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LEGENDplex™: Bead-assisted multiplex cytokine profiling by flow cytometry
CHAPTER NINE
LEGENDplex™: Bead-assisted multiplex cytokine profiling by flow cytometry Jason S. Lehmann*, Priyanka Rughwani, Melissa Kolenovic, Shaoquan Ji, Binggang Sun BioLegend, San Diego, CA, United States *Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 1.1 LEGENDplex™ assay principles 2. Required equipment and materials 2.1 Equipment 2.2 Materials 2.3 Precautions 3. Sample collection 3.1 Serum samples 3.2 Plasma samples 3.3 Cell culture supernatant 3.4 Alternative sample types 3.5 Sample fixation 3.6 Sample dilutions 4. Reagent preparation 4.1 Preparation of antibody-immobilized capture beads 4.2 Preparation of wash buffer 4.3 Preparation of matrix 4.4 Recombinant standard preparation 5. Assay protocol 5.1 Generalized recommendations 5.2 Assay procedure 6. Flow cytometer setup 6.1 Collection of data in appropriate cytometer channels 6.2 Optimal FSC and SSC voltage/sensor gain settings 6.3 Optimal APC voltage/sensor gain settings 6.4 Optimal PE PMT voltage/sensor gain 7. Data acquisition
Methods in Enzymology, Volume 629 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.06.001
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8. Data analysis 8.1 Bead separation 8.2 Bead gating 8.3 Replicate precision 8.4 Bead counts 9. Tips for novice users 9.1 Polypropylene plastics 9.2 Incubation shaker speed 9.3 Flow cytometer settings 10. Example LEGENDplex™ data set 10.1 Materials and methods 10.2 Results 11. Summary Acknowledgments Disclosures References
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Abstract Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunologybased therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.
1. Introduction Clinical advances in cancer immunotherapy have always followed advances in our understanding of tumor immunology. Indeed, over the past two decades in cancer immunotherapy history there have been remarkable advances in CAR-T cell (Feins et al., 2019) and immune checkpoint monotherapies (Chen & Mellman, 2013; Constantinidou et al., 2019; Sharma & Allison, 2015). The success of these therapies relies upon modulating immune effector mechanisms to generate tumor specific cytotoxic T lymphocytes (Durgeau et al., 2018); however, a deeper understanding of human immune responses is needed to realize the full potential of these promising therapies. Immune checkpoint therapy can lead to durable and long-lasting clinical responses, but only in a fraction of patients (Sharma & Allison, 2015). CAR-T therapies have become more potent
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and effective, but the development of cytokine release syndrome in response to treatment risks life threatening toxicity (Acharya et al., 2019; Lee, Gardner, Porter, et al., 2014). Overcoming these challenges will require a greater understanding of immune regulation within the tumor microenvironment. However, this complicated system provides both stimulatory and inhibitory signals to infiltrating lymphocytes. Inflammation, a hallmark of cancer, promotes tumor proliferation, angiogenesis, and metastasis, but also can subvert the immune response (Mantovani et al., 2008). In fact, tumor induced immunosuppression remains one of the major hurdles facing cancer immunotherapy (Fu & Jiang, 2018). Once in the tumor microenvironment, cytotoxic T cells must overcome the suppressive effects of not only the tumor cells, but also regulatory T cells, myeloid-derived suppressor cells, and inhibitory cytokines that together act to mitigate antitumor responses. The inflammatory mediators that drive these immune processes are key constituents of the tumor microenvironment. The role of cytokines and chemokines in cancer is well established (Fuertes et al., 2013; Poeta et al., 2019; Raeber et al., 2018; Roshani et al., 2014; Valilou et al., 2018) and affects many aspects of tumor immunology from immune cell recruitment, modulation of their effects, angiogenesis, and metastasis. In fact, identifying predictive biomarkers that define clinically beneficial immune responses is an area of active research (Sharma & Allison, 2015). The ability to accurately quantify these biomarkers is therefore of critical importance to these efforts. Immunoassays have been able to reliably quantify single analytes for decades; however, as researchers now seek to assess the effectiveness of evolving immune responses, the need to quantify multiple biomarkers simultaneously has become a necessity. Multiplex immunoassays provide researchers the ability to quantitatively test for multiple targets simultaneously from one sample aliquot. In particular, LEGENDplex™ bead-based immunoassays, offered by BioLegend, provide a multiplexing platform to researchers designed to be performed using commonly available flow cytometers. Multiple LEGENDplex™ immunoassays have been cited in the methods sections of several recent publications related to tumor immunology and immunotherapy. These assays have been used to quantify relevant biomarker concentrations from experimental studies involving cancers of the colon (Olguı´n et al., 2018), esophagus (Huang et al., 2017), stomach (Zhao et al., 2019), ovaries (Mlynska et al., 2018), lymphoma (Tiper & Webb, 2016), and malignant melanoma (Yamaguchi et al., 2018). In addition, LEGENDplex™ assays have also been cited in studies investigating
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CAR-T (Han et al., 2018; Sachdeva et al., 2019) and cytokine-induced killer cell immunotherapies (Bremm et al., 2016), as well as dendritic cell and cancer cell-based vaccines (Fromm et al., 2016; L€ ovgren et al., 2017). The particular LEGENDplex™ assays which were cited by these publications are summarized in Table 1.
1.1 LEGENDplex™ assay principles LEGENDplex ™ bead-based immunoassays have been developed based on the same principles as the sandwich immunoassay, and allow users to simultaneously quantify up to 13 different biological molecules from 1 sample aliquot. Capture beads, which can be differentiated based on size and internal fluorescence intensities, are covalently conjugated to analyte-specific antibodies using carbodiimide crosslinking reactions. The LEGENDplex™ assay system uses two sets of beads: A beads and B beads. The A and B bead sets have forward and side-scatter profiles that when run on a flow cytometer, allow for each to be uniquely identified. Bead sets are then further resolved based on the internal APC fluorescence within the beads. There are a total possible six distinct populations of APC intensities within the A beads, and seven within the B beads (Fig. 1). Table 1 LEGENDplex™ references. LEGENDplex™ panel References
Human T Helper Cytokine Panel
Han et al. (2018) and Yamaguchi et al. (2018)
Human Inflammation Panel 1 Sachdeva et al. (2019), Tiper and Webb (2016), and Bremm et al. (2016) Human Inflammation Panel 2 Sachdeva et al. (2019) Human Proinflammatory Chemokine Panel
Mlynska et al. (2018) and Huang et al. (2017)
Human Anti-Virus Response L€ ovgren et al. (2017) Panel Human Type 1/2/3 Interferon Panel
Zhao et al. (2019)
Mouse Th1/Th2 Panel
Fromm et al. (2016)
Mouse Th17 Panel
Olguı´n et al. (2018)
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Fig. 1 LEGENDplex™ beads. (A) Bead size populations: FSC/SSC characteristics of the two bead sizes used in LEGENDplex assays. Clearly visible are the smaller A beads and the larger B beads. (B) A bead ID classification by APC: The six A bead IDs that are used in the assays are displayed on an APC uninvariate histogram. (C) B bead ID classification by APC: The seven bead IDs that are used in the assays are displayed on an APC uninvariate histogram. Bead IDs are distinguished from on another based on internal APC fluorescence signal differences of the beads. Note: The histograms displayed in (B) and (C) were smoothed for esthetic purposes using FlowJo, and are solely presented to familiarize readers with the spectral properties of the fluorescent microsphere reagents employed in the assay.
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Panels of defined capture bead sets are then incubated with recombinant protein standards or the biological samples to be assayed. A second incubation with biotinylated detection antibodies leads to the formation of capture bead-analyte-detection antibody sandwiches. A Streptavidin-PE reagent is then added to bind biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE signal of each capture bead is measured using flow cytometry, and then input into the LEGENDplex™ Data Analysis Software to quantify the analyte concentrations in each sample. This assay platform offers several advantages over traditional ELISA formats including enhanced sensitivity, broader dynamic ranges, lower sample volume requirements, free of charge data analysis software, and multiplexed analysis. In addition, these assays can be performed in the laboratory using any flow cytometer capable of reading both PE and APC fluorescence signals, which obviates the need for capital expenditures on dedicated analysis equipment. LEGENDplex™ bead-based immunoassays have been developed for human, non-human primate, mouse, and rat specificities. For each species, there are pre-defined panels designed to quantify biomarkers related to particular cell types, biological processes, or disease states. Within a given panel, assays can be ordered which quantify only certain targets of interest, should the full pre-defined panel be unsuitable. Additionally, custom panel manufacturing of unique target combinations is available. A full listing of assays as well as ordering information can be found online at https:// www.biolegend.com/legendplex.
2. Required equipment and materials 2.1 Equipment 1. A flow cytometer equipped with a 488 nm blue laser or 532 nm green laser, and a 633–635 nm red laser capable of distinguishing 575 and 660 nm 2. Multichannel pipettes capable of dispensing volumes of 5–200 μL 3. Vortex mixer 4. Bath sonicator (Branson Ultrasonic Cleaner model #B200, or equivalent) 5. Plate shaker (Lab-Line Instruments model #4625, or equivalent)
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6. Benchtop Microcentrifuge (Eppendorf centrifuge 5415C, or equivalent) If the assay is performed in a filter plate: 1. Vacuum filtration unit (Millipore MultiScreen® HTS Vacuum Manifold or equivalent) 2. Vacuum source (mini-vacuum pump or line vacuum) If the assay is performed in a V-bottom plate: 1. Centrifuge with a swinging bucket adaptor for microtiter plates
2.2 Materials 1. 2. 3. 4. 5. 6. 7.
LEGENDplex™ bead-based immunoassay (BioLegend) Pipet tips for multichannel pipette Reagent reservoirs for multichannel pipette Polypropylene microcentrifuge tubes (1.5 mL) Aluminum foil Absorbent pads or paper towels 1.1 mL polypropylene micro FACS tubes, in 96-tube rack (National Scientific Supply TN0946-01R, or equivalent)
2.3 Precautions 1. Sodium azide is used as a preservative in some LEGENDplex™ components. During disposal, flush with a large volume of water to prevent the accumulation of explosive metal azides in laboratory plumbing. 2. The matrix reagent included in LEGENDplex™ kits contains components derived from human or animal blood. The human-based material has been screened for certain infectious diseases using FDA-approved testing methods and is negative for HIV, HBV, and HCV, but should be handled as a potentially hazardous material. 3. The streptavidin-PE and capture bead reagents are UV sensitive. Minimize exposure to light when performing the assay. 4. All tubes and plates used in the assay should be made from low binding polypropylene. Polystyrene ELISA or cell culture plates treated for optimal cell attachment should not be used due to their capacity to absorb recombinant protein standards and affect assay results.
3. Sample collection LEGENDplex™ assays are validated for use with one or more biological samples (panel-specific) including serum, plasma, cerebrospinal fluid,
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urine, saliva, and cell culture supernatant. The assays can potentially be adapted for use with other samples types, but have special considerations that are discussed below in the alternative sample types section.
3.1 Serum samples After collection, allow blood to clot for at least 30 min and then centrifuge at 1000 g for 20 min. Remove serum and proceed to assay immediately or aliquot samples and store at 20 °C.
3.2 Plasma samples Plasma collection using EDTA as an anti-coagulant is recommended. Within 30 min of blood collection, centrifuge sample at 1000 g for 10 min. Remove plasma and proceed to assay immediately or aliquot samples and store at 20 °C. When using frozen serum or plasma samples it is recommended that samples be completely thawed, mixed thoroughly, and centrifuged to remove particulates prior to use. Avoid multiple freeze/thaw cycles.
3.3 Cell culture supernatant After harvesting the supernatant, centrifuge the sample to remove any remaining cellular debris. Proceed to assay immediately or aliquot samples and store at 20 °C.
3.4 Alternative sample types The use of alternative sample inputs has been reported (Mccoll et al., 2016; Nerurkar et al., 2017); however, it is the responsibility of the researcher to establish the compatibility of any sample type for use with the assay. Avoid the use of denaturing chemicals (urea, cholate, etc.) and ionic detergents when preparing buffers for alternative sample types. Minimal amounts (<1%) of non-ionic detergents can be used, provided they are in a neutral pH buffer containing physiological salt concentrations. The inclusion of protease inhibitors is strongly recommended.
3.5 Sample fixation The use of fixatives with LEGENDplex™ reagents is generally discouraged, as prolonged incubations will quench fluorescence signals. If samples must be fixed due to biosafety considerations, it should be performed only after completing the assay. Freshly prepared 1–4% paraformaldehyde in
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1 PBS can be used to fix samples by adding 150 μL per well for 10–15 min. The fixative should be washed out immediately after the incubation by following the wash steps described below.
3.6 Sample dilutions 3.6.1 Cell culture supernatants The concentrations of target analytes in cell culture supernatants can vary greatly across cell lines and culture conditions. A preliminary experiment is recommended to establish an appropriate dilution factor for a particular sample. Any dilutions of samples should be done with either sterile culture media or LEGENDplex™ Assay Buffer to ensure accurate measurement. 3.6.2 Serum and plasma samples Dilution requirements for serum and plasma samples are dependent on the target being assayed, with targets of high endogenous levels requiring greater dilution than those of low abundance. Please refer to the product manual for assay specific details regarding dilution of these sample types.
4. Reagent preparation 4.1 Preparation of antibody-immobilized capture beads If pre-mixed beads are included with the kit: 1. Sonicate the bottle of pre-mixed beads for 60 s in a sonicator bath, and then vortex for 30 s immediately prior to use. Note: Vortex time can be increased to 60 s if access to a sonicator bath is unavailable in the laboratory. If individual bead vials (13) are included with the kit: The beads will need to be diluted from 13 to 1 concentration while being combined in LEGENDplex™ Assay Buffer prior to their use. 1. Sonicate the beads vials for 1 min in a sonicator bath, and then vortex for 30 s to completely resuspend the beads. 2. Calculate the volume of diluted beads needed for the assay. Each assay well will require 25 μL of 1 beads. Multiply the number of wells by 25 μL then add an extra volume (approximately +20% more) to compensate for pipetting loss. 3. Divide this quantity by 13 to determine the volume from each individual bead vial required. Add these volumes into an appropriately sized polypropylene tube, then q.s. with LEGENDplex™ Assay Buffer to reach the necessary total volume calculated in the previous step.
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4.2 Preparation of wash buffer 1. Allow the bottle of 20 Wash Buffer to equilibrate to room temperature and swirl to dissolve any salt crystals that may have formed during storage. 2. Dilute 25 mL of 20 Wash Buffer with 475 mL of deionized water. Unused buffer may be stored for up to 1 month at 2–8 °C.
4.3 Preparation of matrix The matrix reagent is included with LEGENDplex™ immunoassay kits to control for any potential “matrix effects” in serum and plasma sample types. Matrix effects are consistent, but poorly defined non-specific binding interactions caused by endogenous factors in blood derived sample types (Selby, 1999; Wood, 1991). 1. Add 5 mL of LEGENDplex™ Assay Buffer to the bottle containing the lyophilized matrix component provided in the assay kit. 2. Incubate for 15 min and then vortex well to mix. Unused matrix may be stored for up to 1 month at 70 °C.
4.4 Recombinant standard preparation 1. Reconstitute the lyophilized standard cocktail with 250 μL of LEGENDplex™ Assay Buffer. 2. Mix and then allow the vial to incubate at room temperature (20–25 °C) for 15 min. 3. During incubation, label seven polypropylene microcentrifuge tubes C7, C6, C5, C4, C3, C2, C1, respectively. 4. Add 75 μL of LEGENDplex™ Assay Buffer to all tubes except C7 (i.e., C1–C6). 5. After the 15 min has elapsed, transfer the entire volume of reconstituted standard from the vial into the microcentrifuge tube labeled C7. This will be used as the top standard. 6. Perform a 1:4 dilution of the top standard by transferring 25 μL from the C7 tube into the C6 tube and then mix well. 7. Continue this 1:4 dilution series to obtain standards C5, C4, C3, C2, and C1. LEGENDplex™ Assay Buffer will be used as the blank standard (C0).
5. Assay protocol 5.1 Generalized recommendations 1. Read the manual included with the assay prior to performing any experiment.
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2. Standards and samples should be assayed in duplicate. 3. Complete the plate map, found in the back of the assay manual, by assigning each standard and experimental sample to a corresponding well on the document in a manner convenient for data acquisition and analysis. 4. LEGENDplex™ assays can be performed using either filter or V-bottom plates (see Section 2.1 for specific requirements of each). The choice of plate does not affect assay performance, but there are slight variations in protocol between the two formats. 5. Allow all reagents to equilibrate to room temperature (20–25 °C) before use. 6. The assay plate should be placed in the dark or wrapped in aluminum foil during all incubation steps. For assays performed using filter plates: 1. Keep the filter plate upright during the entire assay procedure, including wash steps, to avoid losing beads. 2. Place the filter plate on an inverted plate cover at all times during assay setup and incubation steps. If the bottom of the plate is allowed to contact flat surfaces inadvertently leaking can occur.
5.2 Assay procedure For assays performed using filter plates: 1. Pre-wet the filter plate by adding 100 μL of 1 LEGENDplex™ Assay Buffer to each well and incubate for 1 min. 2. Remove buffer volume by placing the plate on the vacuum manifold and apply vacuum (do not exceed 1000 Hg of pressure) until wells are drained. 3. Press the filter plate onto a stack of clean paper towels to blot excess liquid from the bottom. Assaying cell culture supernatant samples: 1. Add 25 μL of Assay Buffer to all wells. 2. Add 25 μL of C7–C1 standards to appropriate standard wells 3. Add 25 μL of Assay Buffer to C0 wells. 4. Add 25 μL of samples to appropriate wells. Assaying serum and plasma samples: 1. Add 25 μL of Assay Buffer to all sample wells 2. Add 25 μL of Matrix Component to all standard wells 3. Add 25 μL of C7–C1 standards to appropriate standard wells 4. Add 25 μL of Assay Buffer to C0 wells 5. Add 25 μL of samples to appropriate wells.
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For all assay plate wells: 1. Vortex beads for 30 s then add 25 μL of beads to each well. Shake the bottle/tube intermittently to prevent beads from settling. The total volume per well should now be 75 μL. Note: This step will use either the pre-mixed beads bottle or the 1 beads solution depending on the assay format used. 2. Seal the top of the plate with a plate sealer then wrap entire plate (including inverted plate cover if using filter plate) with aluminum foil. 3. Place the wrapped plate on a plate shaker and shake at 500 rpm (filter plate) or 800 rpm (V-bottom plate) for 2 h at room temperature (20–25 °C). 4. Wash the plate according to the following: Filter Plates: Place the plate on the vacuum manifold and apply vacuum. Add 200 μL of 1 Wash Buffer to each well. Remove Wash Buffer with vacuum filtration, and blot excess liquid from plate bottom with paper towels. Repeat this process once more. V-bottom Plates: Centrifuge the plate at 1050 rpm for 5 min. Dump the supernatant into a sink by inverting the plate with one continuous forceful motion. Add 200 μL of 1 Wash Buffer to each well and incubate for 1 min. Repeat centrifugation and dump once more. 5. Add 25 μL of Detection Antibodies to each well. 6. Seal the top of the plate with a new plate sealer then wrap entire plate (including inverted plate cover if using filter plate) with aluminum foil. 7. Place the wrapped plate on a plate shaker and shake at 500 rpm (filter plate) or 800 rpm (V-bottom plate) for 1 h at room temperature (20–25 °C). 8. Add 25 μL SA-PE directly to each well without washing. 9. Seal the top of the plate with a new plate sealer then wrap entire plate (including inverted plate cover if using filter plate) with aluminum foil. 10. Place the wrapped plate on a plate shaker and shake at 500 rpm (filter plate) or 800 rpm (V-bottom plate) for 30 min at room temperature (20–25 °C). 11. Repeat wash step 4 above. 12. Add 150 μL of Wash Buffer to each well. 13. Resuspend the beads into solution by placing the plate on a plate shaker for 1 min. 14. Transfer samples from the assay plate into micro-FACS tubes using a multichannel pipet in order to read samples on a flow cytometer.
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Note: The samples can be kept overnight at 4 °C while being protected from light exposure and read the next day. There may be a decrease in fluorescence signal strength, but overall assay results should not be affected. Storing the samples for extended periods of time is not recommended, as it could lead to further reductions in signal.
6. Flow cytometer setup LEGENDplex™ assays are compatible for use with all flow cytometers capable of detecting PE and APC fluorescence signals; however, to generate the best results it is critical that each instrument be properly set up prior to data acquisition. Users should refer to instrument-specific setup instructions (instrument setup tab, www.biolegend.com/legendplex) when conducting the assay for the first time in order to generate high quality data. Note: Preliminary experiments to establish acceptable cytometer settings are encouraged prior to assaying any experimental samples using the LEGENDplex™ platform. The instrument setup process will obviously vary for users depending on the configuration of their flow cytometer; however, a proper instrument setup will always contain the following critical elements regardless of the instrument used in a particular assay:
6.1 Collection of data in appropriate cytometer channels Ensure that the acquisition software for the cytometer is set to collect data in the following channels: FSC, SSC, APC, and PE. Other fluorescence channels are not required and can be deselected on the instrument.
6.2 Optimal FSC and SSC voltage/sensor gain settings Proper voltage/gain settings for the FCS and SSC parameters will clearly separate the A beads population from the B beads population when viewed on an FSC versus SSC dot plot in linear mode.
6.3 Optimal APC voltage/sensor gain settings Optimal APC settings will clearly delineate all respective bead IDs within a bead region (A or B beads) based on internal APC fluorescence.
6.4 Optimal PE PMT voltage/sensor gain An optimal PE setting will give the widest possible dynamic range for signals in the channel. If set too low, the low end of the standard curve may plateau
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due to a low signal-to-noise ratio. If set too high, the high end of the standard curve may plateau due to signal saturation. Under ideal cytometer settings, standard curve samples will fall onto a sigmoid distribution with the PE-MFI of the C7 standard near the upper limit of detection (i.e., signal saturation), a C0 PE-MFI 50% of the C1 PE-MFI, and well separated standard curve points (C1 through C6) in between.
7. Data acquisition The exact procedures associated with the acquisition of data on a given instrument are dependent on the cytometer’s configuration specifications as well as the interface software used. The instructions below are therefore intended to highlight the essential steps to be followed regardless of the flow cytometer model employed in the assay. Before reading samples, ensure that the cytometer is properly setup (see Section 6). 1. Vortex each sample for 5 s prior to acquisition. 2. If the flow cytometer is equipped for high-throughput screening, then the plate can be read directly using the auto-sampler. Ensure that the auto-sampler is programed to agitate the beads in the well immediately prior to data acquisition. 3. Set the flow rate of the cytometer to low. 4. Using the acquisition software for the cytometer, draw a gate that encompasses both the A and B bead populations (A/B gate). 5. Set the stop condition for acquisition to be equal to 300 events per bead ID included in the particular LEGENDplex™ assay. Do not set acquisition parameters to stop based on total events as these can include debris and other junk signals. Ensure that data acquisition stops based on A/B gated events. 6. Read samples in the same order as the plate map, starting with well A1 and reading through the plate column by column. 7. Export the flow cytometry files from the acquisition software and save them to an appropriate location.
8. Data analysis Exported data files generated should be analyzed using the LEGENDplex™ Data Analysis Software (BioLegend). A license for the software is provided free of charge with each LEGENDplex™ assay. Instructions for the download and use of the software can be found at www. biolegend.com/legendplex, under the software tab. The software,
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compatible with FCS 2.0, FCS 3.0, and FCS 3.1 standard formats as well as . LMD files, is designed to apply a gating strategy that allows for the proper identification of all assay bead IDs, and then fit a 5-parameter logistic curve (Gottschalk & Dunn, 2005) to the PE-reporter signal in the standard curve files. The resulting curve equation is then used to calculate the concentration of assay targets in the experimental samples. The use of other software programs to analyze LEGENDplex™ flow cytometry files is theoretically possible; however, the ultimate suitability of a program would need to be validated by the researcher. Beyond the capacity to analyze the appropriate fluorescence signals from assay files, the most critical feature of any program is its curve fitting algorithm; in particular, the ability to derive and fit 4- or 5-parameter logistic curves to the standard curve files. The use of graphing features found in commonly used spreadsheet programs is particularly discouraged, as these produce inaccurate quantification results. Regardless of the software program used to analyze the LEGENDplex™ data, the researcher should review the following parameters in their results in order to confirm that the assay was successfully performed:
8.1 Bead separation Confirm that there is clear separation of all bead populations in the dot-plots generated by the software. The A beads should be clearly distinguished from B beads in the forward scatter versus side scatter plots, and the APC fluorescence plots should also evidence clearly delineated bead ID populations (Fig. 1).
8.2 Bead gating The gating applied to all assay files should also be reviewed for accuracy, and corrected if necessary. Well defined bead regions and the proper gating of these regions during data analysis are essential to generating accurate results. Although this post hoc review can do nothing to salvage results from the assay should the bead IDs overlap, it does allow the researcher to avoid drawing erroneous conclusions from the data and alert them of the need to adjust their cytometer settings for future experiments.
8.3 Replicate precision The coefficient of variation between replicate samples should also be reviewed, as high values can be indicative of technical errors. All results from replicates that have %CV values >30% should be interpreted with caution.
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Pipetting related errors are by far the most common contributor to these effects. As such, proper calibration and attention to detail are essential requirements for generating reliable quantification results.
8.4 Bead counts As described in the Section 7, approximately 300 events per bead ID should have been collected on the flow cytometer used in the assay. This should be verified by the researcher using the data analysis program. This number of events is suggested as it will provide a large enough sampling of the median PE fluorescence intensity of a given bead ID to establish a 99% confidence interval around this value. Since the PE signals are used to derive calculated concentrations from samples in the assay it is critical to estimate these values as accurately as possible. Certainly, some degree of variation across samples in cytometer-counted events per bead ID should be expected within an assay; however, gross differences (i.e., less than 100 events) should be investigated further. In a given sample, low bead counts for one bead ID but not others can indicate the presence of an inappropriate gate that should be reviewed. Low bead counts across all samples might indicate that the stop parameter on the flow cytometer was inadvertently set on total events (which include debris) versus gated events around just the assay beads.
9. Tips for novice users LEGENDplex™ bead-based immunoassays can be used to reliably quantify soluble mediator levels in biological samples; however, these results are highly dependent on adherence to the assay protocol. There are a few technical considerations regarding the LEGENDplex™ assay platform that new users should give particular attention to when running the assay for the first time:
9.1 Polypropylene plastics The importance of using only polypropylene plastics when performing the assay cannot be overstated. Polystyrene ELISA or tissue culture treated microplates specifically promote the adherence of the assay’s recombinant protein standards to the wells of the plate. The same is true for polystyrene microcentrifuge tubes. The binding of recombinant standards to these
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materials is detrimental to the performance of any quantitative immunoassay, as this effectively removes the very molecules used to generate the standard curve out of solution.
9.2 Incubation shaker speed Proper agitation of the capture beads is required for them to efficiently bind target analytes in solution. Without proper shaking, the binding characteristics of the beads can be severely hindered. In addition, slow shaking speeds can promote the formation of bead aggregates which prevents uniform detection antibody staining. Researchers are thus encouraged to verify that the plate shaker they intend to use during the assay is capable of agitating the plate at the speed stated in the assay manual.
9.3 Flow cytometer settings As discussed above in the Section 6, APC and PE PMT voltages must be adjusted to ensure proper bead separation and broad dynamic ranges for the standard curves. Researchers are again encouraged to perform pilot experiments to optimize instruments settings for the flow cytometer used to interrogate the assay beads.
10. Example LEGENDplex™ data set Cytotoxic T lymphocytes (CTL) are essential for cancer immunosurveillance (Farhood et al., 2018). Despite their critical function in targeting various cancers, our basic understanding of the tenets of CTL immunity is still being refined (Reading et al., 2018). T-cell inhibitory receptors have been one particularly active focus for tumor immunology research as well as clinical immunotherapy (Constantinidou et al., 2019; Durgeau et al., 2018; Farhood et al., 2018; Sharma & Allison, 2015). To demonstrate the utility of LEGENDplex™ immunoassays in quantifying the soluble forms of these key molecules, human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors and cultured with media or media containing anti-CD3 and anti-CD28 antibodies plus recombinant IL-2. After 72 h, the expression levels of immune checkpoint-related molecules on the surface of the PBMCs were analyzed using flow cytometry. Concurrently, the concentrations of the same checkpoint molecules were quantified in the supernatants of the same PBMC cultures using the LEGENDplex™ Human Immune Checkpoint Panel 1.
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10.1 Materials and methods 10.1.1 PBMC isolation and in-vitro culture Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n ¼ 3) using Lymphopure™ (BioLegend: catalog # 426201) density gradient centrifugation, according to manufacturer instructions. Isolated PBMCs were stained with a 0.4% trypan blue solution (Thermo Fisher Scientific) and then enumerated and assessed for viability using a hemocytometer. PBMCs were then cultured in 6-well microtiter plates (6 106 cells/well in 3 mL media) at 37 °C with 5% CO2 for 72 h under the following conditions: Unstimulated: Per well, 6 106 PBMCs in 3 mL RPMI 1640 media (Corning) supplemented with 10% heat-inactivated FBS (GE Life Sciences) and penicillin/streptomycin solution (GE Life Sciences). Stimulated: Per well, 6 106 PBMCs in 3 mL culture media supplemented with anti-human CD3 antibody at 10 μg/mL (BioLegend, clone: HIT3a, Cat. No. 300302), anti-human CD28 antibody at 10 μg/mL (BioLegend, clone: CD28.2, catalog # 302902), IL-2 at 20 ng/mL (BioLegend, catalog # 589104). 10.1.2 Flow cytometry PBMCs were isolated from culture wells by centrifugation (1500 rpm, 5 min), then stained with the following reagents: 7-AAD Viability Staining Solution (BioLegend, catalog # 420403) according to manufacturer instructions and 5 μL of the antibodies listed below in Table 2. Samples were then analyzed using a BD FACSCanto II™ flow cytometer. Resulting data were then processed using FlowJo Software (Becton, Dickinson & Company) and GraphPad Prism (GraphPad Software). 10.1.3 LEGENDplex™ profiling Unstimulated and stimulated PBMC culture supernatants were quantified for soluble immune checkpoint molecules using the LEGENDplex™ Human Immune Checkpoint Panel 1 (BioLegend, catalog # 740867). The supernatants were tested following the assay protocol described in this manuscript. Data were analyzed using the LEGENDplex™ Data Analysis Software (BioLegend, PC version 8) and GraphPad Prism (GraphPad Software).
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Table 2 Flow cytometry reagents. Specificity Format
Clone
Manufacturer
Cat. #
Anti-human CD25
PE
BC96
BioLegend
302605
Anti-human CD86
APC
BU63
BioLegend
374207
Anti-human CTLA-4
APC
L3D10
BioLegend
349907
Anti-human TIM-3
APC
F38-2E2
BioLegend
345011
Anti-human PD-1
APC
EH12.2H7
BioLegend
329907
Anti-human PD-L1
APC
29E.2A3
BioLegend
329707
Anti-human PD-L2
APC
MIH18
BioLegend
345507
Anti-human 4–1BB
APC
4B4-1
BioLegend
309809
Anti-human CD27
APC
M-T271
BioLegend
356409
Anti-human LAG-3
APC
7H2C65
BioLegend
369211
10.2 Results The results of the flow cytometric analysis of donor-matched (n ¼ 3 donors) unstimulated and stimulated PBMCs after 3 days of culture are presented in Fig. 2. The percentage of live PBMCs (7-AAD negative) staining positive for the following markers was calculated using FlowJo (CD25, 4-1BB, CD27, CTLA-4, PD-L1, PD-L2, CD86, PD-1, Tim-3, and LAG-3). There was an observable trend seen in the stimulated cells of increased surface expression, compared to unstimulated cells, for all immune checkpoint markers except CD27. These increases were statistically significant (P-value <0.05, paired student’s t-test) for all markers except CTLA-4 and PD-L1. CTLA-4 and PD-L1 surface expression had a relatively large variability between donors, which likely impacted statistical testing. Representative univariate histograms are also presented to graphically depict the fluorescence staining intensity changes between stimulated and unstimulated PBMCs at the end of culture (Fig. 3). The supernatants obtained from the PBMC cultures were analyzed using the LEGENDplex™ Human Immune Checkpoint Panel 1. The soluble forms of the immune checkpoint molecule targets that were investigated in the flow cytometric phenotyping experiment were quantified and the results are depicted in Fig. 4. An increase in the soluble concentrations of all markers was observed, with statistically significant (P-value <0.05, paired
Fig. 2 Immune checkpoint marker expression on human PBMCs. Human PBMCs from healthy donors (n ¼ 3) were stimulated with anti-CD3 and anti-CD28 antibodies plus recombinant human IL-2 for 72 h. Cells were collected and analyzed for immune checkpoint marker expression using flow cytometry. The percentage of positive cells for each checkpoint marker was calculated and compared to unstimulated PBMC controls. The average percentage positivity + SD are displayed on the bar graphs by marker. The difference in staining positivity across culture conditions was considered statistically significant for P-values <0.05, paired student’s t-test.
Fig. 3 Histogram overlays of immune checkpoint marker expression. Representative histogram overlays comparing immune checkpoint staining intensity of unstimulated PMBCs (gray) to anti-CD3, anti-CD28, IL-2 stimulated PBMCs (white) after 3 days of culture. The positive effect of stimulation on checkpoint marker expression is clearly visible.
Fig. 4 LEGENDplex™ profiling of PBMC supernatants. Culture supernatants obtained from the PBMC cultures described in Figs. 2 and 3 were analyzed using the LEGENDplex™ Human Immune Checkpoint Panel 1 assay. Quantification results are presented in the bar charts as the mean concentration of all three donors + SD. The difference in analyte concentrations across culture conditions was considered statistically significant for P-values <0.05, paired student’s t-test.
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student’s t-test) increases occurring between groups for CD25, 4-1BB, PD-L1, PD-L2, CD86, PD-1, Tim-3, and LAG-3. Generally these results agree with the flow cytometry data, except for the markers that displayed high donor to donor variation, indicating that the LEGENDplex™ assay can serve as a useful tool to interrogate immune checkpoint marker expression in biological samples. The key advantage of analyzing samples using multiplex immunoassays over ELISAs is their ability to simultaneously quantitate multiple targets in a single experiment. This is particularly advantageous when studying complex systems such as the tumor microenvironment, where biological sample volumes available to researchers are often limited. A better understanding of the complex interaction network of cytokines in cancer patients will certainly be critical to defining innovative therapeutic strategies (Costantini et al., 2010), but at the same time this also suggests that biological significance should not be inferred solely from observations of statistically significant concentration differences with respect to any singular analyte. To visually demonstrate the advantage multiplex analysis has over single target assays in evaluating cytokine networks, the results of both the data sets from Figs. 2 and 4 are presented as radar plots in Fig. 5. By representing the data in this format, researchers can easily compare not only the magnitude of single target responses across all experimental groups, but also the relative scale of these responses to those of other analytes in the multiplex assay.
11. Summary LEGENDplex™ assays offer researchers a multiplexed cytokine profiling platform that can be adapted to run on most flow cytometers. This chapter describes the technical details of these assays, and how they can be used as tools to quantitatively investigate the soluble mediators that help coordinate the human immune response in the tumor microenvironment. All LEGENDplex™ assays are fully validated with regards to analyte sensitivity, specificity, linearity of dilution, spike recovery, reproducibility, and detectability of natural target in relevant biological samples. The assays are also backed by a performance guarantee. While there are many commercially available multiplex assays available on the market, users should ensure that they critically evaluate all available validation data prior to selecting any particular platform for their research needs. As researchers seek to advance the promise of durable clinical responses to cancer immunotherapies in all patients, validated and reliable reagents will remain critical to their success.
Fig. 5 Radar plots of flow cytometry and LEGENDplex™ results. (A) The average percentage of cells positive for immune checkpoint marker expression in both treatment groups, as presented in Fig. 2, are presented on a radar plot. Unstimulated cells are denoted by a dashed line, stimulated cells with a solid line. (B) The results of the LEGENDplex™ analysis presented in Fig. 4 presented as a radar plot. Unstimulated cells are denoted by a dashed line, stimulated cells with a solid line. For both (A) and (B) paired student t-test P-values 0.05 are represented as *, 0.01 as **, 0.001 as ***.
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Acknowledgments The authors wish to thank the following BioLegend groups and employees for their technical expertise and input during the drafting of this chapter: The biomarkers and immunoassays product development team, the flow cytometry quality control group, Kellie M. Johnson, and Dr. Ekaterina Zvezdova.
Disclosures The authors are employed by BioLegend, which manufactures the LEGENDplex™ and flow cytometry reagents described in this manuscript.
References Acharya, U. H., et al. (2019). Management of cytokine release syndrome and neurotoxicity in chimeric antigen receptor (CAR) T cell therapy. Expert Review of Hematology, 12(3), 195–205. https://doi.org/10.1080/17474086.2019.1585238. Bremm, M., et al. (2016). In-vitro influence of mycophenolate Mofetil (MMF) and Ciclosporin A (CsA) on cytokine induced killer (CIK) cell immunotherapy. Journal of Translational Medicine, 14(1), 264. https://doi.org/10.1186/s12967-016-1024-4. Chen, D. S., & Mellman, I. (2013). Oncology meets immunology: The Cancer-immunity cycle. Immunity, 39(1), 1–10. https://doi.org/10.1016/j.immuni.2013.07.012. Constantinidou, A., et al. (2019). Targeting programmed cell death -1 (PD-1) and ligand (PD-L1): A new era in Cancer active immunotherapy. Pharmacology & Therapeutics, 194, 84–106. https://doi.org/10.1016/j.pharmthera.2018.09.008. Costantini, S., et al. (2010). Human Cytokinome: A new challenge for systems biology. Bioinformation, 5(4), 166–167. https://doi.org/10.6026/97320630005166. Durgeau, A., et al. (2018). Recent advances in targeting CD8 T-cell immunity for more effective Cancer immunotherapy. Frontiers in Immunology, 9, 14. https://doi.org/ 10.3389/fimmu.2018.00014. Farhood, B., et al. (2018). CD8 cytotoxic T lymphocytes in cancer immunotherapy: A review. Journal of Cellular Physiology, 234, 8509–8521. https://doi.org/10.1002/jcp.27782. Feins, S., et al. (2019). An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human Cancer. American Journal of Hematology, 94, S3–S9. https://doi.org/ 10.1002/ajh.25418. Fromm, G., et al. (2016). Gp96-Ig/Costimulator (OX40L, ICOSL, or 4-1BBL) combination vaccine improves T-cell priming and enhances immunity, memory, and tumor elimination. Cancer Immunology Research, 4(9), 766–778. https://doi.org/10.1158/2326-6066. cir-15-0228. Fu, C., & Jiang, A. (2018). Dendritic cells and CD8 T cell immunity in tumor microenvironment. Frontiers in Immunology, 9, 3059. https://doi.org/10.3389/fimmu.2018.03059. Fuertes, M. B., et al. (2013). Type I interferon response and innate immune sensing of Cancer. Trends in Immunology, 34(2), 67–73. https://doi.org/10.1016/j.it.2012.10.004. Gottschalk, P. G., & Dunn, J. R. (2005). The five-parameter logistic: A characterization and comparison with the four-parameter logistic. Analytical Biochemistry, 343(1), 54–65. https://doi.org/10.1016/j.ab.2005.04.035. Han, Y., et al. (2018). Antitumor effects and persistence of a novel HER2 CAR T cells directed to gastric Cancer in preclinical models. American Journal of Cancer Research, 8(1), 106–119. Huang, L., et al. (2017). WASH overexpression enhances Cancer stem cell properties and correlates with poor prognosis of esophageal carcinoma. Cancer Science, 108(12), 2358–2365. https://doi.org/10.1111/cas.13400. Lee, D. W., Gardner, R., Porter, D. L., et al. (2014). Current concepts in the diagnosis and management of cytokine Release syndrome. Blood, 124(2), 188–195. Blood. 2015; 126 (8): 1048. https://doi.org/10.1182/blood-2015-07-656918.
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L€ ovgren, T., et al. (2017). Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists. Cancer Immunology, Immunotherapy, 66(10), 1333–1344. https://doi.org/10.1007/ s00262-017-2029-4. Mantovani, A., et al. (2008). Cancer-related inflammation. Nature, 454(7203), 436–444. https://doi.org/10.1038/nature07205. Mccoll, A., et al. (2016). TLR7-mediated skin inflammation remotely triggers chemokine expression and leukocyte accumulation in the brain. Journal of Neuroinflammation, 13(1), 102. https://doi.org/10.1186/s12974-016-0562-2. Mlynska, A., et al. (2018). Chemokine profiling in serum from patients with ovarian Cancer reveals candidate biomarkers for recurrence and immune infiltration. Oncology Reports, 41, 1238–1252. https://doi.org/10.3892/or.2018.6886. Nerurkar, L., et al. (2017). The systemic response to topical Aldara treatment is mediated through direct TLR7 stimulation as Imiquimod enters the circulation. Scientific Reports, 7(1). https://doi.org/10.1038/s41598-017-16707-5. Olguı´n, J. E., et al. (2018). Early and partial reduction in CD4 Foxp3 regulatory T cells during colitis-associated Colon Cancer induces CD4 and CD8 T cell activation inhibiting tumorigenesis. Journal of Cancer, 9(2), 239–249. https://doi.org/10.7150/jca.21336. Poeta, V. M., et al. (2019). Chemokines and chemokine receptors: New targets for Cancer immunotherapy. Frontiers in Immunology, 10, 379. https://doi.org/10.3389/ fimmu.2019.00379. Raeber, M. E., et al. (2018). The role of cytokines in T-cell memory in health and disease. Immunological Reviews, 283(1), 176–193. https://doi.org/10.1111/imr.12644. Reading, J. L., et al. (2018). The function and dysfunction of memory CD8 T cells in tumor immunity. Immunological Reviews, 283(1), 194–212. https://doi.org/10.1111/imr.12657. Roshani, R., et al. (2014). Inflammatory cytokines in human pancreatic Cancer. Cancer Letters, 345(2), 157–163. https://doi.org/10.1016/j.canlet.2013.07.014. Sachdeva, M., et al. (2019). Granulocyte-macrophage Colony-stimulating factor inactivation in CAR T-cells prevents monocyte-dependent release of key cytokine release syndrome mediators. Journal of Biological Chemistry, 294, 5430–5437. https://doi.org/10.1074/jbc. ac119.007558. Selby, C. (1999). Interference in immunoassay. Annals of Clinical Biochemistry: An International Journal of Biochemistry and Laboratory Medicine, 36(6), 704–721. https://doi.org/ 10.1177/000456329903600603. Sharma, P., & Allison, J. P. (2015). The future of immune checkpoint therapy. Science, 348(6230), 56–61. https://doi.org/10.1126/science.aaa8172. Tiper, I. V., & Webb, T. J. (2016). Histone deacetylase inhibitors enhance CD1d-dependent NKT cell responses to lymphoma. Cancer Immunology, Immunotherapy, 65(11), 1411–1421. https://doi.org/10.1007/s00262-016-1900-z. Valilou, S. F., et al. (2018). The role of inflammatory cytokines and tumor associated macrophages (TAMs) in microenvironment of pancreatic cancer. Cytokine & Growth Factor Reviews, 39, 46–61. https://doi.org/10.1016/j.cytogfr.2018.01.007. Wood, W. G. (1991). Matrix effects’ in immunoassays. Scandinavian Journal of Clinical and Laboratory Investigation, 51(Suppl. 205), 105–112. https://doi.org/10.3109/ 00365519109104608. Yamaguchi, K., et al. (2018). Activation of central/effector memory T cells and T-helper 1 polarization in malignant melanoma patients treated with anti-programmed Death-1 antibody. Cancer Science, 109(10), 3032–3042. https://doi.org/10.1111/cas.13758. Zhao, H., et al. (2019). Clinical significance of serum type III interferons in patients with gastric Cancer. Journal of Interferon & Cytokine Research, 39(3), 155–163. https://doi. org/10.1089/jir.2018.0119.
LEGENDplex™: Bead-assisted multiplex cytokine profiling by flow cytometry Jason S. Lehmann*, Priyanka Rughwani, Melissa Kolenovic, Shaoquan Ji, Binggang Sun BioLegend, San Diego, CA, United States *Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 1.1 LEGENDplex™ assay principles 2. Required equipment and materials 2.1 Equipment 2.2 Materials 2.3 Precautions 3. Sample collection 3.1 Serum samples 3.2 Plasma samples 3.3 Cell culture supernatant 3.4 Alternative sample types 3.5 Sample fixation 3.6 Sample dilutions 4. Reagent preparation 4.1 Preparation of antibody-immobilized capture beads 4.2 Preparation of wash buffer 4.3 Preparation of matrix 4.4 Recombinant standard preparation 5. Assay protocol 5.1 Generalized recommendations 5.2 Assay procedure 6. Flow cytometer setup 6.1 Collection of data in appropriate cytometer channels 6.2 Optimal FSC and SSC voltage/sensor gain settings 6.3 Optimal APC voltage/sensor gain settings 6.4 Optimal PE PMT voltage/sensor gain 7. Data acquisition
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8. Data analysis 8.1 Bead separation 8.2 Bead gating 8.3 Replicate precision 8.4 Bead counts 9. Tips for novice users 9.1 Polypropylene plastics 9.2 Incubation shaker speed 9.3 Flow cytometer settings 10. Example LEGENDplex™ data set 10.1 Materials and methods 10.2 Results 11. Summary Acknowledgments Disclosures References
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Abstract Over the past two decades there have been tremendous advances in our understanding of tumor immunology, which have in turn led to new and exciting immunologybased therapeutics. However, further research is needed into the dynamics and regulation of the immune response in the tumor microenvironment in order to achieve the full potential of these agents in treating all cancer patients. Defining the role of cytokines, chemokines, and other soluble mediators will be essential to this endeavor. This chapter describes, in detail, the technical protocol and applicability of LEGENDplex™ bead-based multiplex assays in quantifying these critical signaling molecules.
1. Introduction Clinical advances in cancer immunotherapy have always followed advances in our understanding of tumor immunology. Indeed, over the past two decades in cancer immunotherapy history there have been remarkable advances in CAR-T cell (Feins et al., 2019) and immune checkpoint monotherapies (Chen & Mellman, 2013; Constantinidou et al., 2019; Sharma & Allison, 2015). The success of these therapies relies upon modulating immune effector mechanisms to generate tumor specific cytotoxic T lymphocytes (Durgeau et al., 2018); however, a deeper understanding of human immune responses is needed to realize the full potential of these promising therapies. Immune checkpoint therapy can lead to durable and long-lasting clinical responses, but only in a fraction of patients (Sharma & Allison, 2015). CAR-T therapies have become more potent
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and effective, but the development of cytokine release syndrome in response to treatment risks life threatening toxicity (Acharya et al., 2019; Lee, Gardner, Porter, et al., 2014). Overcoming these challenges will require a greater understanding of immune regulation within the tumor microenvironment. However, this complicated system provides both stimulatory and inhibitory signals to infiltrating lymphocytes. Inflammation, a hallmark of cancer, promotes tumor proliferation, angiogenesis, and metastasis, but also can subvert the immune response (Mantovani et al., 2008). In fact, tumor induced immunosuppression remains one of the major hurdles facing cancer immunotherapy (Fu & Jiang, 2018). Once in the tumor microenvironment, cytotoxic T cells must overcome the suppressive effects of not only the tumor cells, but also regulatory T cells, myeloid-derived suppressor cells, and inhibitory cytokines that together act to mitigate antitumor responses. The inflammatory mediators that drive these immune processes are key constituents of the tumor microenvironment. The role of cytokines and chemokines in cancer is well established (Fuertes et al., 2013; Poeta et al., 2019; Raeber et al., 2018; Roshani et al., 2014; Valilou et al., 2018) and affects many aspects of tumor immunology from immune cell recruitment, modulation of their effects, angiogenesis, and metastasis. In fact, identifying predictive biomarkers that define clinically beneficial immune responses is an area of active research (Sharma & Allison, 2015). The ability to accurately quantify these biomarkers is therefore of critical importance to these efforts. Immunoassays have been able to reliably quantify single analytes for decades; however, as researchers now seek to assess the effectiveness of evolving immune responses, the need to quantify multiple biomarkers simultaneously has become a necessity. Multiplex immunoassays provide researchers the ability to quantitatively test for multiple targets simultaneously from one sample aliquot. In particular, LEGENDplex™ bead-based immunoassays, offered by BioLegend, provide a multiplexing platform to researchers designed to be performed using commonly available flow cytometers. Multiple LEGENDplex™ immunoassays have been cited in the methods sections of several recent publications related to tumor immunology and immunotherapy. These assays have been used to quantify relevant biomarker concentrations from experimental studies involving cancers of the colon (Olguı´n et al., 2018), esophagus (Huang et al., 2017), stomach (Zhao et al., 2019), ovaries (Mlynska et al., 2018), lymphoma (Tiper & Webb, 2016), and malignant melanoma (Yamaguchi et al., 2018). In addition, LEGENDplex™ assays have also been cited in studies investigating
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CAR-T (Han et al., 2018; Sachdeva et al., 2019) and cytokine-induced killer cell immunotherapies (Bremm et al., 2016), as well as dendritic cell and cancer cell-based vaccines (Fromm et al., 2016; L€ ovgren et al., 2017). The particular LEGENDplex™ assays which were cited by these publications are summarized in Table 1.
1.1 LEGENDplex™ assay principles LEGENDplex ™ bead-based immunoassays have been developed based on the same principles as the sandwich immunoassay, and allow users to simultaneously quantify up to 13 different biological molecules from 1 sample aliquot. Capture beads, which can be differentiated based on size and internal fluorescence intensities, are covalently conjugated to analyte-specific antibodies using carbodiimide crosslinking reactions. The LEGENDplex™ assay system uses two sets of beads: A beads and B beads. The A and B bead sets have forward and side-scatter profiles that when run on a flow cytometer, allow for each to be uniquely identified. Bead sets are then further resolved based on the internal APC fluorescence within the beads. There are a total possible six distinct populations of APC intensities within the A beads, and seven within the B beads (Fig. 1). Table 1 LEGENDplex™ references. LEGENDplex™ panel References
Human T Helper Cytokine Panel
Han et al. (2018) and Yamaguchi et al. (2018)
Human Inflammation Panel 1 Sachdeva et al. (2019), Tiper and Webb (2016), and Bremm et al. (2016) Human Inflammation Panel 2 Sachdeva et al. (2019) Human Proinflammatory Chemokine Panel
Mlynska et al. (2018) and Huang et al. (2017)
Human Anti-Virus Response L€ ovgren et al. (2017) Panel Human Type 1/2/3 Interferon Panel
Zhao et al. (2019)
Mouse Th1/Th2 Panel
Fromm et al. (2016)
Mouse Th17 Panel
Olguı´n et al. (2018)
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Fig. 1 LEGENDplex™ beads. (A) Bead size populations: FSC/SSC characteristics of the two bead sizes used in LEGENDplex assays. Clearly visible are the smaller A beads and the larger B beads. (B) A bead ID classification by APC: The six A bead IDs that are used in the assays are displayed on an APC uninvariate histogram. (C) B bead ID classification by APC: The seven bead IDs that are used in the assays are displayed on an APC uninvariate histogram. Bead IDs are distinguished from on another based on internal APC fluorescence signal differences of the beads. Note: The histograms displayed in (B) and (C) were smoothed for esthetic purposes using FlowJo, and are solely presented to familiarize readers with the spectral properties of the fluorescent microsphere reagents employed in the assay.
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Panels of defined capture bead sets are then incubated with recombinant protein standards or the biological samples to be assayed. A second incubation with biotinylated detection antibodies leads to the formation of capture bead-analyte-detection antibody sandwiches. A Streptavidin-PE reagent is then added to bind biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE signal of each capture bead is measured using flow cytometry, and then input into the LEGENDplex™ Data Analysis Software to quantify the analyte concentrations in each sample. This assay platform offers several advantages over traditional ELISA formats including enhanced sensitivity, broader dynamic ranges, lower sample volume requirements, free of charge data analysis software, and multiplexed analysis. In addition, these assays can be performed in the laboratory using any flow cytometer capable of reading both PE and APC fluorescence signals, which obviates the need for capital expenditures on dedicated analysis equipment. LEGENDplex™ bead-based immunoassays have been developed for human, non-human primate, mouse, and rat specificities. For each species, there are pre-defined panels designed to quantify biomarkers related to particular cell types, biological processes, or disease states. Within a given panel, assays can be ordered which quantify only certain targets of interest, should the full pre-defined panel be unsuitable. Additionally, custom panel manufacturing of unique target combinations is available. A full listing of assays as well as ordering information can be found online at https:// www.biolegend.com/legendplex.
2. Required equipment and materials 2.1 Equipment 1. A flow cytometer equipped with a 488 nm blue laser or 532 nm green laser, and a 633–635 nm red laser capable of distinguishing 575 and 660 nm 2. Multichannel pipettes capable of dispensing volumes of 5–200 μL 3. Vortex mixer 4. Bath sonicator (Branson Ultrasonic Cleaner model #B200, or equivalent) 5. Plate shaker (Lab-Line Instruments model #4625, or equivalent)
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6. Benchtop Microcentrifuge (Eppendorf centrifuge 5415C, or equivalent) If the assay is performed in a filter plate: 1. Vacuum filtration unit (Millipore MultiScreen® HTS Vacuum Manifold or equivalent) 2. Vacuum source (mini-vacuum pump or line vacuum) If the assay is performed in a V-bottom plate: 1. Centrifuge with a swinging bucket adaptor for microtiter plates
2.2 Materials 1. 2. 3. 4. 5. 6. 7.
LEGENDplex™ bead-based immunoassay (BioLegend) Pipet tips for multichannel pipette Reagent reservoirs for multichannel pipette Polypropylene microcentrifuge tubes (1.5 mL) Aluminum foil Absorbent pads or paper towels 1.1 mL polypropylene micro FACS tubes, in 96-tube rack (National Scientific Supply TN0946-01R, or equivalent)
2.3 Precautions 1. Sodium azide is used as a preservative in some LEGENDplex™ components. During disposal, flush with a large volume of water to prevent the accumulation of explosive metal azides in laboratory plumbing. 2. The matrix reagent included in LEGENDplex™ kits contains components derived from human or animal blood. The human-based material has been screened for certain infectious diseases using FDA-approved testing methods and is negative for HIV, HBV, and HCV, but should be handled as a potentially hazardous material. 3. The streptavidin-PE and capture bead reagents are UV sensitive. Minimize exposure to light when performing the assay. 4. All tubes and plates used in the assay should be made from low binding polypropylene. Polystyrene ELISA or cell culture plates treated for optimal cell attachment should not be used due to their capacity to absorb recombinant protein standards and affect assay results.
3. Sample collection LEGENDplex™ assays are validated for use with one or more biological samples (panel-specific) including serum, plasma, cerebrospinal fluid,
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urine, saliva, and cell culture supernatant. The assays can potentially be adapted for use with other samples types, but have special considerations that are discussed below in the alternative sample types section.
3.1 Serum samples After collection, allow blood to clot for at least 30 min and then centrifuge at 1000 g for 20 min. Remove serum and proceed to assay immediately or aliquot samples and store at 20 °C.
3.2 Plasma samples Plasma collection using EDTA as an anti-coagulant is recommended. Within 30 min of blood collection, centrifuge sample at 1000 g for 10 min. Remove plasma and proceed to assay immediately or aliquot samples and store at 20 °C. When using frozen serum or plasma samples it is recommended that samples be completely thawed, mixed thoroughly, and centrifuged to remove particulates prior to use. Avoid multiple freeze/thaw cycles.
3.3 Cell culture supernatant After harvesting the supernatant, centrifuge the sample to remove any remaining cellular debris. Proceed to assay immediately or aliquot samples and store at 20 °C.
3.4 Alternative sample types The use of alternative sample inputs has been reported (Mccoll et al., 2016; Nerurkar et al., 2017); however, it is the responsibility of the researcher to establish the compatibility of any sample type for use with the assay. Avoid the use of denaturing chemicals (urea, cholate, etc.) and ionic detergents when preparing buffers for alternative sample types. Minimal amounts (<1%) of non-ionic detergents can be used, provided they are in a neutral pH buffer containing physiological salt concentrations. The inclusion of protease inhibitors is strongly recommended.
3.5 Sample fixation The use of fixatives with LEGENDplex™ reagents is generally discouraged, as prolonged incubations will quench fluorescence signals. If samples must be fixed due to biosafety considerations, it should be performed only after completing the assay. Freshly prepared 1–4% paraformaldehyde in
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1 PBS can be used to fix samples by adding 150 μL per well for 10–15 min. The fixative should be washed out immediately after the incubation by following the wash steps described below.
3.6 Sample dilutions 3.6.1 Cell culture supernatants The concentrations of target analytes in cell culture supernatants can vary greatly across cell lines and culture conditions. A preliminary experiment is recommended to establish an appropriate dilution factor for a particular sample. Any dilutions of samples should be done with either sterile culture media or LEGENDplex™ Assay Buffer to ensure accurate measurement. 3.6.2 Serum and plasma samples Dilution requirements for serum and plasma samples are dependent on the target being assayed, with targets of high endogenous levels requiring greater dilution than those of low abundance. Please refer to the product manual for assay specific details regarding dilution of these sample types.
4. Reagent preparation 4.1 Preparation of antibody-immobilized capture beads If pre-mixed beads are included with the kit: 1. Sonicate the bottle of pre-mixed beads for 60 s in a sonicator bath, and then vortex for 30 s immediately prior to use. Note: Vortex time can be increased to 60 s if access to a sonicator bath is unavailable in the laboratory. If individual bead vials (13) are included with the kit: The beads will need to be diluted from 13 to 1 concentration while being combined in LEGENDplex™ Assay Buffer prior to their use. 1. Sonicate the beads vials for 1 min in a sonicator bath, and then vortex for 30 s to completely resuspend the beads. 2. Calculate the volume of diluted beads needed for the assay. Each assay well will require 25 μL of 1 beads. Multiply the number of wells by 25 μL then add an extra volume (approximately +20% more) to compensate for pipetting loss. 3. Divide this quantity by 13 to determine the volume from each individual bead vial required. Add these volumes into an appropriately sized polypropylene tube, then q.s. with LEGENDplex™ Assay Buffer to reach the necessary total volume calculated in the previous step.
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4.2 Preparation of wash buffer 1. Allow the bottle of 20 Wash Buffer to equilibrate to room temperature and swirl to dissolve any salt crystals that may have formed during storage. 2. Dilute 25 mL of 20 Wash Buffer with 475 mL of deionized water. Unused buffer may be stored for up to 1 month at 2–8 °C.
4.3 Preparation of matrix The matrix reagent is included with LEGENDplex™ immunoassay kits to control for any potential “matrix effects” in serum and plasma sample types. Matrix effects are consistent, but poorly defined non-specific binding interactions caused by endogenous factors in blood derived sample types (Selby, 1999; Wood, 1991). 1. Add 5 mL of LEGENDplex™ Assay Buffer to the bottle containing the lyophilized matrix component provided in the assay kit. 2. Incubate for 15 min and then vortex well to mix. Unused matrix may be stored for up to 1 month at 70 °C.
4.4 Recombinant standard preparation 1. Reconstitute the lyophilized standard cocktail with 250 μL of LEGENDplex™ Assay Buffer. 2. Mix and then allow the vial to incubate at room temperature (20–25 °C) for 15 min. 3. During incubation, label seven polypropylene microcentrifuge tubes C7, C6, C5, C4, C3, C2, C1, respectively. 4. Add 75 μL of LEGENDplex™ Assay Buffer to all tubes except C7 (i.e., C1–C6). 5. After the 15 min has elapsed, transfer the entire volume of reconstituted standard from the vial into the microcentrifuge tube labeled C7. This will be used as the top standard. 6. Perform a 1:4 dilution of the top standard by transferring 25 μL from the C7 tube into the C6 tube and then mix well. 7. Continue this 1:4 dilution series to obtain standards C5, C4, C3, C2, and C1. LEGENDplex™ Assay Buffer will be used as the blank standard (C0).
5. Assay protocol 5.1 Generalized recommendations 1. Read the manual included with the assay prior to performing any experiment.
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2. Standards and samples should be assayed in duplicate. 3. Complete the plate map, found in the back of the assay manual, by assigning each standard and experimental sample to a corresponding well on the document in a manner convenient for data acquisition and analysis. 4. LEGENDplex™ assays can be performed using either filter or V-bottom plates (see Section 2.1 for specific requirements of each). The choice of plate does not affect assay performance, but there are slight variations in protocol between the two formats. 5. Allow all reagents to equilibrate to room temperature (20–25 °C) before use. 6. The assay plate should be placed in the dark or wrapped in aluminum foil during all incubation steps. For assays performed using filter plates: 1. Keep the filter plate upright during the entire assay procedure, including wash steps, to avoid losing beads. 2. Place the filter plate on an inverted plate cover at all times during assay setup and incubation steps. If the bottom of the plate is allowed to contact flat surfaces inadvertently leaking can occur.
5.2 Assay procedure For assays performed using filter plates: 1. Pre-wet the filter plate by adding 100 μL of 1 LEGENDplex™ Assay Buffer to each well and incubate for 1 min. 2. Remove buffer volume by placing the plate on the vacuum manifold and apply vacuum (do not exceed 1000 Hg of pressure) until wells are drained. 3. Press the filter plate onto a stack of clean paper towels to blot excess liquid from the bottom. Assaying cell culture supernatant samples: 1. Add 25 μL of Assay Buffer to all wells. 2. Add 25 μL of C7–C1 standards to appropriate standard wells 3. Add 25 μL of Assay Buffer to C0 wells. 4. Add 25 μL of samples to appropriate wells. Assaying serum and plasma samples: 1. Add 25 μL of Assay Buffer to all sample wells 2. Add 25 μL of Matrix Component to all standard wells 3. Add 25 μL of C7–C1 standards to appropriate standard wells 4. Add 25 μL of Assay Buffer to C0 wells 5. Add 25 μL of samples to appropriate wells.
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For all assay plate wells: 1. Vortex beads for 30 s then add 25 μL of beads to each well. Shake the bottle/tube intermittently to prevent beads from settling. The total volume per well should now be 75 μL. Note: This step will use either the pre-mixed beads bottle or the 1 beads solution depending on the assay format used. 2. Seal the top of the plate with a plate sealer then wrap entire plate (including inverted plate cover if using filter plate) with aluminum foil. 3. Place the wrapped plate on a plate shaker and shake at 500 rpm (filter plate) or 800 rpm (V-bottom plate) for 2 h at room temperature (20–25 °C). 4. Wash the plate according to the following: Filter Plates: Place the plate on the vacuum manifold and apply vacuum. Add 200 μL of 1 Wash Buffer to each well. Remove Wash Buffer with vacuum filtration, and blot excess liquid from plate bottom with paper towels. Repeat this process once more. V-bottom Plates: Centrifuge the plate at 1050 rpm for 5 min. Dump the supernatant into a sink by inverting the plate with one continuous forceful motion. Add 200 μL of 1 Wash Buffer to each well and incubate for 1 min. Repeat centrifugation and dump once more. 5. Add 25 μL of Detection Antibodies to each well. 6. Seal the top of the plate with a new plate sealer then wrap entire plate (including inverted plate cover if using filter plate) with aluminum foil. 7. Place the wrapped plate on a plate shaker and shake at 500 rpm (filter plate) or 800 rpm (V-bottom plate) for 1 h at room temperature (20–25 °C). 8. Add 25 μL SA-PE directly to each well without washing. 9. Seal the top of the plate with a new plate sealer then wrap entire plate (including inverted plate cover if using filter plate) with aluminum foil. 10. Place the wrapped plate on a plate shaker and shake at 500 rpm (filter plate) or 800 rpm (V-bottom plate) for 30 min at room temperature (20–25 °C). 11. Repeat wash step 4 above. 12. Add 150 μL of Wash Buffer to each well. 13. Resuspend the beads into solution by placing the plate on a plate shaker for 1 min. 14. Transfer samples from the assay plate into micro-FACS tubes using a multichannel pipet in order to read samples on a flow cytometer.
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Note: The samples can be kept overnight at 4 °C while being protected from light exposure and read the next day. There may be a decrease in fluorescence signal strength, but overall assay results should not be affected. Storing the samples for extended periods of time is not recommended, as it could lead to further reductions in signal.
6. Flow cytometer setup LEGENDplex™ assays are compatible for use with all flow cytometers capable of detecting PE and APC fluorescence signals; however, to generate the best results it is critical that each instrument be properly set up prior to data acquisition. Users should refer to instrument-specific setup instructions (instrument setup tab, www.biolegend.com/legendplex) when conducting the assay for the first time in order to generate high quality data. Note: Preliminary experiments to establish acceptable cytometer settings are encouraged prior to assaying any experimental samples using the LEGENDplex™ platform. The instrument setup process will obviously vary for users depending on the configuration of their flow cytometer; however, a proper instrument setup will always contain the following critical elements regardless of the instrument used in a particular assay:
6.1 Collection of data in appropriate cytometer channels Ensure that the acquisition software for the cytometer is set to collect data in the following channels: FSC, SSC, APC, and PE. Other fluorescence channels are not required and can be deselected on the instrument.
6.2 Optimal FSC and SSC voltage/sensor gain settings Proper voltage/gain settings for the FCS and SSC parameters will clearly separate the A beads population from the B beads population when viewed on an FSC versus SSC dot plot in linear mode.
6.3 Optimal APC voltage/sensor gain settings Optimal APC settings will clearly delineate all respective bead IDs within a bead region (A or B beads) based on internal APC fluorescence.
6.4 Optimal PE PMT voltage/sensor gain An optimal PE setting will give the widest possible dynamic range for signals in the channel. If set too low, the low end of the standard curve may plateau
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due to a low signal-to-noise ratio. If set too high, the high end of the standard curve may plateau due to signal saturation. Under ideal cytometer settings, standard curve samples will fall onto a sigmoid distribution with the PE-MFI of the C7 standard near the upper limit of detection (i.e., signal saturation), a C0 PE-MFI 50% of the C1 PE-MFI, and well separated standard curve points (C1 through C6) in between.
7. Data acquisition The exact procedures associated with the acquisition of data on a given instrument are dependent on the cytometer’s configuration specifications as well as the interface software used. The instructions below are therefore intended to highlight the essential steps to be followed regardless of the flow cytometer model employed in the assay. Before reading samples, ensure that the cytometer is properly setup (see Section 6). 1. Vortex each sample for 5 s prior to acquisition. 2. If the flow cytometer is equipped for high-throughput screening, then the plate can be read directly using the auto-sampler. Ensure that the auto-sampler is programed to agitate the beads in the well immediately prior to data acquisition. 3. Set the flow rate of the cytometer to low. 4. Using the acquisition software for the cytometer, draw a gate that encompasses both the A and B bead populations (A/B gate). 5. Set the stop condition for acquisition to be equal to 300 events per bead ID included in the particular LEGENDplex™ assay. Do not set acquisition parameters to stop based on total events as these can include debris and other junk signals. Ensure that data acquisition stops based on A/B gated events. 6. Read samples in the same order as the plate map, starting with well A1 and reading through the plate column by column. 7. Export the flow cytometry files from the acquisition software and save them to an appropriate location.
8. Data analysis Exported data files generated should be analyzed using the LEGENDplex™ Data Analysis Software (BioLegend). A license for the software is provided free of charge with each LEGENDplex™ assay. Instructions for the download and use of the software can be found at www. biolegend.com/legendplex, under the software tab. The software,
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compatible with FCS 2.0, FCS 3.0, and FCS 3.1 standard formats as well as . LMD files, is designed to apply a gating strategy that allows for the proper identification of all assay bead IDs, and then fit a 5-parameter logistic curve (Gottschalk & Dunn, 2005) to the PE-reporter signal in the standard curve files. The resulting curve equation is then used to calculate the concentration of assay targets in the experimental samples. The use of other software programs to analyze LEGENDplex™ flow cytometry files is theoretically possible; however, the ultimate suitability of a program would need to be validated by the researcher. Beyond the capacity to analyze the appropriate fluorescence signals from assay files, the most critical feature of any program is its curve fitting algorithm; in particular, the ability to derive and fit 4- or 5-parameter logistic curves to the standard curve files. The use of graphing features found in commonly used spreadsheet programs is particularly discouraged, as these produce inaccurate quantification results. Regardless of the software program used to analyze the LEGENDplex™ data, the researcher should review the following parameters in their results in order to confirm that the assay was successfully performed:
8.1 Bead separation Confirm that there is clear separation of all bead populations in the dot-plots generated by the software. The A beads should be clearly distinguished from B beads in the forward scatter versus side scatter plots, and the APC fluorescence plots should also evidence clearly delineated bead ID populations (Fig. 1).
8.2 Bead gating The gating applied to all assay files should also be reviewed for accuracy, and corrected if necessary. Well defined bead regions and the proper gating of these regions during data analysis are essential to generating accurate results. Although this post hoc review can do nothing to salvage results from the assay should the bead IDs overlap, it does allow the researcher to avoid drawing erroneous conclusions from the data and alert them of the need to adjust their cytometer settings for future experiments.
8.3 Replicate precision The coefficient of variation between replicate samples should also be reviewed, as high values can be indicative of technical errors. All results from replicates that have %CV values >30% should be interpreted with caution.
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Pipetting related errors are by far the most common contributor to these effects. As such, proper calibration and attention to detail are essential requirements for generating reliable quantification results.
8.4 Bead counts As described in the Section 7, approximately 300 events per bead ID should have been collected on the flow cytometer used in the assay. This should be verified by the researcher using the data analysis program. This number of events is suggested as it will provide a large enough sampling of the median PE fluorescence intensity of a given bead ID to establish a 99% confidence interval around this value. Since the PE signals are used to derive calculated concentrations from samples in the assay it is critical to estimate these values as accurately as possible. Certainly, some degree of variation across samples in cytometer-counted events per bead ID should be expected within an assay; however, gross differences (i.e., less than 100 events) should be investigated further. In a given sample, low bead counts for one bead ID but not others can indicate the presence of an inappropriate gate that should be reviewed. Low bead counts across all samples might indicate that the stop parameter on the flow cytometer was inadvertently set on total events (which include debris) versus gated events around just the assay beads.
9. Tips for novice users LEGENDplex™ bead-based immunoassays can be used to reliably quantify soluble mediator levels in biological samples; however, these results are highly dependent on adherence to the assay protocol. There are a few technical considerations regarding the LEGENDplex™ assay platform that new users should give particular attention to when running the assay for the first time:
9.1 Polypropylene plastics The importance of using only polypropylene plastics when performing the assay cannot be overstated. Polystyrene ELISA or tissue culture treated microplates specifically promote the adherence of the assay’s recombinant protein standards to the wells of the plate. The same is true for polystyrene microcentrifuge tubes. The binding of recombinant standards to these
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materials is detrimental to the performance of any quantitative immunoassay, as this effectively removes the very molecules used to generate the standard curve out of solution.
9.2 Incubation shaker speed Proper agitation of the capture beads is required for them to efficiently bind target analytes in solution. Without proper shaking, the binding characteristics of the beads can be severely hindered. In addition, slow shaking speeds can promote the formation of bead aggregates which prevents uniform detection antibody staining. Researchers are thus encouraged to verify that the plate shaker they intend to use during the assay is capable of agitating the plate at the speed stated in the assay manual.
9.3 Flow cytometer settings As discussed above in the Section 6, APC and PE PMT voltages must be adjusted to ensure proper bead separation and broad dynamic ranges for the standard curves. Researchers are again encouraged to perform pilot experiments to optimize instruments settings for the flow cytometer used to interrogate the assay beads.
10. Example LEGENDplex™ data set Cytotoxic T lymphocytes (CTL) are essential for cancer immunosurveillance (Farhood et al., 2018). Despite their critical function in targeting various cancers, our basic understanding of the tenets of CTL immunity is still being refined (Reading et al., 2018). T-cell inhibitory receptors have been one particularly active focus for tumor immunology research as well as clinical immunotherapy (Constantinidou et al., 2019; Durgeau et al., 2018; Farhood et al., 2018; Sharma & Allison, 2015). To demonstrate the utility of LEGENDplex™ immunoassays in quantifying the soluble forms of these key molecules, human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors and cultured with media or media containing anti-CD3 and anti-CD28 antibodies plus recombinant IL-2. After 72 h, the expression levels of immune checkpoint-related molecules on the surface of the PBMCs were analyzed using flow cytometry. Concurrently, the concentrations of the same checkpoint molecules were quantified in the supernatants of the same PBMC cultures using the LEGENDplex™ Human Immune Checkpoint Panel 1.
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10.1 Materials and methods 10.1.1 PBMC isolation and in-vitro culture Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n ¼ 3) using Lymphopure™ (BioLegend: catalog # 426201) density gradient centrifugation, according to manufacturer instructions. Isolated PBMCs were stained with a 0.4% trypan blue solution (Thermo Fisher Scientific) and then enumerated and assessed for viability using a hemocytometer. PBMCs were then cultured in 6-well microtiter plates (6 106 cells/well in 3 mL media) at 37 °C with 5% CO2 for 72 h under the following conditions: Unstimulated: Per well, 6 106 PBMCs in 3 mL RPMI 1640 media (Corning) supplemented with 10% heat-inactivated FBS (GE Life Sciences) and penicillin/streptomycin solution (GE Life Sciences). Stimulated: Per well, 6 106 PBMCs in 3 mL culture media supplemented with anti-human CD3 antibody at 10 μg/mL (BioLegend, clone: HIT3a, Cat. No. 300302), anti-human CD28 antibody at 10 μg/mL (BioLegend, clone: CD28.2, catalog # 302902), IL-2 at 20 ng/mL (BioLegend, catalog # 589104). 10.1.2 Flow cytometry PBMCs were isolated from culture wells by centrifugation (1500 rpm, 5 min), then stained with the following reagents: 7-AAD Viability Staining Solution (BioLegend, catalog # 420403) according to manufacturer instructions and 5 μL of the antibodies listed below in Table 2. Samples were then analyzed using a BD FACSCanto II™ flow cytometer. Resulting data were then processed using FlowJo Software (Becton, Dickinson & Company) and GraphPad Prism (GraphPad Software). 10.1.3 LEGENDplex™ profiling Unstimulated and stimulated PBMC culture supernatants were quantified for soluble immune checkpoint molecules using the LEGENDplex™ Human Immune Checkpoint Panel 1 (BioLegend, catalog # 740867). The supernatants were tested following the assay protocol described in this manuscript. Data were analyzed using the LEGENDplex™ Data Analysis Software (BioLegend, PC version 8) and GraphPad Prism (GraphPad Software).
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Table 2 Flow cytometry reagents. Specificity Format
Clone
Manufacturer
Cat. #
Anti-human CD25
PE
BC96
BioLegend
302605
Anti-human CD86
APC
BU63
BioLegend
374207
Anti-human CTLA-4
APC
L3D10
BioLegend
349907
Anti-human TIM-3
APC
F38-2E2
BioLegend
345011
Anti-human PD-1
APC
EH12.2H7
BioLegend
329907
Anti-human PD-L1
APC
29E.2A3
BioLegend
329707
Anti-human PD-L2
APC
MIH18
BioLegend
345507
Anti-human 4–1BB
APC
4B4-1
BioLegend
309809
Anti-human CD27
APC
M-T271
BioLegend
356409
Anti-human LAG-3
APC
7H2C65
BioLegend
369211
10.2 Results The results of the flow cytometric analysis of donor-matched (n ¼ 3 donors) unstimulated and stimulated PBMCs after 3 days of culture are presented in Fig. 2. The percentage of live PBMCs (7-AAD negative) staining positive for the following markers was calculated using FlowJo (CD25, 4-1BB, CD27, CTLA-4, PD-L1, PD-L2, CD86, PD-1, Tim-3, and LAG-3). There was an observable trend seen in the stimulated cells of increased surface expression, compared to unstimulated cells, for all immune checkpoint markers except CD27. These increases were statistically significant (P-value <0.05, paired student’s t-test) for all markers except CTLA-4 and PD-L1. CTLA-4 and PD-L1 surface expression had a relatively large variability between donors, which likely impacted statistical testing. Representative univariate histograms are also presented to graphically depict the fluorescence staining intensity changes between stimulated and unstimulated PBMCs at the end of culture (Fig. 3). The supernatants obtained from the PBMC cultures were analyzed using the LEGENDplex™ Human Immune Checkpoint Panel 1. The soluble forms of the immune checkpoint molecule targets that were investigated in the flow cytometric phenotyping experiment were quantified and the results are depicted in Fig. 4. An increase in the soluble concentrations of all markers was observed, with statistically significant (P-value <0.05, paired
Fig. 2 Immune checkpoint marker expression on human PBMCs. Human PBMCs from healthy donors (n ¼ 3) were stimulated with anti-CD3 and anti-CD28 antibodies plus recombinant human IL-2 for 72 h. Cells were collected and analyzed for immune checkpoint marker expression using flow cytometry. The percentage of positive cells for each checkpoint marker was calculated and compared to unstimulated PBMC controls. The average percentage positivity + SD are displayed on the bar graphs by marker. The difference in staining positivity across culture conditions was considered statistically significant for P-values <0.05, paired student’s t-test.
Fig. 3 Histogram overlays of immune checkpoint marker expression. Representative histogram overlays comparing immune checkpoint staining intensity of unstimulated PMBCs (gray) to anti-CD3, anti-CD28, IL-2 stimulated PBMCs (white) after 3 days of culture. The positive effect of stimulation on checkpoint marker expression is clearly visible.
Fig. 4 LEGENDplex™ profiling of PBMC supernatants. Culture supernatants obtained from the PBMC cultures described in Figs. 2 and 3 were analyzed using the LEGENDplex™ Human Immune Checkpoint Panel 1 assay. Quantification results are presented in the bar charts as the mean concentration of all three donors + SD. The difference in analyte concentrations across culture conditions was considered statistically significant for P-values <0.05, paired student’s t-test.
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student’s t-test) increases occurring between groups for CD25, 4-1BB, PD-L1, PD-L2, CD86, PD-1, Tim-3, and LAG-3. Generally these results agree with the flow cytometry data, except for the markers that displayed high donor to donor variation, indicating that the LEGENDplex™ assay can serve as a useful tool to interrogate immune checkpoint marker expression in biological samples. The key advantage of analyzing samples using multiplex immunoassays over ELISAs is their ability to simultaneously quantitate multiple targets in a single experiment. This is particularly advantageous when studying complex systems such as the tumor microenvironment, where biological sample volumes available to researchers are often limited. A better understanding of the complex interaction network of cytokines in cancer patients will certainly be critical to defining innovative therapeutic strategies (Costantini et al., 2010), but at the same time this also suggests that biological significance should not be inferred solely from observations of statistically significant concentration differences with respect to any singular analyte. To visually demonstrate the advantage multiplex analysis has over single target assays in evaluating cytokine networks, the results of both the data sets from Figs. 2 and 4 are presented as radar plots in Fig. 5. By representing the data in this format, researchers can easily compare not only the magnitude of single target responses across all experimental groups, but also the relative scale of these responses to those of other analytes in the multiplex assay.
11. Summary LEGENDplex™ assays offer researchers a multiplexed cytokine profiling platform that can be adapted to run on most flow cytometers. This chapter describes the technical details of these assays, and how they can be used as tools to quantitatively investigate the soluble mediators that help coordinate the human immune response in the tumor microenvironment. All LEGENDplex™ assays are fully validated with regards to analyte sensitivity, specificity, linearity of dilution, spike recovery, reproducibility, and detectability of natural target in relevant biological samples. The assays are also backed by a performance guarantee. While there are many commercially available multiplex assays available on the market, users should ensure that they critically evaluate all available validation data prior to selecting any particular platform for their research needs. As researchers seek to advance the promise of durable clinical responses to cancer immunotherapies in all patients, validated and reliable reagents will remain critical to their success.
Fig. 5 Radar plots of flow cytometry and LEGENDplex™ results. (A) The average percentage of cells positive for immune checkpoint marker expression in both treatment groups, as presented in Fig. 2, are presented on a radar plot. Unstimulated cells are denoted by a dashed line, stimulated cells with a solid line. (B) The results of the LEGENDplex™ analysis presented in Fig. 4 presented as a radar plot. Unstimulated cells are denoted by a dashed line, stimulated cells with a solid line. For both (A) and (B) paired student t-test P-values 0.05 are represented as *, 0.01 as **, 0.001 as ***.
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Acknowledgments The authors wish to thank the following BioLegend groups and employees for their technical expertise and input during the drafting of this chapter: The biomarkers and immunoassays product development team, the flow cytometry quality control group, Kellie M. Johnson, and Dr. Ekaterina Zvezdova.
Disclosures The authors are employed by BioLegend, which manufactures the LEGENDplex™ and flow cytometry reagents described in this manuscript.
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