only provide qualitative information, while others can be unnecessarily time-consuming and expensive. We suggest an experimental pipeline that in our opinion is most informative yet time- and cost-efficient; of course, it can be modified, if there is a need, to address specific biochemical or cell biology questions, or as new technologies arise. 1. When testing purified effector proteins, we suggest starting with a shortterm (4 h) 51Cr release assay that will determine synergy between perforin and granzymes and, if there is enhanced cytotoxicity, this can be followed by FACS-based assay/s that will confirm (or rule out) cell death by apoptosis. This may be followed up by live cell microscopy, if a more detailed and/or kinetic analysis is required. 2. For CL-based assays, we also suggest starting with the 51Cr release assay—this is generally sufficient to provide an accurate and reliable assessment of cytotoxic activity. If the assay fails to reveal cell death, it is important to next assess degranulation of the CLs using CD107a externalization, to confirm the formation of a functional immunological synapse and rule out congenital defect/s of the secretory machinery—for example, in suspected cases of congenital immunodeficiency. If the CLs undergo normal degranulation, but do not kill the target, it would then be important to confirm the expression of active perforin and granzymes by intracellular staining of activated CLs. These tests can be followed by time-lapse microscopy that should elucidate target cell membrane disruption by perforin (PI fluorescence), synapse dwell time and the fate of the target cell. For example, single cell microscopy has led

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to the discovery of an unusual and unsuspected caspase-independent mechanism of cell death mediated by murine granzyme A (Susanto et al., 2013). Understanding the mechanisms of CL cytotoxicity is an important biological issue, and more recently, with the development of CL-based immunotherapies, research in this field underwent a renaissance in which significant efforts have been made to improve the function of genetically modified cells (e.g., for CAR T cell based adoptive immunotherapy) (Davenport et al., 2018, 2015). To determine whether the resultant cells indeed have enhanced cytotoxic activity, they need to be rigorously characterized, ideally by using a mix of functional assays. The current paper offers a validated framework of relatively straightforward in vitro tests that will be useful for fundamental research and also for the applied diagnostic and therapyoriented research into CL function.

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