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The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity
Methods 31 (2003) 135–142 www.elsevier.com/locate/ymeth
The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity Karin Fischer and Andreas Mackensen* Department of Hematology/Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg D-93042, Germany Accepted 7 April 2003
Abstract We present a rapid flow cytometric and non-radioactive functional assay developed for the determination of the cytotoxic activity of T lymphocytes, natural killer cells, and lymphokine-activated killer cells. In contrast to indirect evaluation of cytotoxicity using radioactive assays, this assay is based on the quantitative and qualitative flow cytometric analysis of cell damage on a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane, without disturbing its surface marker expression. It, thus, permits the distinction between target and effector cells. After short term in vitro incubation (1.5–3 h), AnnexinV-FITC (ann-FITC) staining allows to discriminate between apoptotic and non-apoptotic target cells. Data analysis is performed first by gating on PKH-26 positive target cells, followed by the analysis of the ann-FITC positive subpopulation. The percentage of cytotoxicity in the PKH-26 gated cell population is calculated by subtracting unspecific ann-FITC positive target cells, measured in appropriate controls without effector cells. Using in vitro generated antigen-specific cytotoxic T lymphocytes, we demonstrate that this flow cytometric assay is sensitive, correlates well with the standard 51 Cr release assay, and is easy to handle. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Cytotoxicity; Flow cytometry; Cytotoxic T lymphocytes; Apoptosis
1. Introduction Cellular cytotoxicity can be mediated by two major classes of contact dependent mechanisms. The first class is based on receptor–ligand interactions (e.g., Fas(Apo1)/Fas-ligand), which is known to be one of the central mechanisms for activation-induced cell death (AICD) in the homeostasis of lymphocytes [1]. The second class includes the granule secretion pathway that activates both caspase- independent (granzyme A) and dependent (granzyme B) death programs in the target cell. This activation appears to require the direct intracellular delivery of a family of granule-associated serine proteases. Multimeric complexes of serglycin, perforin, and granzymes are responsible for mediation and targeting of the apoptosis signal [2]. *
Corresponding author. Fax: +49-941-944-5581. E-mail address: [email protected] (A. Mackensen).
Effector cell-mediated cytolysis is usually determined by methods based on the release of different tracers from lysed target cells. In general, these markers are compounds containing radioactive isotopes such as 51 Chromium (51 Cr), 75 Selenium (75 Se), or Tritium (3 H) [3,4]. The 51 Cr release assay is the most widely used method [5] for measuring CTL responses in vitro due to its reliability and simplicity. Nevertheless, as the usage of radioactive material is potentially hazardous, it is desirable to find easy and reliable alternatives, which are safer and more cost efficient. Several non-radioactive assays based on the labelling with markers such as Europium (Eu3þ ) [6], bisbenzamid dye [7], and Calcein-AM (acetoxymethyl ester of calcein) [8] have been developed. However, the indirect measurement of the supernatant limits the accuracy of these methods. More recently, novel assays have been developed using fluorescent dyes [9–11]. The assessment of cell damage by flow cytometry aims to a more exact
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00123-3
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characterization of the death pathway via the detection of the percentage of apoptotic and dead cells [12]. One of the characteristic processes of the apoptosis pathway is a change in the plasma membrane architecture. During early apoptosis, typical membrane compounds such as phosphatidyl serine (PS) molecules turn from the inner to the outer leaflet of the cell membrane, without any loss of membrane integrity [13,14]. AnnexinV-FITC (ann-FITC), a molecule with high affinity to PS, can be used to label cells in the early apoptotic state, while propidium iodide (PI) indicates late apoptosis or cell death. We present here the development of a three color flow cytometric assay that uses the lipophilic membrane dye PKH-26, allowing a fine characterization of the specific effector cell cytotoxicity. PKH-26 has previously been used both in proliferation analysis, cell tracking studies [15,16], and flow cytometric analysis [17–19], and has been found to be a stable tracer. A recently described three color flow cytometric assay [12] proposed the staining of effector cells with PKH-26 for the further analysis of unstained target cells. However, this may lead to an accumulation of unlabelled effector cells in the target cell gate at high effector-to-target cell (E:T) ratios. To avoid this problem, we decided to label the target cells instead. We have demonstrated that the loading of target cells with PKH-26 is stable and does not impair viability and expression of surface molecules such as adhesion and major histocompatibilty (MHC) antigens [20]. The PHK-26 assay correlates well with the conventional 51 Cr release assay and provides additional information on target and effector cells. The current manuscript details the cell staining and flow cytometric procedures of the PKH-26 assay with special reference to the best application of this technique and troubleshooting at different situations.
2. Description of method 2.1. Principle The PKH-26 assay is a simple, fast, and reliable functional assay to determine cell-mediated cytotoxicity and is therefore a reliable alternative to the standard 51 Cr release assay. Instead of loading cells with radioactive 51 Cr, target cells are labelled with PKH-26, a fluorescent dye that stably integrates into the cell membrane. This step is necessary to distinguish between target and effector cells. Coincubation of target and effector cells can be performed in 96 well plates for 3 h or less. After the coincubation of PKH-26 labelled target cells together with effector cells, staining with AnnexinV-FITC allows the determination of apoptotic and non-apoptotic target cells.
2.2. Materials and experimental set-up 2.2.1. Target and effector cells As target cells, both cells growing in suspension as well as adherent cells can be used. Trypsination does not hamper subsequent membrane loading with PKH-26. In our experiments, we used human melanoma cell lines MeR190 and MeI493 and primary leukemic AML blasts. T2 cells are HLA*0201 human lymphoid cells that are defective in antigen processing, but efficiently present in exogenously supplied peptides [21]. T2 cells were loaded with 30 lg/ml of adequate peptide and were incubated overnight at 37 °C in serum-free medium. For the permeabilization of the plasma membrane in some experiments, paraformaldehyde fixed T2 cells were washed with 0.1% saponine solution (containing 0.1% bovine serum) and then stained with 5 ll ann-FITC for 30 min on ice in the presence of saponine. As effector cells we used HLA-A2 restricted Melan-A26-35 specific CTL lines generated in vitro by repetitive stimulation with autologous peptide-loaded dendritic cells [22]. Detection of antigen-specific CTL was performed with HLA-A2-Melan-A tetramers. PE-labelled HLA-A*0201 TM that had been folded around ELAGIGILTV (Melan-A26-35L ) were synthesized by Beckman Coulter (Fullerton, CA). Staining with tetramers was carried out for 30 min at 37 °C. In some experiments, the perforin pathway in CTL was blocked by concanamycin A (CMA, Sigma, St. Louis, MO) for 20 h before the PKH26 assay as well as during the assay incubation in a concentration of 100 ng/ml. 2.2.2. Fluorescence labelling Various target cells are transferred to polystyrene tubes and washed twice with serum-free medium before staining. The cells are then resuspended in a loading buffer (an aqueous, osmolarity-regulating solution containing no Ca2þ or other physiological salts; Sigma) and incubated for 40 min with freshly prepared PKH-26 (stock 1 mM in ethanol, Sigma) at room temperature. For the loading of tumor cells, it is recommended to use 0.8 ll, for small cells (lymphocytes) 0.5 ll PKH-26 in 200 ll loading buffer. This dye solution is then added slowly, while agitating the cell suspension on a shaker, in order to avoid overstained cells. After 30–40 min of shaking at room temperature, the loading of target cells is completed. The staining reaction is stopped by the incubation with 500 ll human serum (PAN Biotech GmbH, Aidenbach, Germany) for 30 s at room temperature. This step is recommended to bind most of the residual lipophilic PKH-26 dye to serum proteins. After centrifugation, the cell pellet is transferred into a fresh 50 ml tube (Falcon, France) and washed twice with 40 ml RPMI containing 10% human serum. It is important to abide by the washing procedure in order to remove all PKH-26 in the supernatant completely. The
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dye stock is dissolved in ethanol and can be stored light protected at room temperature, when sealed tightly to prevent evaporation. 2.2.3. Coincubation of target and effector cells During the incubation for fluorescence labelling and washing of the target cells, the effector cells are counted, washed, and seeded in a volume of 100 ll medium into 96-well-V-bottom plates (Costar, Corning, NY) at different effector:target ratios. Six to eight wells per effector:target ratio are applied. Higher effector: target cell ratios require a large amount of effector cells, in this case 2 wells should be sufficient. Coincubation of effector and target cells should be performed in effector cell medium. PKH-26 labelled target cells (5 103 ) are plated in 100 ll medium/well and define the assay start. V-bottom plates allow rapid contact between target and effector cell, therefore additional centrifugation is not necessary. The recommended standard incubation time for cocultivation is 3 h. 2.2.4. Harvesting of cells and staining with ann-FITC and PI After the co-incubation, cells are harvested from the microtiter plates into separate polystyrene tubes (Falcon) for each effector:target ratio. One ml phosphate buffered saline (PBS, Gibco, Germany) is added and cells are centrifuged. Cells are then resuspended in 100 ll high calcium annexinV-binding buffer (1-fold concentrated, Pharmingen, Germany). Staining with 5 ll annFITC (BD PharMingen, USA) is performed for 10 min at room temperature in the dark. Immediately before flow cytometric analysis, 0.1 lg PI (Calbiochem, Germany) per 100 ll binding buffer is added. If more washing steps are planned, it is essential to prepare more wells per effector/target ratio, to compensate for cell loss during centrifugation. 2.2.5. FACS analysis and compensation FACS analysis was carried out on a FACScan Cytometer (Becton–Dickinson, Immunocytometry Systems, San Jose, USA). The excitation is at 488 nm, the fluorescence of the three fluorochromes is recorded at 530 nm for FITC (FL1), at 585 nm for PKH-26 (FL2), and at >675 nm for PI (FL3), respectively. For compensation, both unstained and PKH-26 stained cells should be prepared. The small amount of spontaneously apoptotic cells in the cell suspension is sufficient for compensation. As ann-FITC is known to increase unspecific fluorescence, compensation is carried out in the presence of ann-FITC: Compensation between FL1 and FL2: Vial 1: unlabelled target cells stained with ann-FITC (compensation of specific ann-FITC positive cells). Vial 2: PKH-26 labelled cells with ann-FITC (compensation of ann-negative cells with neglection of
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the small population of specific ann-FITC positive cells). Approximate values are: FL1—0.5% FL2; FL2—78% FL1. Fluorescence analysis is quantified using the Cellquest software (Becton–Dickinson). At low effector:target cell ratios, usually a smaller amount of cells is acquired than at higher effector:target ratios. Once compensated, the instrument settings can be used for further experiments, especially if no PI staining is carried out. Nevertheless the measurement of unstained cells is advisable. 2.2.6. 51 Cr release assay Conventional 51 Cr release assay is performed as previously described [23]. Briefly, the cytotoxic activity of T cell lines is measured using triplicate cultures in Vbottomed plates. Target cells are labelled with 200 lCi for 1 h. Target cells are then washed twice and seeded into the plates at E:T ratios of 25:1, 5:1, and 1:1 at 2000 target cells/well. For spontaneous release, targets are plated without T cells in MÔ medium plus 5% autologous plasma. For maximum release, target cells in 100 ll medium are plated with 100 ll of 0.15% Triton X-100 (Sigma). Triplicate wells are averaged and the percentage of specific cytotoxicity was calculated as follows: cpmsample cpmspontaneous release 100 cpmmaximum release cpmspontaneous release ¼ % specific lysis:
3. Detection of T cell mediated cytotoxicity by the PKH26 assay 3.1. Stability of PKH-26 in the cell membrane Flow cytometric analysis of cell-mediated target cell damage is based on the stable loading of target cells combined with a low percentage of spontaneous release. We tested the membrane dye PKH-26 (red fluorescence) for its ability to stain different vital primary tumor cells, such as melanoma cells and leukemic blasts. Staining with the optimal concentration of 2 lM PKH-26 results in stable loading with high fluorescence intensity in several primary tumor cells. One representative experiment is shown in Fig. 1A. In contrast, overstaining of target cells with 10 lM leads to the accumulation of intensively stained single cells, resulting in an artificial light emission in channel 1 [20]. This potentially interferes with actual double-positive target cells and may lead to false-positive results in the cytotoxic assay. The concentration of 2 lM used in the experiments allows complete compensation in the absence of overstained cells. Of note, leukemic blasts, which generally show a rather weak uptake of compounds from the external medium, reveals a high staining intensity with PKH-26
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Fig. 1. Labelling of target cells with PKH-26. Panels represent either one color histograms (A) with the x-axis showing log scale red fluorescence intensity (FL-2) and with the y-axis showing the relative cell number or two color dot plots (B). (A) Leukemic blasts were loaded for 40 min with 2 lM PKH-26 (lower plots) and co-stained with propidium iodide (PI). Unstained cells were used as control (upper plots). (B) Apoptotic PKH-26 labelled target cells (R2) exhibit a slightly decreased fluorescence intensity compared to vital PKH-26 stained target cells (R1). Separation from unstained effector cells (R3) is clearly possible.
upon loading. Labelling with PKH-26 remains stable in all vital cells tested for a minimum of 72 h without any loss of fluorescence intensity. In contrast, labelling of primary leukemic blasts with 51 Cr reveals a high spontaneous release (>25%) after 4 h incubation (data not shown). Apoptotic target cells exhibit a slightly decreased PKH-26 intensity but can be clearly discriminated as PKH-26 positive target cells (Fig. 1B). As the PKH-26 assay is a snap-shot which detects cell damage, it is recommended to choose a co-incubation time which accounts for the killing kinetics of effector cells. Strongly prolonged coincubation time will result in the death of apoptotic target cells and will, thus, generate artificial results. 3.2. Analysis of viability and surface marker expression in PKH-26 labelled target cells We tested whether staining with PKH-26 may alter the viability or the surface marker expression in labelled target cells. Apoptotic cell death of target cells measured by ann-FITC/PI staining before and 4 h after loading with PKH-26 did not show any changes in the percentage of either ann-FITC (data not shown) or PI-
positive cells (Fig. 1A). Moreover, we assessed if staining with PKH-26 may alter the expression of cell surface antigens required for functional effector/target interactions. Target cells were stained before and 4 h after PKH-26 labelling with fluorescence-conjugated mAbs against CD54, CD58, HLA-ABC, HLA-DR, and CD33, CD34 Mel-1, and Mel-2, for leukemic blasts and melanoma cells, respectively. Analysis of the surface markers did not show any significant changes after PKH-26 staining [20]. 3.3. Analysis and interpretation of results obtained from the PKH-26 assay 3.3.1. Information on the apoptotic state of target cells The PKH-26 assay provides fast and reliable information on specific target cell damage. The determination of target cell killing is mainly based on ann-FITC binding on PKH-26 gated cells. As shown in Fig. 2A, concurrent PI staining does not add to the information obtained with ann-FITC staining alone. PI single positive target cells are rare, because PIþ affected target cells are prevailing ann-FITCþ . The reason for this effect may be the diffusion of ann-FITC through impaired cell
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Fig. 2. Analysis of specific cytotoxicity using the PKH-26 assay. Target cells were selected by gating on the PKH26-positive cell population and further analyzed for different subpopulations. (A) Percentage of different apoptotic target cell subpopulations calculated according to the formula indicated in Section 3.3.4. (B) Treatment of T2 target cells with saponine leads to diffusion of ann-FITC into the cytoplasm of damaged cells and mainly results in ann-FITCþ /PIþ double positive target cells.
membranes and subsequent binding to phosphatidylserine residues on the inner leaflet of the cell membrane. In order to verify this hypothesis, we treated T2 target cells with saponine, a reagent that permeabilizes the cell membrane, and determined ann-FITC binding on these cells. As shown in Fig. 2B, cells treated with saponine are 94% double positive for PI and ann-FITC, despite the absence of classical apoptotic stimuli. Comparable results were obtained when target cells were submitted to one freeze and thaw cycle (data not shown). Therefore, we conclude that a separation of late apoptosis and necrosis in the PKH-26 assay is principally impossible. The distinction of ann-FITC single (early apoptosis) and ann-FITC double positive (late apoptosis/necrosis) is feasible, but not necessary. As shown in Fig. 2A, the correlation of early target cell damage with ann-FITC is very high, and only few cells are in a late apoptotic/necrotic state. We, therefore, suggest that no additional PI staining beside the ann-FITC staining is necessary in the PKH-26 assay. 3.3.2. Information on the frequency of specific T cells The information concerning the frequency of specific effector T cells is very limited. We carried out the PKH26 assay in parallel with Melan-A tetramer staining. In the first experiment we used effector cells containing 76%
Melan-A-specific tetramer positive T cells, in the second setting we diluted the Melan-A specific T cells with effector cells of a different specificity, but from the same donor, to a frequency of 26% Melan-A specific T cells (Fig. 3A). Both effector cell cultures result in a similar specific lysis curve (Fig. 3B). This finding reveals that the result of a PKH-26 assay does not correlate necessarily with the frequency of antigen-specific CTL. Other methods should be used in order to determine the content of antigen specific T cells, for example tetramer staining. 3.3.3. Phenotypic information on target cells The PKH-26 assay is performed on the flow cytometer and, therefore, allows any additional surface marker staining. In the described setting, fluorescence channels 1 and 2 are required, while any other channels can be used for further phenotypic analysis of the target or effector cells. However, it should be noted that an additional antibody staining prolongs incubation time. Furthermore, supplements in the antibody solution should be avoided, if they increase apoptosis. 3.3.4. Data analysis and cytotoxicity quantification In order to distinguish between target and effector cells, gating of PKH-26 positive target cells is performed.
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The ratio of corrected ann-FITC positive events ( ¼ ann-FITC positive eventssample ) ann-FITC positive eventscontrol ) to corrected total events ( ¼ total eventssample ) ann-FITC positive eventscontrol ) 100 reflects the real percentage of ann-FITC-positive cytotoxicity. Note that if the number of acquired total events differs at various E/T ratios, the amount of unspecific positive events has to be determined separately for each E/T ratio in the formula: % specific ann-positive cells ¼ ð½ann positive cellssample ann positive cellscontrol =½total eventssample ann positive cellscontrol Þ 100:
3.4. Incubation time and standardization
Fig. 3. The cytotoxic activity measured with the PKH-26 assay does not correlate with the frequency of antigen-specific T cells. (A) PKH-26 assays were carried out with two different CTL lines, which contain a frequency of 74.6 or 26.4% Melan-A specific T cells, respectively. The frequency of antigen-specific CTL was determined by HLA-A2-Melan-A tetramer staining. (B) The cytotoxic activity of both Melan-A specific T cell lines containing different frequencies of Melan-A tetramerþ CTL was measured in an 3 h PKH-26 assay. Data demonstrate no correlation between the frequency of antigenspecific CTL and the amount of cytotoxic activity measured with the Pkh-26 assay. Similar results were obtained with 51 Cr release assay (data not shown).
Ann-FITC single-positive cells represent target cells in early apoptotic state, ann-FITC/PI doublepositive cells include cells in the later apoptotic state, and PI single-positive cells represent dead/necrotic target cells. With escalating E/T ratios, increasing numbers of ann-FITC single-positive or ann-FITC/PI double-positive cells are observed. For the quantification of the specific cytotoxicity, the amount of unspecific positive cells (determined in the control sample without effector cells) in the corresponding quadrant must be subtracted. The percentage of unspecific positive cells must not be subtracted, because otherwise 100% cytotoxicity could principally not be reached. The calculation is to be carried out separately for each quadrant and is exemplified for the ann-FITC single-positive fraction.
One of the most important advantages of the PKH26 assay is its short incubation time. We analyzed the cytotoxic activity of Melan-A specific CTL, measured with PKH-26/ann-FITC double-positive target cells at three different co-incubation times, at 1.5, 2.5, and 3.5 h respectively [20]. The cytotoxic activity of effector cells at higher E/T ratios (>5:1) was found to be relatively constant over the analyzed co-incubation times from 1.5 to 3.5 h, reaching a plateau at a ratio of 20:1. In contrast, at lower E:T ratios (0.2 to 5:1), the percentage of cytotoxicity correlated well with the time of incubation. Despite the detection of specific lysis even after 1.5 h of coincubation, an incubation time of about 3 h is recommended in order to maintain the comparability of results from PKH-26 and 51 Cr release assay. Please note that the results from different PKH-26 assays can only be compared if the incubation time is standardized. Moreover, in our test system with Melan-A specific T cells, an incubation time of over 6 h definitely does not show a further increase in detected target cell damage (Fig. 4A). To ascertain if this rapid cytotoxicity is mainly mediated by the perforin/granzyme pathway, blocking experiments with concanamycin-A (CMA) were performed. As shown in Fig. 4B CMA, an efficient blocker of the perforin pathway, is able to completely block the cytotoxic activity of Melan-A specific CTL measured in the PKH-26 assay. Please note that the experimental setup and timing can influence the incubation time. For example, the usage of flat bottom plates instead of the recommended V-bottom plates may shorten the effector/target cell contact time. Moreover, an extensive delay between harvesting the cells and flow cytometric analysis should be avoided, as the killing in the cell effector/target cell suspension can proceed slightly at room temperature. Working on ice may minimize this effect. In summary, standardization of the working procedure will achieve optimal PKH-26 assay results.
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Melan-A specific CTL. Incubation of target cells with anti-HMW-MAA mAb, recognizing an irrelevant epitope on melanoma cells, had no effect [20].
4. Practical considerations and trouble shooting
Fig. 4. PKH-26 assay kinetics and inhibition of cytotoxicity by blocking the perforin pathway. (A) PKH-26 labelled Melan-A expressing target cells were incubated with Melan-A antigen-specific CTL for 3, 6, and 9 h at various effector:target ratios prior to the PKH26 assay. Data are presented as mean cytotoxicity (n ¼ 3) of Melan-A specific CTL, as determined by % ann-FITC positive cells. (B) Cytotoxicity of in vitro generated Melan-A specific T cells can be inhibited by the pretreatment of CMA, a blocker of the perforin pathway.
3.5. Comparison between the PKH-26 and assays
51
Cr release
The PKH-26 flow cytometric assay was directly compared to the standard 51 Cr release assay [20]. MelanA specific CTL generated in vitro were tested simultaneously by both methods against HLA-A2þ Melan-A expressing target cells and HLA-A2þ Melan-A control cells, using E/T ratios of 25:1, 5:1, and 1:1. The two different assays showed comparable results at all E/T ratios. In addition, cytotoxicity against the control cell line was negative in both assays. Data demonstrated a good correlation between results obtained from the PKH-26 assay and those obtained by the standard 51 Cr release assay (r2 ¼ 0:9777). Interestingly, results obtained with the PKH-26 assay reveal a slightly higher percentage of cytotoxicity as compared to the 51 Cr release assay. Furthermore, it was tested if the addition of blocking mAbs can inhibit the percentage of specific cytotoxicity measured with the PKH-26 assay. Blocking mAbs, either against effector cells (CD3, CD8) or target cells (HLA-ABC), are able to inhibit the cytotoxicity of
Analysis of the cytolytic activity of CTL is central in monitoring antigen-specific immune responses. Traditionally, the cytotoxicity of T cells has been measured using 51 Cr release assays [5]. Here, we have described an easy flow cytometry based killing assay, which permits exact evaluation of target cell death (early apoptosis, membrane damage) occurring during antigen-specific CTL responses. This method exhibits several advantages over standard radioactive functional assays like the 51 Cr release assay. First, the potentially harmful handling of radioactive isotopes can be avoided. Expensive laboratory equipment and waste disposal is not necessary for the PKH26 assay. Second, an outstanding advantage is the ability to distinguish clearly between target and effector cells. It should be mentioned that, if a subpopulation of target cells is overstained it is recommended to load the PKH26 dye more slowly onto the target cells by adding the dye solution in drops. If the whole cell suspension is overstained, a lower concentration of PKH-26 dye is recommended. As the basis of the PKH-26 assay is not the release of a marker, ‘‘minimal release’’ or ‘‘maximal release’’—as ascertained in the 51 Cr release assay—needs not to be determined. The maximum effect is determined by the count of vital cells that can potentially be killed in the assay. The background apoptosis without effector cells corresponds to the ‘‘minimal release’’ in the 51 Cr release assay. Notably, primary leukemic blasts, showing inefficient labelling and high spontaneous release after incubation with 51 Cr [24], demonstrated high and stable staining of the PKH-26 dye without increased spontaneous apoptosis (see Fig. 1A). In contrast to the 51 Cr release assay, which is based on the relatively unelucidated process of 51 Cr release in affected target cells, AnnexinV binding—the basis for the PKH-26 assay—is a well described process. Another striking advantage of the PKH-26 assay is the shortened incubation time needed to produce reliable results. The sensitivity of standard 51 Cr release assays requires an incubation time of 4 h. Since optimal killing is generally reached soon after the effector and target cells are brought into contact [25], a reliable measurement of killing should be detectable at an earlier time point. Optimal killing in the PKH-26 assay was observed even within an incubation period of 1.5–2.5 h [20]. The fourth advantage is the ability to directly evaluate the effector/target cell suspension in the flow cytometer, instead of indirectly concluding the target cell
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damage from the supernatant of the 51 Cr release assay. It is very useful to get information about the physiological condition, the viability, and the phenotype of the effector cells. Moreover, any further phenotypic surface marker staining on target cells (e.g., MHC expression) can be carried out simultaneously. As early cell damage mediated by effector cells correlates mainly with annFITC staining, an additional PI staining is not necessary for optimal results. In addition, we were able to demonstrate that a more detailed separation of late apoptotic (ann-FITCþ /PI ) and necrotic (PIþ ) cells is virtually impossible: it is strongly suggested that membrane impairment in the affected target cells is responsible for the diffusion of PI as well as ann-FITC into the cytoplasm. The most important advantage of the PKH-26 assay is its flexibility. The flow cytometric assay principle can be adapted to various conditions. It is, therefore, strictly recommended to standardize the PKH-26 assay in order to allow comparison between several PKH-26 assay results. Our results demonstrate that the staining of both effector and target cells [10] is not necessary for the accurate discrimination between the two cell types. Aubry et al. [12] have shown recently that the labelling of effector instead of target cells may represent an alternative method for the assessment of cellular cytotoxicity. However, staining of effector cells may be critical, since small percentages of cells may remain unstained. Especially if high E/T ratios (50:1, 100:1) are applied, unstained effector cells being counted as target cells may lead to false-positive or false-negative results. Data demonstrate strong lysis of Melan-A expressing target cells by Melan-A specific CTL lines, while maintaining excellent specificity and HLA restriction. A definitive comparison of the PKH-26 assay to the standard 51 Cr release assay was performed and showed a very good correlation between the two methods. However, results obtained with the flow cytometric assay showed a higher percentage of cytotoxicity as compared to the 51 Cr release assay. In conclusion, the PKH-26 assay is a quantitative method for the detection of cell-mediated cytotoxicity and does avoid the use of radioactive labels. The reliability and reproducibility of this assay makes it a valuable alternative to the standard 51 Cr release assay. allowing a greater insight into the mechanisms of cellmediated cytolysis by different effector populations.
References [1] N. Moulian, S. Berrih-Aknin, Sem. Immunol. 10 (1998) 449– 456. [2] S.S. Metkar, B. Wang, M. Aguilar-Santelises, S.M. Raja, L. Uhlin- Hansen, E. Podack, J.A. Trapani, J.C. Froelich, Immunity 16 (2002) 417–428. [3] J.-C. Cerottini, K.T. Brunner, Adv. Immunol. 18 (1974) 67–132. [4] A. Mantovani, T.R. Jerrells, J.H. Dean, R.B. Herberman, Int. J. Cancer 23 (1979) 18–27. [5] K.T. Brunner, J. Mauel, J.C. Cerottini, B. Chapuis, Immunology 14 (1968) 181–196. [6] A.K. Patel, P.N. Boyd, J. Immunol. Methods 184 (1995) 29–38. [7] F.N. Toka, M.G. Niemialtowski, I. Spohr de Faundez, M. Gierynska, Acta Virol. 40 (1996) 239–244. [8] R. Lichtenfels, W.E. Biddison, H. Schulz, A.B. Vogt, R. Martin, J. Immunol. Methods 172 (1994) 227–239. [9] A.E. Mattis, G. Bernhardt, M. Lipp, R. Forster, J. Immunol. Methods 204 (1997) 135–142. [10] D. Flieger, R. Gruber, G. Schlimok, C. Reiter, K. Pantel, G. Riethmuller, J. Immunol. Methods 180 (1995) 1–13. [11] S. Johann, J.S. Blumel, M. Lipp, R. Forster, J. Immunol. Methods 185 (1995) 209–216. [12] J.-P. Aubry, A. Blaecke, S. Lecoanet-Henchoz, P. Jeannin, N. Herbault, G. Caron, V. Moine, J.-Y. Bonnefoy, Cytometry 37 (1999) 197–204. [13] S.J. Martin, C.P. Reutelingsperger, A.J. McGahon, J.A. Rader, R.C. Van-Schie, D.M. LaFace, D.R. Green, J. Exp. Med. 182 (1995) 1545–1556. [14] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, J. Immunol. Methods 184 (1995) 39–51. [15] A.J. Young, J.B. Hay, Int. Immunol. 7 (1995) 1607–1615. [16] N. Rosenblatt-Velin, J.F. Arrighi, P.Y. Dietrich, V. Schnuriger, I. Masouye, C. Hauser, J. Invest. Dermatol. 109 (1997) 744–750. [17] Y. Yamamura, N. Rodriguez, A. Schwartz, E. Eylar, B. Bagwell, N. Yano, Cell Mol. Biol. 41 (1995) 121–132. [18] D. Flieger, U. Spengler, I. Beier, R. Keinschmidt, A. Hoff, M. Varvenne, T. Sauerbruch, I. Schmidt-Wolf, Hybridoma 18 (1999) 63–68. [19] M.E. Sheehy, A.B. McDermott, S.N. Furlan, P. Klenerman, D.F. Nixon, J. Immunol. Methods 249 (2001) 99–110. [20] K. Fischer, R. Andreesen, A. Mackensen, J. Immunol. Methods 259 (2002) 159–169. [21] R.D. Salter, D.N. Howell, P. Cresswell, Immunogenetics 21 (1985) 235–246. [22] N. Meidenbauer, J. Marienhagen, M. Laumer, S. Vogl, J. Heymann, R. Andreesen, A. Mackensen, J. Immunol. 170 (2003) 2161–2169. [23] A. Mackensen, G. Carcelain, S. Viel, M.C. Raynal, H. Michalaki, F. Triebel, J. Bosq, T. Hercend, J. Clin. Invest. 93 (1994) 1397– 1401. [24] E. Weidmann, J. Brieger, B. Jahn, D. Hoelzer, L. Bergmann, P.S. Mitrou, Ann. Hematol. 70 (1995) 153–158. [25] J.C. Hiserodt, L.J. Britvan, S.R. Targan, J. Immunol. 129 (1982) 1782–1787.
The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity Karin Fischer and Andreas Mackensen* Department of Hematology/Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg D-93042, Germany Accepted 7 April 2003
Abstract We present a rapid flow cytometric and non-radioactive functional assay developed for the determination of the cytotoxic activity of T lymphocytes, natural killer cells, and lymphokine-activated killer cells. In contrast to indirect evaluation of cytotoxicity using radioactive assays, this assay is based on the quantitative and qualitative flow cytometric analysis of cell damage on a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane, without disturbing its surface marker expression. It, thus, permits the distinction between target and effector cells. After short term in vitro incubation (1.5–3 h), AnnexinV-FITC (ann-FITC) staining allows to discriminate between apoptotic and non-apoptotic target cells. Data analysis is performed first by gating on PKH-26 positive target cells, followed by the analysis of the ann-FITC positive subpopulation. The percentage of cytotoxicity in the PKH-26 gated cell population is calculated by subtracting unspecific ann-FITC positive target cells, measured in appropriate controls without effector cells. Using in vitro generated antigen-specific cytotoxic T lymphocytes, we demonstrate that this flow cytometric assay is sensitive, correlates well with the standard 51 Cr release assay, and is easy to handle. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Cytotoxicity; Flow cytometry; Cytotoxic T lymphocytes; Apoptosis
1. Introduction Cellular cytotoxicity can be mediated by two major classes of contact dependent mechanisms. The first class is based on receptor–ligand interactions (e.g., Fas(Apo1)/Fas-ligand), which is known to be one of the central mechanisms for activation-induced cell death (AICD) in the homeostasis of lymphocytes [1]. The second class includes the granule secretion pathway that activates both caspase- independent (granzyme A) and dependent (granzyme B) death programs in the target cell. This activation appears to require the direct intracellular delivery of a family of granule-associated serine proteases. Multimeric complexes of serglycin, perforin, and granzymes are responsible for mediation and targeting of the apoptosis signal [2]. *
Corresponding author. Fax: +49-941-944-5581. E-mail address: [email protected] (A. Mackensen).
Effector cell-mediated cytolysis is usually determined by methods based on the release of different tracers from lysed target cells. In general, these markers are compounds containing radioactive isotopes such as 51 Chromium (51 Cr), 75 Selenium (75 Se), or Tritium (3 H) [3,4]. The 51 Cr release assay is the most widely used method [5] for measuring CTL responses in vitro due to its reliability and simplicity. Nevertheless, as the usage of radioactive material is potentially hazardous, it is desirable to find easy and reliable alternatives, which are safer and more cost efficient. Several non-radioactive assays based on the labelling with markers such as Europium (Eu3þ ) [6], bisbenzamid dye [7], and Calcein-AM (acetoxymethyl ester of calcein) [8] have been developed. However, the indirect measurement of the supernatant limits the accuracy of these methods. More recently, novel assays have been developed using fluorescent dyes [9–11]. The assessment of cell damage by flow cytometry aims to a more exact
1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00123-3
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characterization of the death pathway via the detection of the percentage of apoptotic and dead cells [12]. One of the characteristic processes of the apoptosis pathway is a change in the plasma membrane architecture. During early apoptosis, typical membrane compounds such as phosphatidyl serine (PS) molecules turn from the inner to the outer leaflet of the cell membrane, without any loss of membrane integrity [13,14]. AnnexinV-FITC (ann-FITC), a molecule with high affinity to PS, can be used to label cells in the early apoptotic state, while propidium iodide (PI) indicates late apoptosis or cell death. We present here the development of a three color flow cytometric assay that uses the lipophilic membrane dye PKH-26, allowing a fine characterization of the specific effector cell cytotoxicity. PKH-26 has previously been used both in proliferation analysis, cell tracking studies [15,16], and flow cytometric analysis [17–19], and has been found to be a stable tracer. A recently described three color flow cytometric assay [12] proposed the staining of effector cells with PKH-26 for the further analysis of unstained target cells. However, this may lead to an accumulation of unlabelled effector cells in the target cell gate at high effector-to-target cell (E:T) ratios. To avoid this problem, we decided to label the target cells instead. We have demonstrated that the loading of target cells with PKH-26 is stable and does not impair viability and expression of surface molecules such as adhesion and major histocompatibilty (MHC) antigens [20]. The PHK-26 assay correlates well with the conventional 51 Cr release assay and provides additional information on target and effector cells. The current manuscript details the cell staining and flow cytometric procedures of the PKH-26 assay with special reference to the best application of this technique and troubleshooting at different situations.
2. Description of method 2.1. Principle The PKH-26 assay is a simple, fast, and reliable functional assay to determine cell-mediated cytotoxicity and is therefore a reliable alternative to the standard 51 Cr release assay. Instead of loading cells with radioactive 51 Cr, target cells are labelled with PKH-26, a fluorescent dye that stably integrates into the cell membrane. This step is necessary to distinguish between target and effector cells. Coincubation of target and effector cells can be performed in 96 well plates for 3 h or less. After the coincubation of PKH-26 labelled target cells together with effector cells, staining with AnnexinV-FITC allows the determination of apoptotic and non-apoptotic target cells.
2.2. Materials and experimental set-up 2.2.1. Target and effector cells As target cells, both cells growing in suspension as well as adherent cells can be used. Trypsination does not hamper subsequent membrane loading with PKH-26. In our experiments, we used human melanoma cell lines MeR190 and MeI493 and primary leukemic AML blasts. T2 cells are HLA*0201 human lymphoid cells that are defective in antigen processing, but efficiently present in exogenously supplied peptides [21]. T2 cells were loaded with 30 lg/ml of adequate peptide and were incubated overnight at 37 °C in serum-free medium. For the permeabilization of the plasma membrane in some experiments, paraformaldehyde fixed T2 cells were washed with 0.1% saponine solution (containing 0.1% bovine serum) and then stained with 5 ll ann-FITC for 30 min on ice in the presence of saponine. As effector cells we used HLA-A2 restricted Melan-A26-35 specific CTL lines generated in vitro by repetitive stimulation with autologous peptide-loaded dendritic cells [22]. Detection of antigen-specific CTL was performed with HLA-A2-Melan-A tetramers. PE-labelled HLA-A*0201 TM that had been folded around ELAGIGILTV (Melan-A26-35L ) were synthesized by Beckman Coulter (Fullerton, CA). Staining with tetramers was carried out for 30 min at 37 °C. In some experiments, the perforin pathway in CTL was blocked by concanamycin A (CMA, Sigma, St. Louis, MO) for 20 h before the PKH26 assay as well as during the assay incubation in a concentration of 100 ng/ml. 2.2.2. Fluorescence labelling Various target cells are transferred to polystyrene tubes and washed twice with serum-free medium before staining. The cells are then resuspended in a loading buffer (an aqueous, osmolarity-regulating solution containing no Ca2þ or other physiological salts; Sigma) and incubated for 40 min with freshly prepared PKH-26 (stock 1 mM in ethanol, Sigma) at room temperature. For the loading of tumor cells, it is recommended to use 0.8 ll, for small cells (lymphocytes) 0.5 ll PKH-26 in 200 ll loading buffer. This dye solution is then added slowly, while agitating the cell suspension on a shaker, in order to avoid overstained cells. After 30–40 min of shaking at room temperature, the loading of target cells is completed. The staining reaction is stopped by the incubation with 500 ll human serum (PAN Biotech GmbH, Aidenbach, Germany) for 30 s at room temperature. This step is recommended to bind most of the residual lipophilic PKH-26 dye to serum proteins. After centrifugation, the cell pellet is transferred into a fresh 50 ml tube (Falcon, France) and washed twice with 40 ml RPMI containing 10% human serum. It is important to abide by the washing procedure in order to remove all PKH-26 in the supernatant completely. The
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dye stock is dissolved in ethanol and can be stored light protected at room temperature, when sealed tightly to prevent evaporation. 2.2.3. Coincubation of target and effector cells During the incubation for fluorescence labelling and washing of the target cells, the effector cells are counted, washed, and seeded in a volume of 100 ll medium into 96-well-V-bottom plates (Costar, Corning, NY) at different effector:target ratios. Six to eight wells per effector:target ratio are applied. Higher effector: target cell ratios require a large amount of effector cells, in this case 2 wells should be sufficient. Coincubation of effector and target cells should be performed in effector cell medium. PKH-26 labelled target cells (5 103 ) are plated in 100 ll medium/well and define the assay start. V-bottom plates allow rapid contact between target and effector cell, therefore additional centrifugation is not necessary. The recommended standard incubation time for cocultivation is 3 h. 2.2.4. Harvesting of cells and staining with ann-FITC and PI After the co-incubation, cells are harvested from the microtiter plates into separate polystyrene tubes (Falcon) for each effector:target ratio. One ml phosphate buffered saline (PBS, Gibco, Germany) is added and cells are centrifuged. Cells are then resuspended in 100 ll high calcium annexinV-binding buffer (1-fold concentrated, Pharmingen, Germany). Staining with 5 ll annFITC (BD PharMingen, USA) is performed for 10 min at room temperature in the dark. Immediately before flow cytometric analysis, 0.1 lg PI (Calbiochem, Germany) per 100 ll binding buffer is added. If more washing steps are planned, it is essential to prepare more wells per effector/target ratio, to compensate for cell loss during centrifugation. 2.2.5. FACS analysis and compensation FACS analysis was carried out on a FACScan Cytometer (Becton–Dickinson, Immunocytometry Systems, San Jose, USA). The excitation is at 488 nm, the fluorescence of the three fluorochromes is recorded at 530 nm for FITC (FL1), at 585 nm for PKH-26 (FL2), and at >675 nm for PI (FL3), respectively. For compensation, both unstained and PKH-26 stained cells should be prepared. The small amount of spontaneously apoptotic cells in the cell suspension is sufficient for compensation. As ann-FITC is known to increase unspecific fluorescence, compensation is carried out in the presence of ann-FITC: Compensation between FL1 and FL2: Vial 1: unlabelled target cells stained with ann-FITC (compensation of specific ann-FITC positive cells). Vial 2: PKH-26 labelled cells with ann-FITC (compensation of ann-negative cells with neglection of
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the small population of specific ann-FITC positive cells). Approximate values are: FL1—0.5% FL2; FL2—78% FL1. Fluorescence analysis is quantified using the Cellquest software (Becton–Dickinson). At low effector:target cell ratios, usually a smaller amount of cells is acquired than at higher effector:target ratios. Once compensated, the instrument settings can be used for further experiments, especially if no PI staining is carried out. Nevertheless the measurement of unstained cells is advisable. 2.2.6. 51 Cr release assay Conventional 51 Cr release assay is performed as previously described [23]. Briefly, the cytotoxic activity of T cell lines is measured using triplicate cultures in Vbottomed plates. Target cells are labelled with 200 lCi for 1 h. Target cells are then washed twice and seeded into the plates at E:T ratios of 25:1, 5:1, and 1:1 at 2000 target cells/well. For spontaneous release, targets are plated without T cells in MÔ medium plus 5% autologous plasma. For maximum release, target cells in 100 ll medium are plated with 100 ll of 0.15% Triton X-100 (Sigma). Triplicate wells are averaged and the percentage of specific cytotoxicity was calculated as follows: cpmsample cpmspontaneous release 100 cpmmaximum release cpmspontaneous release ¼ % specific lysis:
3. Detection of T cell mediated cytotoxicity by the PKH26 assay 3.1. Stability of PKH-26 in the cell membrane Flow cytometric analysis of cell-mediated target cell damage is based on the stable loading of target cells combined with a low percentage of spontaneous release. We tested the membrane dye PKH-26 (red fluorescence) for its ability to stain different vital primary tumor cells, such as melanoma cells and leukemic blasts. Staining with the optimal concentration of 2 lM PKH-26 results in stable loading with high fluorescence intensity in several primary tumor cells. One representative experiment is shown in Fig. 1A. In contrast, overstaining of target cells with 10 lM leads to the accumulation of intensively stained single cells, resulting in an artificial light emission in channel 1 [20]. This potentially interferes with actual double-positive target cells and may lead to false-positive results in the cytotoxic assay. The concentration of 2 lM used in the experiments allows complete compensation in the absence of overstained cells. Of note, leukemic blasts, which generally show a rather weak uptake of compounds from the external medium, reveals a high staining intensity with PKH-26
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Fig. 1. Labelling of target cells with PKH-26. Panels represent either one color histograms (A) with the x-axis showing log scale red fluorescence intensity (FL-2) and with the y-axis showing the relative cell number or two color dot plots (B). (A) Leukemic blasts were loaded for 40 min with 2 lM PKH-26 (lower plots) and co-stained with propidium iodide (PI). Unstained cells were used as control (upper plots). (B) Apoptotic PKH-26 labelled target cells (R2) exhibit a slightly decreased fluorescence intensity compared to vital PKH-26 stained target cells (R1). Separation from unstained effector cells (R3) is clearly possible.
upon loading. Labelling with PKH-26 remains stable in all vital cells tested for a minimum of 72 h without any loss of fluorescence intensity. In contrast, labelling of primary leukemic blasts with 51 Cr reveals a high spontaneous release (>25%) after 4 h incubation (data not shown). Apoptotic target cells exhibit a slightly decreased PKH-26 intensity but can be clearly discriminated as PKH-26 positive target cells (Fig. 1B). As the PKH-26 assay is a snap-shot which detects cell damage, it is recommended to choose a co-incubation time which accounts for the killing kinetics of effector cells. Strongly prolonged coincubation time will result in the death of apoptotic target cells and will, thus, generate artificial results. 3.2. Analysis of viability and surface marker expression in PKH-26 labelled target cells We tested whether staining with PKH-26 may alter the viability or the surface marker expression in labelled target cells. Apoptotic cell death of target cells measured by ann-FITC/PI staining before and 4 h after loading with PKH-26 did not show any changes in the percentage of either ann-FITC (data not shown) or PI-
positive cells (Fig. 1A). Moreover, we assessed if staining with PKH-26 may alter the expression of cell surface antigens required for functional effector/target interactions. Target cells were stained before and 4 h after PKH-26 labelling with fluorescence-conjugated mAbs against CD54, CD58, HLA-ABC, HLA-DR, and CD33, CD34 Mel-1, and Mel-2, for leukemic blasts and melanoma cells, respectively. Analysis of the surface markers did not show any significant changes after PKH-26 staining [20]. 3.3. Analysis and interpretation of results obtained from the PKH-26 assay 3.3.1. Information on the apoptotic state of target cells The PKH-26 assay provides fast and reliable information on specific target cell damage. The determination of target cell killing is mainly based on ann-FITC binding on PKH-26 gated cells. As shown in Fig. 2A, concurrent PI staining does not add to the information obtained with ann-FITC staining alone. PI single positive target cells are rare, because PIþ affected target cells are prevailing ann-FITCþ . The reason for this effect may be the diffusion of ann-FITC through impaired cell
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Fig. 2. Analysis of specific cytotoxicity using the PKH-26 assay. Target cells were selected by gating on the PKH26-positive cell population and further analyzed for different subpopulations. (A) Percentage of different apoptotic target cell subpopulations calculated according to the formula indicated in Section 3.3.4. (B) Treatment of T2 target cells with saponine leads to diffusion of ann-FITC into the cytoplasm of damaged cells and mainly results in ann-FITCþ /PIþ double positive target cells.
membranes and subsequent binding to phosphatidylserine residues on the inner leaflet of the cell membrane. In order to verify this hypothesis, we treated T2 target cells with saponine, a reagent that permeabilizes the cell membrane, and determined ann-FITC binding on these cells. As shown in Fig. 2B, cells treated with saponine are 94% double positive for PI and ann-FITC, despite the absence of classical apoptotic stimuli. Comparable results were obtained when target cells were submitted to one freeze and thaw cycle (data not shown). Therefore, we conclude that a separation of late apoptosis and necrosis in the PKH-26 assay is principally impossible. The distinction of ann-FITC single (early apoptosis) and ann-FITC double positive (late apoptosis/necrosis) is feasible, but not necessary. As shown in Fig. 2A, the correlation of early target cell damage with ann-FITC is very high, and only few cells are in a late apoptotic/necrotic state. We, therefore, suggest that no additional PI staining beside the ann-FITC staining is necessary in the PKH-26 assay. 3.3.2. Information on the frequency of specific T cells The information concerning the frequency of specific effector T cells is very limited. We carried out the PKH26 assay in parallel with Melan-A tetramer staining. In the first experiment we used effector cells containing 76%
Melan-A-specific tetramer positive T cells, in the second setting we diluted the Melan-A specific T cells with effector cells of a different specificity, but from the same donor, to a frequency of 26% Melan-A specific T cells (Fig. 3A). Both effector cell cultures result in a similar specific lysis curve (Fig. 3B). This finding reveals that the result of a PKH-26 assay does not correlate necessarily with the frequency of antigen-specific CTL. Other methods should be used in order to determine the content of antigen specific T cells, for example tetramer staining. 3.3.3. Phenotypic information on target cells The PKH-26 assay is performed on the flow cytometer and, therefore, allows any additional surface marker staining. In the described setting, fluorescence channels 1 and 2 are required, while any other channels can be used for further phenotypic analysis of the target or effector cells. However, it should be noted that an additional antibody staining prolongs incubation time. Furthermore, supplements in the antibody solution should be avoided, if they increase apoptosis. 3.3.4. Data analysis and cytotoxicity quantification In order to distinguish between target and effector cells, gating of PKH-26 positive target cells is performed.
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The ratio of corrected ann-FITC positive events ( ¼ ann-FITC positive eventssample ) ann-FITC positive eventscontrol ) to corrected total events ( ¼ total eventssample ) ann-FITC positive eventscontrol ) 100 reflects the real percentage of ann-FITC-positive cytotoxicity. Note that if the number of acquired total events differs at various E/T ratios, the amount of unspecific positive events has to be determined separately for each E/T ratio in the formula: % specific ann-positive cells ¼ ð½ann positive cellssample ann positive cellscontrol =½total eventssample ann positive cellscontrol Þ 100:
3.4. Incubation time and standardization
Fig. 3. The cytotoxic activity measured with the PKH-26 assay does not correlate with the frequency of antigen-specific T cells. (A) PKH-26 assays were carried out with two different CTL lines, which contain a frequency of 74.6 or 26.4% Melan-A specific T cells, respectively. The frequency of antigen-specific CTL was determined by HLA-A2-Melan-A tetramer staining. (B) The cytotoxic activity of both Melan-A specific T cell lines containing different frequencies of Melan-A tetramerþ CTL was measured in an 3 h PKH-26 assay. Data demonstrate no correlation between the frequency of antigenspecific CTL and the amount of cytotoxic activity measured with the Pkh-26 assay. Similar results were obtained with 51 Cr release assay (data not shown).
Ann-FITC single-positive cells represent target cells in early apoptotic state, ann-FITC/PI doublepositive cells include cells in the later apoptotic state, and PI single-positive cells represent dead/necrotic target cells. With escalating E/T ratios, increasing numbers of ann-FITC single-positive or ann-FITC/PI double-positive cells are observed. For the quantification of the specific cytotoxicity, the amount of unspecific positive cells (determined in the control sample without effector cells) in the corresponding quadrant must be subtracted. The percentage of unspecific positive cells must not be subtracted, because otherwise 100% cytotoxicity could principally not be reached. The calculation is to be carried out separately for each quadrant and is exemplified for the ann-FITC single-positive fraction.
One of the most important advantages of the PKH26 assay is its short incubation time. We analyzed the cytotoxic activity of Melan-A specific CTL, measured with PKH-26/ann-FITC double-positive target cells at three different co-incubation times, at 1.5, 2.5, and 3.5 h respectively [20]. The cytotoxic activity of effector cells at higher E/T ratios (>5:1) was found to be relatively constant over the analyzed co-incubation times from 1.5 to 3.5 h, reaching a plateau at a ratio of 20:1. In contrast, at lower E:T ratios (0.2 to 5:1), the percentage of cytotoxicity correlated well with the time of incubation. Despite the detection of specific lysis even after 1.5 h of coincubation, an incubation time of about 3 h is recommended in order to maintain the comparability of results from PKH-26 and 51 Cr release assay. Please note that the results from different PKH-26 assays can only be compared if the incubation time is standardized. Moreover, in our test system with Melan-A specific T cells, an incubation time of over 6 h definitely does not show a further increase in detected target cell damage (Fig. 4A). To ascertain if this rapid cytotoxicity is mainly mediated by the perforin/granzyme pathway, blocking experiments with concanamycin-A (CMA) were performed. As shown in Fig. 4B CMA, an efficient blocker of the perforin pathway, is able to completely block the cytotoxic activity of Melan-A specific CTL measured in the PKH-26 assay. Please note that the experimental setup and timing can influence the incubation time. For example, the usage of flat bottom plates instead of the recommended V-bottom plates may shorten the effector/target cell contact time. Moreover, an extensive delay between harvesting the cells and flow cytometric analysis should be avoided, as the killing in the cell effector/target cell suspension can proceed slightly at room temperature. Working on ice may minimize this effect. In summary, standardization of the working procedure will achieve optimal PKH-26 assay results.
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Melan-A specific CTL. Incubation of target cells with anti-HMW-MAA mAb, recognizing an irrelevant epitope on melanoma cells, had no effect [20].
4. Practical considerations and trouble shooting
Fig. 4. PKH-26 assay kinetics and inhibition of cytotoxicity by blocking the perforin pathway. (A) PKH-26 labelled Melan-A expressing target cells were incubated with Melan-A antigen-specific CTL for 3, 6, and 9 h at various effector:target ratios prior to the PKH26 assay. Data are presented as mean cytotoxicity (n ¼ 3) of Melan-A specific CTL, as determined by % ann-FITC positive cells. (B) Cytotoxicity of in vitro generated Melan-A specific T cells can be inhibited by the pretreatment of CMA, a blocker of the perforin pathway.
3.5. Comparison between the PKH-26 and assays
51
Cr release
The PKH-26 flow cytometric assay was directly compared to the standard 51 Cr release assay [20]. MelanA specific CTL generated in vitro were tested simultaneously by both methods against HLA-A2þ Melan-A expressing target cells and HLA-A2þ Melan-A control cells, using E/T ratios of 25:1, 5:1, and 1:1. The two different assays showed comparable results at all E/T ratios. In addition, cytotoxicity against the control cell line was negative in both assays. Data demonstrated a good correlation between results obtained from the PKH-26 assay and those obtained by the standard 51 Cr release assay (r2 ¼ 0:9777). Interestingly, results obtained with the PKH-26 assay reveal a slightly higher percentage of cytotoxicity as compared to the 51 Cr release assay. Furthermore, it was tested if the addition of blocking mAbs can inhibit the percentage of specific cytotoxicity measured with the PKH-26 assay. Blocking mAbs, either against effector cells (CD3, CD8) or target cells (HLA-ABC), are able to inhibit the cytotoxicity of
Analysis of the cytolytic activity of CTL is central in monitoring antigen-specific immune responses. Traditionally, the cytotoxicity of T cells has been measured using 51 Cr release assays [5]. Here, we have described an easy flow cytometry based killing assay, which permits exact evaluation of target cell death (early apoptosis, membrane damage) occurring during antigen-specific CTL responses. This method exhibits several advantages over standard radioactive functional assays like the 51 Cr release assay. First, the potentially harmful handling of radioactive isotopes can be avoided. Expensive laboratory equipment and waste disposal is not necessary for the PKH26 assay. Second, an outstanding advantage is the ability to distinguish clearly between target and effector cells. It should be mentioned that, if a subpopulation of target cells is overstained it is recommended to load the PKH26 dye more slowly onto the target cells by adding the dye solution in drops. If the whole cell suspension is overstained, a lower concentration of PKH-26 dye is recommended. As the basis of the PKH-26 assay is not the release of a marker, ‘‘minimal release’’ or ‘‘maximal release’’—as ascertained in the 51 Cr release assay—needs not to be determined. The maximum effect is determined by the count of vital cells that can potentially be killed in the assay. The background apoptosis without effector cells corresponds to the ‘‘minimal release’’ in the 51 Cr release assay. Notably, primary leukemic blasts, showing inefficient labelling and high spontaneous release after incubation with 51 Cr [24], demonstrated high and stable staining of the PKH-26 dye without increased spontaneous apoptosis (see Fig. 1A). In contrast to the 51 Cr release assay, which is based on the relatively unelucidated process of 51 Cr release in affected target cells, AnnexinV binding—the basis for the PKH-26 assay—is a well described process. Another striking advantage of the PKH-26 assay is the shortened incubation time needed to produce reliable results. The sensitivity of standard 51 Cr release assays requires an incubation time of 4 h. Since optimal killing is generally reached soon after the effector and target cells are brought into contact [25], a reliable measurement of killing should be detectable at an earlier time point. Optimal killing in the PKH-26 assay was observed even within an incubation period of 1.5–2.5 h [20]. The fourth advantage is the ability to directly evaluate the effector/target cell suspension in the flow cytometer, instead of indirectly concluding the target cell
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damage from the supernatant of the 51 Cr release assay. It is very useful to get information about the physiological condition, the viability, and the phenotype of the effector cells. Moreover, any further phenotypic surface marker staining on target cells (e.g., MHC expression) can be carried out simultaneously. As early cell damage mediated by effector cells correlates mainly with annFITC staining, an additional PI staining is not necessary for optimal results. In addition, we were able to demonstrate that a more detailed separation of late apoptotic (ann-FITCþ /PI ) and necrotic (PIþ ) cells is virtually impossible: it is strongly suggested that membrane impairment in the affected target cells is responsible for the diffusion of PI as well as ann-FITC into the cytoplasm. The most important advantage of the PKH-26 assay is its flexibility. The flow cytometric assay principle can be adapted to various conditions. It is, therefore, strictly recommended to standardize the PKH-26 assay in order to allow comparison between several PKH-26 assay results. Our results demonstrate that the staining of both effector and target cells [10] is not necessary for the accurate discrimination between the two cell types. Aubry et al. [12] have shown recently that the labelling of effector instead of target cells may represent an alternative method for the assessment of cellular cytotoxicity. However, staining of effector cells may be critical, since small percentages of cells may remain unstained. Especially if high E/T ratios (50:1, 100:1) are applied, unstained effector cells being counted as target cells may lead to false-positive or false-negative results. Data demonstrate strong lysis of Melan-A expressing target cells by Melan-A specific CTL lines, while maintaining excellent specificity and HLA restriction. A definitive comparison of the PKH-26 assay to the standard 51 Cr release assay was performed and showed a very good correlation between the two methods. However, results obtained with the flow cytometric assay showed a higher percentage of cytotoxicity as compared to the 51 Cr release assay. In conclusion, the PKH-26 assay is a quantitative method for the detection of cell-mediated cytotoxicity and does avoid the use of radioactive labels. The reliability and reproducibility of this assay makes it a valuable alternative to the standard 51 Cr release assay. allowing a greater insight into the mechanisms of cellmediated cytolysis by different effector populations.
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