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 comm…

Representative 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

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