Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Summary

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

Abstract

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells is regulated by their expression of germline-encoded inhibitory and activating receptors. The engagement of these receptors by their cognate ligands on target cells determines whether the intercellular interaction will result in NK cell killing. This protocol details the design and optimization of two complementary mass cytometry (CyTOF) panels. One panel was designed to phenotype NK cells based on receptor expression. The other panel was designed to interrogate expression of known ligands for NK cell receptors on several immune cell subsets. Together, these two panels allow for the profiling of the human NK cell receptor-ligand repertoire. Furthermore, this protocol also details the process by which we stain samples for CyTOF. This process has been optimized for improved reproducibility and standardization. An advantage of CyTOF is its ability to measure over 40 markers in each panel, with minimal signal overlap, allowing researchers to capture the breadth of the NK cell receptor-ligand repertoire. Palladium barcoding also reduces inter-sample variation, as well as consumption of reagents, making it easier to stain samples with each panel in parallel. Limitations of this protocol include the relatively low throughput of CyTOF and the inability to recover cells after analysis. These panels were designed for the analysis of clinical samples from patients suffering from acute and chronic viral infections, including dengue virus, human immunodeficiency virus (HIV), and influenza. However, they can be utilized in any setting to investigate the human NK cell receptor-ligand repertoire. Importantly, these methods can be applied broadly to the design and execution of future CyTOF panels.

Introduction

Natural killer (NK) cells are innate immune cells whose primary role is to target and kill malignant, infected, or otherwise stressed cells. Through their secretion of cytokines such as IFNγ and TNFα, as well as their cytotoxic activity, NK cells can also shape the adaptive immune response to pathogens and malignancies. The NK response is mediated in part by the combinatorial signaling of germline-encoded inhibitory and activating receptors, which bind a myriad of ligands expressed on potential target cells. Several NK cell receptors have more than one ligand with new receptor-ligand pairs being identified regularly.

There is a particular interest in studying NK cells in the context of viral infections, where their ability to rapidly respond to stressed cells may limit viral spread or promote the development of NK cell evasion strategies. This interest in NK cell biology extends to the field of cancer immunotherapy where researchers are investigating the role of NK cells in tumor immunosurveillance and in the tumor microenvironment¹. However, the ability to profile NK cell-target cell interactions is complicated by the fact that human NK cells can express over 30 receptors which in turn can interact with over 30 known ligands². The simultaneous detection of multiple NK cell receptors and their cognate ligands is, therefore, necessary to capture the complexity of the receptor-ligand interactions that control NK function. Consequently, we turned to mass cytometry (CyTOF), which allows for the simultaneous detection of over 40 markers at the single cell level. Our goal was to create two CyTOF panels to profile the NK cell receptor-ligand repertoire. We also wanted to design a protocol for effective processing and staining of clinical samples. Clinical human samples provide a wealth of information on how the body responds to viral infection. Therefore, we developed this protocol to investigate expression of NK cell receptors and their cognate ligands in parallel for better standardization, improved recovery, reduced reagent consumption, and limited batch effects.

Several flow cytometry panels designed to characterize the phenotype of human NK cells have been published previously^3,4,5,6,7,8. Most of these panels are limited in their ability to capture the breadth of the receptor-ligand repertoire, only allowing for the detection of a limited selection of markers. Moreover, these panels are limited by signal overlap between fluorochromes. CyTOF uses antibodies conjugated to metal isotopes, which are read out by time-of-flight mass spectrometry, thus dramatically reducing spillover between channels.

Like us, other researchers have turned to CyTOF to study NK cells^{9,10,11,12,13,14}, though generally with fewer NK cell markers, which reduces the depth of phenotyping. While the general staining protocols used by these groups are similar to ours, there are some key differences. Other protocols do not involve isolating NK cells prior to staining even though the researchers are only interested in that subset^13,14. Given that NK cells only make up 5-20% of peripheral blood mononuclear cells (PBMCs), staining whole PBMCs rather than isolated NK cells means that most of the collected events will not be NK cells. This reduces the amount of data generated on the subset of interest and results in inefficient use of machine time. Additionally, while many of these panels interrogate expression of NK cell receptors such as killer Ig-like receptors (KIRs), NKG2A/C/D, and the natural cytotoxicity receptors (NKp30, NKp44, and NKp46), expression of these markers is not put into a broader context due to the absence of data on expression of their respective ligands. Consequently, while these previously published methods for investigating NK cells via CyTOF are sufficient for broad NK cell phenotyping, used in isolation, they cannot provide a comprehensive picture of NK cell activity. This brings us to the major advantage of the methods described here, which is that up to this point there are no published flow cytometry or CyTOF panels focused on exploring the expression of ligands for NK cell receptors. Importantly, our ligand panel has several open channels to allow for the addition of markers to suit the unique needs of each experiment.

Considering that one of the main limitations of CyTOF is the inability to recover the sample after analysis, this method may not be appropriate for researchers who have limited samples with which they are interested in performing additional experiments. Additionally, the low throughput nature of CyTOF means that the data generated will be of poor quality if the starting number of cells is low. Barring these two limitations, this method will perform well in any setting to investigate receptor-ligand interactions between NK cells and target cells.

Protocol

Anonymized healthy adult PBMCs were obtained from leukoreduction system chambers purchased from the Stanford Blood Center. PBMCs from de-identified healthy pediatric donors and pediatric acute dengue patients were obtained from Gorgas Memorial Institute of Health Studies in Panama City, Panama and hospitals belonging to the Ministry of Health, the Social Security System in Panama City, and suburban areas. The dengue study protocol was approved by the IRB of Hospital del Niño (CBIHN-M-0634), then approved by the committees of ICGES, CSS, Santo Tomas Hospital, and Stanford University. PBMCs from HIV-infected patients on antiretroviral treatment were obtained from ACTG study A5321.

1. Antibody labeling, panel preparation, and storage

1. Antibody labeling with metal isotopes
   ​NOTE: To increase inter-experimental standardization of staining, it is recommended to perform multiple conjugations for each antibody and then combine the products into a single master mix for long-term storage as described below.
   1. Determine the concentration of each antibody by measuring the absorbance at 280 nm prior to conjugation. Antibodies used for this protocol are commercially available and were purchased from the vendors listed in the Table of Materials.
   2. Label antibodies with metal isotopes using commercially available antibody labeling kits according to the manufacturer's instructions. Use 100 µg of antibody for each reaction.
   3. Determine the final concentration of the recovered antibody by measuring the absorbance at 280 nm. Store antibodies for short-term at 4 °C.
2. Antibody titrations
   ​NOTE: CyTOF technology is very sensitive to potential contaminating signals from environmental metals. Therefore, all buffers/reagents used should be prepared with ultrapure water and stored in plastic or glass containers that have never been washed with soap.
   1. Prepare centrifuge tubes for each donor containing 9 mL of warm, complete RPMI (RPMI 1640, 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin) and 20 µL of benzonase per vial of PBMCs to be thawed. Thaw the PBMCs in a water bath and add to tubes.
      ​NOTE: Benzonase decreases viscosity and background from free DNA from lysed cells.
   2. Centrifuge at 300 x g at room temperature for 5 min. Resuspend the PBMCs in 5 mL of complete RPMI media and count.
   3. For each panel titration, plate 2-4 million PBMCs/well in 6 wells of a round bottom 96-well plate (one well for each titer and one for unstained). Centrifuge the plate at 600 x g at room temperature for 3 min. Flick the plate to remove the supernatant. Resuspend each well in 200 µL of CyPBS.
   4. Perform cisplatin viability staining as described below.
      ​NOTE: Cisplatin is used to discriminate live from dead cells in mass cytometry.
      1. Resuspend cells in 100 µL of 25 µM cisplatin stock. Incubate at room temperature for 1 min.
      2. Quench the cisplatin reaction by adding 100 µL of FBS to each well and mixing. Centrifuge and flick the plate.
         NOTE: Perform all subsequent centrifuge steps at 4 °C.
      3. Wash cells twice with 200 µL of CyFACS (1x PBS without heavy metal contaminants in ultrapure water with 0.1% BSA, 0.05% sodium azide). Centrifuge and flick the plate each time.
   5. Titrate the surface antibody panel as described below.
      ​NOTE: Separate master mixes should be made for the NK surface panel and the ligand panel.
      1. Make a master mix of all the surface antibodies at a concentration of 10 µg/mL using CyFACS. Aim for a final volume of 150 µL. Make serial 1:2 dilutions using CyFACS, to obtain the following concentrations: 10, 5, 2.5, 1.25, and 0.625 µg/mL.
      2. Filter antibody cocktails through a centrifugal filter unit (0.1 µm pore size) at 10,600 x g for 3 min prior to staining.
      3. Resuspend the plated cells in 50 µL of the surface antibody cocktail at the respective titer. Resuspend the unstained well in CyFACS. Incubate at 4 °C for 30 min.
      4. Wash cells with 150 µL of CyFACS. Centrifuge and flick the plate.
      5. Wash cells with 200 µL of CyFACS. Centrifuge and flick the plate.
   6. Perform fixation of cells by resuspending each well in 100 µL of 2% paraformaldehyde (PFA) in CyPBS. Incubate the plate at room temperature in the dark for 20 min. Wash cells with 100 µL of CyFACS. Centrifuge at 700 x g for 5 min.
      CAUTION: PFA is suspected of causing genetic defects as well as cancer. Additionally, it is harmful if it gets in contact with the eyes, skin, or is inhaled. Handle appropriately by ensuring good ventilation, opening the receptacle carefully, and preventing the formation of aerosols.
      NOTE: All subsequent centrifuge spins are performed at 700 x g for 5 min at 4 °C.
   7. Permeabilize cells by resuspending in 200 µL of 1x Permeabilization Buffer (Perm buffer) diluted in ultrapure water. Centrifuge and flick the plate. Wash cells with 200 µL of Perm buffer. Centrifuge and flick the plate.
      NOTE: Incubation in the Perm buffer is not necessary.
   8. Intracellular antibody panel titration
      1. Make a master mix of all the intracellular antibodies at a concentration of 10 µg/mL using Perm Buffer. Aim for a final volume of 150 µL. Make serial 1:2 dilutions using Perm Buffer to obtain the following concentrations: 10, 5, 2.5, 1.25, and 0.625 µg/mL.
      2. Filter antibody cocktails through a centrifugal filter unit (0.1 µm pore size) at 10,600 x g for 3 min prior to staining.
      3. Resuspend the plated cells in 50 µL of the intracellular antibody cocktail at the respective titer. Resuspend the unstained well in Perm Buffer. Incubate at 4 °C for 45 min.
         NOTE: If an intracellular panel is not going to be titrated, resuspend wells in 50 µL of the Perm buffer.
      4. Wash cells with 150 µL of Perm buffer. Centrifuge and flick the plate.
      5. Wash cells with 200 µL of Perm buffer. Centrifuge and flick the plate.
      6. Wash cells twice with 200 µL of CyFACS. Centrifuge and flick the plate.
   9. DNA intercalator staining
      ​NOTE: Intercalator binds to cellular nucleic acid and is used to identify nucleated cells in mass cytometry.
      1. Resuspend cells in 200 µL of intercalator diluted 1:10,000 in CyPBS and 2% PFA. Incubate the plate overnight at 4 °C.
      2. Store plates, if needed, at 4 °C covered with paraffin film for up to a week.
         NOTE: Perform all subsequent centrifuge steps at 700 x g for 5 min at 4 °C.
      3. Before running the samples on CyTOF, remove the paraffin film from the plate and centrifuge at 700 x g for 5 min at 4 °C. Flick the plate. Wash cells once with 200 µL of CyFACS. Centrifuge and flick the plate.
      4. Wash cells three times with 200 µL of ultrapure water. Centrifuge and flick the plate. Resuspend cells in 200 µL of ultrapure water. Immediately before running the sample, adjust the concentration to approximately 6 x 10⁵ cells/mL in normalization beads diluted to a 1x concentration in ultrapure water.
   10. Run the samples on CyTOF.
   11. Analyze data and choose appropriate titers for each antibody by selecting the lowest antibody titer which results in the highest signal intensity and the best separation between positive and negative populations based on visual assessment.
       NOTE: Titrations for the NK and ligand panels are shown in Figure 1 and Figure 2 respectively. If a clear distinction between positive and negative populations is not identified, titers can be assessed on multiple cell types or on cell lines, to allow for identification of both positive and negative cell populations.
   12. Antibody panel storage
       1. Combine titrated antibodies into a master mix and filter through a sterile 0.1 µm syringe filter unit. Separate master mixes should be made for the NK surface panel, the NK intracellular panel, and the ligand panel.
       2. For the long-term storage of panels follow one of the two acceptable options:
       3. Send master mix to a third-party company for lyophilization. This method is used for the NK panel. Antibodies not conjugated in-house cannot be lyophilized, due to the presence of antibody stabilizer, which interferes with the lyophilization process. These antibodies are added to the panel on the day of staining.
       4. Use a repeater pipette to make 50 µL aliquots of each master mix and store them at -80 °C.

2. Staining protocol

1. Thaw peripheral blood mononuclear cells (PBMCs) as described in steps 1.2.1 and 1.2.2. Set aside at least 1 million PBMCs for ligand panel staining in a 15 mL centrifuge tube. Keep these PBMCs on ice during the NK cell isolation.
2. NK cell isolation
   ​NOTE: The following NK cell isolation steps are a modified version of a specific vendor's protocol. However, any kit or protocol that performs magnetic-based negative selection of NK cells would be a suitable alternative. Additionally, this step is optional as this protocol is also suitable for the phenotyping of NK cells from whole PBMCs.
   1. Spin the remaining PBMCs at 450 x g for 5 min. Resuspend cell pellet in 40 µL of MACS buffer (PBS, 0.5% BSA, 2 mM EDTA) per 10⁷ total cells.
   2. Add 10 µL of NK cell Biotin-Antibody Cocktail per 10⁷ total cells. Mix well and incubate for 5 min on ice.
   3. Add 30 µL of MACS buffer per 10⁷ total cells and 20 µL of NK cell MicroBead Cocktail per 10⁷ total cells. Mix well and incubate for 10 min on ice.
   4. Prepare elution columns by rinsing with 500 µL of MACS buffer. Add 2 mL of cold complete RPMI to 15 mL collection tubes.
   5. Add MACS buffer to tubes containing cells to bring the volume up to 500 µL. Pipette the entire 500 µL volume onto the prepared elution column. Rinse out the tube with another 500 µL of MACS buffer and transfer to the column.
   6. After flow has stopped, rinse the elution column with 500 µL MACS buffer twice. After flow has stopped, count NK cells.
3. Plate and wash cells.
   1. Centrifuge isolated NK cells and PBMCs at 300 x g at room temperature for 10 min. Resuspend cells at a concentration of 5 million cells/mL in CyPBS (1x PBS without heavy metal contaminants in ultrapure water). Plate cells in a U-bottom, 96-well plate.
      NOTE: Each aliquot of the NK cell panel can stain up to 3 million cells. If six individual NK cell samples are being barcoded and pooled prior to staining, the combined total number of NK cells should not exceed 3 million. The ligand panel can stain up to 6 million PBMCs per sample. If six individual samples are being barcoded and pooled prior to staining, the combined total number of PBMCs should not exceed 6 million.
   2. Centrifuge plate at 600 x g at room temperature for 3 min. Flick the plate to remove supernatant.
      ​NOTE: Perform all subsequent centrifuge spins at 600 x g for 3 min until step 2.7.
   3. Resuspend cells in 200 µL of CyPBS. Centrifuge and flick the plate.
4. Perform cisplatin viability staining as described in step 1.2.4.
5. Barcoding staining
   ​NOTE: These panels are used in conjunction with a modified two-of-four, Palladium-based barcoding method on live unfixed cells to minimize batch effects and maximize cell recovery¹⁵. However, this step is optional as barcoding is not necessary to obtain quality data.
   1. Resuspend each well in 50 µL of respective premixed barcode and incubate at 4 °C for 30 min. Wash cells with 150 µL of CyFACS. Centrifuge and flick the plate.
   2. Wash cells twice with 200 µL of CyFACS. Centrifuge and flick the plate. Resuspend all wells in 30 µL of CyFACS. Combine up to six wells of cells stained with unique barcodes into one well and perform centrifugation and flicking of the plate.
6. Surface staining
   1. Dissolve the surface NK panel lyosphere in 50 µL of CyFACS with additional surface antibodies spiked in (anti-CD16, anti-HLA-DR, anti-LILRB1). Thaw ligand panel stored at -80 °C and spin down tube using a mini-centrifuge. Spike in additional ligand panel surface markers (anti-CD16, anti-CD19).
      NOTE: Any antibody cocktail that has not been previously filtered (i.e., prior to lyophilization or freezing) should be filtered through a centrifugal filter unit (0.1 µm pore size) at 10,600 x g for 3 min prior to staining.
   2. Resuspend each well in 50 µL of the respective panel. Incubate at 4 °C for 30 min. Wash cells with 150 µL of CyFACS. Centrifuge and flick the plate.Wash cells again with 200 µL of CyFACS. Centrifuge and flick the plate.
7. Fix cells as described in step 1.2.6.
   ​NOTE: Perform all subsequent centrifuge spins at 700 x g for 5 min at 4 °C.
8. Permeabilize cells as described in step 1.2.7.
9. Intracellular staining
   1. Dissolve the intracellular NK panel lyosphere in 50 µL of Perm buffer. Prepare an intracellular antibody cocktail for PBMC samples if so desired.
      ​NOTE: Any antibody cocktail that has not been previously filtered (i.e., prior to lyophilization or freezing) should be filtered through a centrifugal filter unit (0.1 µm pore size) at 10,600 x g for 3 min prior to staining.
   2. Resuspend wells in 50 µL of the respective intracellular panels. If an intracellular panel is not used in conjunction with the ligand surface panel, resuspend PBMC wells in 50 µL of the Perm buffer. Incubate at 4 °C for 45 min.
   3. Wash cells with 150 µL of Perm buffer. Centrifuge and flick plate. Wash cells with 200 µL of Perm buffer. Centrifuge and flick the plate.
   4. Wash cells twice with 200 µL of CyFACS. Centrifuge and flick the plate.
10. DNA intercalator staining. Perform DNA intercalator staining as described in step 1.2.9.1. Incubate plate overnight at 4 °C.
    NOTE: Intercalator binds to cellular nucleic acid and is used to identify nucleated cells in mass cytometry. Plates can be stored covered with paraffin film for up to a week at 4 °C.
11. Before running the samples on CyTOF, wash cells as described in steps 1.2.9.3 and 1.2.9.4.
12. Run samples on CyTOF.

Results

Antibodies were conjugated to metal isotopes using commercially available labeling kits, according to the manufacturer's instructions. Antibody clones were validated by flow cytometry and mass cytometry prior to use in this panel. An initial list of clones was selected based on review of the literature and antibody availability. The expression levels of some ligands for NK cell receptors are low or undetectable on healthy PBMCs. Therefore, positive staining for some antibodies was val...

Discussion

Here we describe the design and application of two complimentary CyTOF panels aimed at profiling the NK cell receptor-ligand repertoire. This protocol includes several steps that are critical to obtaining quality data. CyTOF uses heavy metal ions, rather than fluorochromes, as label probes for antibodies¹⁹. This technology is therefore subject to potential contaminating signals from environmental metals²⁰. Potential sources of metal impurities include laboratory dish soap (...

Materials

Name	Company	Catalog Number	Comments
89Y	Sigma-Aldrich	204919
102-Palladium nitrate	Trace Sciences International	Special Order
104-Palladium nitrate	Trace Sciences International	Special Order
106-Palladium nitrate	Trace Sciences International	Special Order
108-Palladium nitrate	Trace Sciences International	Special Order
115In	Trace Sciences International	Special Order
141Pr	Fluidigm	201141A
142Nd	Fluidigm	201142A
143Nd	Fluidigm	201143A
144Nd	Fluidigm	201144A
145Nd	Fluidigm	201145A
146Nd	Fluidigm	201146A
147Sm	Fluidigm	201147A
148Nd	Fluidigm	201148A
149Sm	Fluidigm	201149A
150Nd	Fluidigm	201150A
151Eu	Fluidigm	201151A
152Sm	Fluidigm	201152A
153Eu	Fluidigm	201153A
154Sm	Fluidigm	201154A
155Gd	Fluidigm	201155A
156Gd	Fluidigm	201156A
157Gd	Trace Sciences International	N/A
158Gd	Fluidigm	201158A
159Tb	Fluidigm	201159A
160Gd	Fluidigm	201160A
161Dy	Fluidigm	201161A
162Dy	Fluidigm	201162A
163Dy	Fluidigm	201163A
164Dy	Fluidigm	201164A
165Ho	Fluidigm	201165A
166Er	Fluidigm	201166A
167Er	Fluidigm	201167A
168Er	Fluidigm	201168A
169Tm	Fluidigm	201169A
170Er	Fluidigm	201170A
171Yb	Fluidigm	201171A
172Yb	Fluidigm	201172A
173Yb	Fluidigm	201173A
174Yb	Fluidigm	201174A
175Lu	Fluidigm	201175A
176Yb	Fluidigm	201176A
209Bi anti-CD16	Fluidigm	3209002B	Clone 3G8. Used at a 1:50 dilution.
697 cells	Creative Bioarray	CSC-C0217
Amicon Ultra Centrifugal Filter Units 0.5 with Ultracel-30 Membrane, 30 kDa	Millipore	UFC503096
Anhydrous acetonitrile	Fisher Scientific	BP1165-50
anti-2B4	Biolegend	329502	Clone C1.7.
anti-B7-H6	R&D Systems	MAB7144	Clone 875001.
anti-CCR2	Biolegend	357202	Clone K036C2.
anti-CD2	Biolegend	300202	Clone RPA-2.10.
anti-CD3	Biolegend	300402	Clone UCHT1.
anti-CD4	Biolegend	317402	Clone OKT4.
anti-CD4	Biolegend	344602	Clone SK3.
anti-CD7	Biolegend	343102	Clone CD7-6B7.
anti-CD8	Biolegend	344702	Clone SK1.
anti-CD11b	Biolegend	301302	Clone ICRF44.
anti-CD14	Biolegend	301802	Clone M5E2.
anti-CD19	Biolegend	302202	Clone HIB19.
anti-CD33	Biolegend	303402	Clone WM53.
anti-CD38	Biolegend	303502	Clone HIT2.
anti-CD48	Biolegend	336702	Clone BJ40.
anti-CD56	BD Pharmingen	559043	Clone NCAM16.2.
anti-CD57	Biolegend	322302	Clone HCD57.
anti-CD62L	Biolegend	304802	Clone DREG-56.
anti-CD69	Biolegend	310902	Clone FN50.
anti-CD94	Biolegend	305502	Clone DX22.
anti-CD95	Biolegend	305602	Clone DX2.
anti-CD155	Biolegend	337602	Clone SKII.4.
anti-CXCR6	Biolegend	356002	Clone K041E5.
anti-DNAM-1	BD Biosciences	559787	Clone DX11.
anti-DR4	Biolegend	307202	Clone DJR1.
anti-DR5	Biolegend	307302	Clone DJR2-2.
anti-FAS-L	Biolegend	306402	Clone NOK-1.
anti-FcRg	Millipore	06-727	Polyclonal antibody.
anti-HLA-C,E	Millipore	MABF233	Clone DT9.
anti-HLA-Bw4	Miltenyi Biotec	Special Order	Clone REA274.
anti-HLA-Bw6	Miltenyi Biotec	130-124-530	Clone REA143.
anti-HLA-DR	Biolegend	307602	Clone L243.
anti-HLA-E	Biolegend	342602	Clone 3D12.
anti-ICAM-1	Biolegend	353102	Clone HA58.
anti-Ki-67	Biolegend	350502	Clone Ki-67.
anti-KIR2DL1/KIR2DS5	R&D Systems	MAB1844	Clone 143211.
anti-KIR2DL3	R&D Systems	MAB2014	Clone 180701.
anti-KIR2DL5	Miltenyi Biotec	130-096-200	Clone UP-R1.
anti-KIR2DS4	R&D Systems	MAB1847	Clone 179315.
anti-KIR3DL1	BD Biosciences	555964	Clone DX-9.
anti-LFA-3	Biolegend	330902	Clone TS2/9.
anti-LILRB1	R&D Systems	292319	Clone MAB20172.
anti-LLT-1	R&D Systems	AF3480	Clone 402659.
anti-MICA	R&D Systems	MAB1300-100	Clone 159227.
anti-MICB	R&D Systems	MAB1599-100	Clone 236511.
anti-Nectin-1	Biolegend	340402	Clone R1.302.
anti-Nectin-2	Biolegend	337402	Clone TX31.
anti-NKG2A	R&D Systems	MAB1059	Clone 131411.
anti-NKG2C	R&D Systems	MAB1381	Clone 134522.
anti-NKG2D	Biolegend	320802	Clone 1D11.
anti-NKp30	Biolegend	325202	Clone P30-15.
anti-NKp44	Biolegend	325102	Clone P44-8.
anti-NKp46	Biolegend	331902	Clone 9E2.
anti-NTB-A	Biolegend	317202	Clone NT-7.
anti-Pan HLA class I	Biolegend	311402	Clone W6/32.
anti-PD1	Biolegend	329902	Clone EH12.2H7.
anti-Perforin	Abcam	ab47225	Clone B-D48.
anti-Siglec-7	Biolegend	347702	Clone S7.7.
anti-Syk	Biolegend	644302	Clone 4D10.2.
anti-TACTILE	Biolegend	338402	Clone NK92.39.
anti-TIGIT	R&D Systems	MAB7898	Clone 741182.
anti-ULBP-1	R&D Systems	MAB1380-100	Clone 170818.
anti-ULBP-2, 5, 6	R&D Systems	MAB1298-100	Clone 165903.
Antibody Stabilizer	Candor Bioscience	131 050
Benzonase Nuclease	Millipore	70664
Bond-Breaker TCEP Solution	Thermo Fisher Scientific	77720
Bovine Serum Albumin solution	Sigma-Aldrich	A9576
Calcium chloride dihydrate (CaCl2+2H2O)	Sigma-Aldrich	223506-25G
Cis-Platinum(II)diamine dichloride (cisplatin)	Enzo Life Sciences	ALX-400-040-M250	A 100 mM stock solution was prepared in DMSO and divided into 25 µL aliquots. Used at a 25 µM dilution for live/dead stain. Signal appears in 194Pt and 195Pt channels.
DMSO	Sigma-Aldrich	D2650
eBioscience Permeabilization Buffer	Thermo Fisher Scientific	00-8333-56
EDTA (0.5 M)	Hoefer	GR123-100	A double-concentrated HEPES buffer with EDTA was made according to the following recipe: 1.3 g NaCl (Thermo Fisher Scientific), 27 mg CaCl2+2H2O (Sigma-Aldrich), 23 mg MgCl2 (Sigma-Aldrich), 83.6 mg KH2PO4 (Thermo Fisher Scientific), 4 mL of 1M HEPES (Thermo Fisher Scientific), 2 mL of 0.5M EDTA (Hoefer, Holliston, MA, USA), and 100mL H2O. The pH of this double-concentrated HEPES buffer was adjusted to a pH of 7.3 using 1M HCl and 1M NaOH.
EQ Four Element Calibration Beads	Fluidigm	201078
Fetal Bovine Serum	Thermo Fisher Scientific	N/A
Helios mass cytometer	Fluidigm	N/A
HEPES (1M)	Thermo Fisher Scientific	15630080
HyClone Antibiotic/Antimycotic Solution (Pen/Strep/Fungiezone) solution	Fisher Scientific	SV3007901
Iridium - 191Ir/193Ir intercalator	DVS Sciences (Fluidigm)	201192B	Used at a 1:10000 dilution.
Isothiocyanobenzyl-EDTA (ITCB-EDTA)	Dojindo Molecular Technologies, Inc.	M030-10	Diluted to 1.25 mg/mL in anhydrous acetonitrile.
K562 cells	American Type Culture Collection (ATCC)	ATCC CCL-243
L-Glutamine (200 mM)	Thermo Fisher Scientific	SH30034
Magnesium chloride (MgCl2)	Sigma-Aldrich	208337-100G
Maxpar X8 Antibody Labeling Kits	Fluidigm	N/A	No catalog number as kits come with metals.
Millex-VV Syringe Filter Unit, 0.1 µm	Millipore	SLVV033RS
Milli-Q Advantage A10 Water Purification System	Millipore	Z00Q0V0WW
MS Columns	Miltenyi Biotec
NALM6 cells	American Type Culture Collection (ATCC)	ATCC CRL-3273
Nanosep Centrifugal Devices with Omega Membrane 3K	Pall Corporation	OD003C35
NK Cell Isolation Kit, human	Miltenyi Biotec	130-092-657
Paraformaldehyde (16%)	Electron Microscopy Sciences	15710
PBS	Thermo Fisher Scientific	10010023
Potassium Phosphate Monobasic (KH2PO4)	Fisher Scientific	MP021954531
Qdot 655 anti-CD19	Thermo Fisher Scientific	Q10179	Clone SJ25-C1. Used at a 1:50 dilution. Signal appears in 112Cd-114Cd channels.
Qdot 655 anti-HLA-DR	Thermo Fisher Scientific	Q22158	Clone Tü36. Used at a 1:200 dilution.
Rockland PBS	Rockland Immunochemicals, Inc.	MB-008	Used to make CyPBS (10X Rockland PBS diluted to 1X in Milli-Q water) and CyFACS buffers (10X Rockland PBS diluted to 1X in Milli-Q water with 0.1% BSA and 0.05% sodium azide). Buffers were sterile-filtered through a 0.22 µM filter and sotred at 4°C in Stericup bottles.
RPMI 1640	Thermo Fisher Scientific	21870092
Sodium azide (NaN3)	Sigma-Aldrich	S2002
Sodium chloride (NaCl)	Fisher Scientific	S271-500
Stericup Quick Release-GP Sterile Vacuum Filtration System	Millipore Sigma	S2GPU10RE
Tuning solution	Fluidigm	201072
Washing solution	Fluidigm	201070