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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 pa....

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 (.......

Acknowledgements

The authors would like to thank all current and former members of the Blish Laboratory who contributed to this panel. Thank you to the AIDS Clinical Trials Group and the ACTG A5321 team as well as Dr. Sandra López-Vergès and Davis Beltrán at Gorgas Memorial Institute for Health Studies for sample curation. Finally, thank you to Michael Leipold, Holden Maecker, and the Stanford Human Immune Monitoring Center for use of their Helios machines. This work was supported by NIH U19AI057229, NIH R21 AI135287, NIH R21 AI130532, NIH DP1 DA046089, and Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687 to CB, NIH Ruth L. ....

Materials

NameCompanyCatalog NumberComments
89YSigma-Aldrich204919
102-Palladium nitrateTrace Sciences InternationalSpecial Order
104-Palladium nitrateTrace Sciences InternationalSpecial Order
106-Palladium nitrateTrace Sciences InternationalSpecial Order
108-Palladium nitrateTrace Sciences InternationalSpecial Order
115InTrace Sciences InternationalSpecial Order
141PrFluidigm201141A
142NdFluidigm201142A
143NdFluidigm201143A
144NdFluidigm201144A
145NdFluidigm201145A
146NdFluidigm201146A
147SmFluidigm201147A
148NdFluidigm201148A
149SmFluidigm201149A
150NdFluidigm201150A
151EuFluidigm201151A
152SmFluidigm201152A
153EuFluidigm201153A
154SmFluidigm201154A
155GdFluidigm201155A
156GdFluidigm201156A
157GdTrace Sciences InternationalN/A
158GdFluidigm201158A
159TbFluidigm201159A
160GdFluidigm201160A
161DyFluidigm201161A
162DyFluidigm201162A
163DyFluidigm201163A
164DyFluidigm201164A
165HoFluidigm201165A
166ErFluidigm201166A
167ErFluidigm201167A
168ErFluidigm201168A
169TmFluidigm201169A
170ErFluidigm201170A
171YbFluidigm201171A
172YbFluidigm201172A
173YbFluidigm201173A
174YbFluidigm201174A
175LuFluidigm201175A
176YbFluidigm201176A
209Bi anti-CD16Fluidigm3209002BClone 3G8. Used at a 1:50 dilution. 
697 cellsCreative BioarrayCSC-C0217
Amicon Ultra Centrifugal Filter Units 0.5 with Ultracel-30 Membrane, 30 kDaMilliporeUFC503096
Anhydrous acetonitrileFisher ScientificBP1165-50
anti-2B4Biolegend329502Clone C1.7.
anti-B7-H6R&D SystemsMAB7144Clone 875001.
anti-CCR2Biolegend357202Clone K036C2.
anti-CD2Biolegend300202Clone RPA-2.10.
anti-CD3Biolegend300402Clone UCHT1.
anti-CD4Biolegend317402Clone OKT4.
anti-CD4Biolegend344602Clone SK3.
anti-CD7Biolegend343102Clone CD7-6B7.
anti-CD8Biolegend344702Clone SK1.
anti-CD11bBiolegend301302Clone ICRF44.
anti-CD14Biolegend301802Clone M5E2.
anti-CD19Biolegend302202Clone HIB19.
anti-CD33Biolegend303402Clone WM53.
anti-CD38Biolegend303502Clone HIT2.
anti-CD48Biolegend336702Clone BJ40.
anti-CD56BD Pharmingen559043Clone NCAM16.2.
anti-CD57Biolegend322302Clone HCD57.
anti-CD62LBiolegend304802Clone DREG-56.
anti-CD69Biolegend310902Clone FN50.
anti-CD94Biolegend305502Clone DX22.
anti-CD95Biolegend305602Clone DX2.
anti-CD155Biolegend337602Clone SKII.4.
anti-CXCR6Biolegend356002Clone K041E5.
anti-DNAM-1BD Biosciences559787Clone DX11.
anti-DR4Biolegend307202Clone DJR1.
anti-DR5Biolegend307302Clone DJR2-2.
anti-FAS-LBiolegend306402Clone NOK-1.
anti-FcRgMillipore06-727Polyclonal antibody.
anti-HLA-C,EMilliporeMABF233Clone DT9.
anti-HLA-Bw4Miltenyi BiotecSpecial OrderClone REA274.
anti-HLA-Bw6Miltenyi Biotec130-124-530Clone REA143.
anti-HLA-DRBiolegend307602Clone L243.
anti-HLA-EBiolegend342602Clone 3D12.
anti-ICAM-1Biolegend353102Clone HA58.
anti-Ki-67Biolegend350502Clone Ki-67.
anti-KIR2DL1/KIR2DS5R&D SystemsMAB1844Clone 143211.
anti-KIR2DL3R&D SystemsMAB2014Clone 180701.
anti-KIR2DL5Miltenyi Biotec130-096-200Clone UP-R1.
anti-KIR2DS4R&D SystemsMAB1847Clone 179315.
anti-KIR3DL1BD Biosciences555964Clone DX-9.
anti-LFA-3Biolegend330902Clone TS2/9.
anti-LILRB1R&D Systems292319Clone MAB20172.
anti-LLT-1R&D SystemsAF3480Clone 402659.
anti-MICAR&D SystemsMAB1300-100Clone 159227.
anti-MICBR&D SystemsMAB1599-100Clone 236511.
anti-Nectin-1Biolegend340402Clone R1.302.
anti-Nectin-2Biolegend337402Clone TX31.
anti-NKG2AR&D SystemsMAB1059Clone 131411.
anti-NKG2CR&D SystemsMAB1381Clone 134522.
anti-NKG2DBiolegend320802Clone 1D11.
anti-NKp30Biolegend325202Clone P30-15.
anti-NKp44Biolegend325102Clone P44-8.
anti-NKp46Biolegend331902Clone 9E2.
anti-NTB-ABiolegend317202Clone NT-7.
anti-Pan HLA class IBiolegend311402Clone W6/32.
anti-PD1Biolegend329902Clone EH12.2H7.
anti-PerforinAbcamab47225Clone B-D48.
anti-Siglec-7Biolegend347702Clone S7.7.
anti-SykBiolegend644302Clone 4D10.2.
anti-TACTILEBiolegend338402Clone NK92.39.
anti-TIGITR&D SystemsMAB7898Clone 741182.
anti-ULBP-1R&D SystemsMAB1380-100Clone 170818.
anti-ULBP-2, 5, 6R&D SystemsMAB1298-100Clone 165903.
Antibody StabilizerCandor Bioscience131 050
Benzonase NucleaseMillipore70664
Bond-Breaker TCEP SolutionThermo Fisher Scientific77720
Bovine Serum Albumin solutionSigma-AldrichA9576
Calcium chloride dihydrate (CaCl2+2H2O)Sigma-Aldrich223506-25G
Cis-Platinum(II)diamine dichloride (cisplatin)Enzo Life SciencesALX-400-040-M250A 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.
DMSOSigma-AldrichD2650
eBioscience Permeabilization BufferThermo Fisher Scientific00-8333-56
EDTA (0.5 M)HoeferGR123-100A 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 BeadsFluidigm201078
Fetal Bovine SerumThermo Fisher ScientificN/A
Helios mass cytometerFluidigmN/A
HEPES (1M)Thermo Fisher Scientific15630080
HyClone Antibiotic/Antimycotic Solution (Pen/Strep/Fungiezone) solutionFisher ScientificSV3007901
Iridium - 191Ir/193Ir intercalatorDVS Sciences (Fluidigm)201192BUsed at a 1:10000 dilution.
Isothiocyanobenzyl-EDTA (ITCB-EDTA)Dojindo Molecular Technologies, Inc.M030-10Diluted to 1.25 mg/mL in anhydrous acetonitrile.
K562 cellsAmerican Type Culture Collection (ATCC)ATCC CCL-243
L-Glutamine (200 mM)Thermo Fisher ScientificSH30034
Magnesium chloride (MgCl2)Sigma-Aldrich208337-100G
Maxpar X8 Antibody Labeling KitsFluidigmN/ANo catalog number as kits come with metals. 
Millex-VV Syringe Filter Unit, 0.1 µmMilliporeSLVV033RS
Milli-Q Advantage A10 Water Purification SystemMilliporeZ00Q0V0WW
MS ColumnsMiltenyi Biotec
NALM6 cellsAmerican Type Culture Collection (ATCC)ATCC CRL-3273
Nanosep Centrifugal Devices with Omega Membrane 3KPall CorporationOD003C35
NK Cell Isolation Kit, humanMiltenyi Biotec130-092-657
Paraformaldehyde (16%)Electron Microscopy Sciences15710
PBSThermo Fisher Scientific10010023
Potassium Phosphate Monobasic (KH2PO4)Fisher ScientificMP021954531
Qdot 655 anti-CD19Thermo Fisher ScientificQ10179Clone SJ25-C1. Used at a 1:50 dilution. Signal appears in 112Cd-114Cd channels. 
Qdot 655 anti-HLA-DRThermo Fisher ScientificQ22158Clone Tü36. Used at a 1:200 dilution.
Rockland PBSRockland 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 1640Thermo Fisher Scientific21870092
Sodium azide (NaN3)Sigma-AldrichS2002
Sodium chloride (NaCl)Fisher ScientificS271-500
Stericup Quick Release-GP Sterile Vacuum Filtration SystemMillipore SigmaS2GPU10RE
Tuning solutionFluidigm201072
Washing solution Fluidigm201070

References

  1. Shimasaki, N., Jain, A., Campana, D. NK cells for cancer immunotherapy. Nature Reviews. Drug Discovery. 19 (3), 200-218 (2020).
  2. Vivier, E., Tomasello, E., Baratin, M., Walzer, T., Ugolini, S. Functions of natural killer cells. Nature Immunology.....

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Natural Killer CellsNK Cell ReceptorsNK Cell LigandsMass CytometryCyTOFVirus InfectionImmune Cell ProfilingReceptor ligand Repertoire

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