Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

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

Summary

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Abstract

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

Introduction

NK cells are important effectors of the innate immune system that can recognize and eliminate malignant cells or stressed healthy cells (e.g., by a viral infection) without prior antigen stimulation1. This process is tightly regulated via a complex repertoire of activating receptors —such as natural cytotoxicity receptors (NCRs), NKG2D and CD16— and inhibitory receptors that are largely represented by killer immunoglobulin-like receptors (KIRs)2. Binding of KIRs to human leukocyte antigen (HLA) class I molecules on somatic cells ensures self-recognition and conveys NK cell tolerance. On the other hand, absenc....

Protocol

Patient samples were collected following approval from the Ethics Review Board of the University Hospitals of Basel and Tuebingen.

1. Biotinylation of the NKG2D fusion protein

NOTE: This step is performed with a biotinylation kit (see Table of Materials) according to the manufacturer’s instruction. This step of the protocol must be performed at least 24 h prior to the staining. The biotinylated NKG2D fusion protein should be stored at -20 °.......

Representative Results

Both the protocols presented here allow the enrichment of AML LSC by flow cytometric analyses using CD34, a known marker of LSC17, in combination with NKG2DL surface expression by either utilizing pan-ligand recognition or staining with pooled antibodies against individual ligands. In Figure 1, we show that the analyzed AML samples are positive for CD34 and NKG2DL, but negative subpopulations also exist, which is shown by the presence of four different populations in .......

Discussion

Here we present two flow cytometric methods that can detect NKG2DL surface expression on human primary AML cells. We show that both detection methods can be used in conjunction with other antibody stainings (e.g., detecting CD34 expression). Similar stainings may also be performed on other primary cell types and cell lines.

We recently showed that the absence of NKG2DL on the surface of AML patient blasts can enrich LSC14. In AML, NKG2DL negative but not NKG2DL positive.......

Acknowledgements

This study was supported by grants from the Swiss National Science Foundation (179239), the Foundation for Fight Against Cancer (Zuerich), the Wilhelm Sander Foundation to CL (2019.042.1), and the Novartis Foundation for medical-biological research to C.L. Furthermore, this project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 765104. We thank the Flow Cytometry Facility in Basel for support.

....

Materials

NameCompanyCatalog NumberComments
7-amminoactinomycin D (7-AAD)InvitrogenA1310Viability dye
96 well plate U bottomSarstedt83392550096 Well plate for our Flow cytometer
APC Mouse Anti-Human cd34BD555824Antibody detecting CD34
RRID: AB_398614
Bovine Serum AlbuminPanReac AppliChemA1391,0050Component of the staining buffer
Ethylenediaminetetraacetic acidRoth8043.1Component of the staining buffer
Fetal Calf Serum (FCS)BioConcept2-01F10-IComponent of the supplemented RPMI medium
FlowJo 10.2BD/Software enabling data analysis for flow cytometry experiment
Goat- anti-Rabbit IgG (H+L) Alexa Fluor 488Thermo ScientificA21222Secondary antibody detecting the primary antibodies for MICA and MICB
RRID: AB_1037853
Human NKG2D Fc Chimera Protein, CFR&D1299-NK-050Fusion Protein detecting all NKG2DLs
RRID:
Human ULBP-1 AntibodyR&DAF1380Antibody detecting ULBP1
RRID: AB_354765
Human ULBP-2/5/6 AntibodyR&DAF1298Antibody detecting ULBP2/5/6
RRID: AB_354725
Human ULBP-3 AntibodyR&DAF1517Antibody detecting ULBP3
RRID: AB_354835
MICA Polyclonal AntibodyThermo ScientificPA5-35346Antibody detecting MICA
RRID: AB_2552656
MICB Polyclonal AntibodyThermo ScientificPA5-66698Antibody detecting MICB
RRID: AB_2663413
One-step Antibody Biotinylation Kit 1 strip, for 8 reactionsMiltenyibiotec130-093-385Biotinylation kit for the NKG2DL fusion protein
Phosphate Buffered SalineSigma AldrichD8537-500MLComponent of the staining buffer
Rabbit anti-Goat IgG (H+L) Alexa Fluor 488Thermo ScientificA11034Secondary antibody detecting the primary antibodies for the ULBPs
RRID: AB_2576217
Rainbow Calibration Particles (8-peaks) 3.0 umSpherotech Inc.RCP-30-20ABeads used for flow cytometry device maintainance
RPMI mediumSigma AldrichR8758-500MLCell culture medium
RayBright Universal Compensation BeadsRaybiotech137-00013-100Beads used to create the compensation matrix
Streptavidin, R-Phycoerythrin Conjugate (SAPE) - 1 mg/mLInvitrogenS866Seondary step for the biotinylated NKG2DL fusion protein detection

References

  1. Topham, N. J., Hewitt, E. W. Natural killer cell cytotoxicity: how do they pull the trigger. Immunology. 128 (1), 7-15 (2009).
  2. Campbell, K. S., Hasegawa, J. Natural killer cell biology: an update and future directions.

Explore More Articles

NKG2D LigandsFlow CytometryAcute Myeloid LeukemiaLeukemic Stem CellsCell Surface DetectionBiotinylated NKG2D Fusion ProteinStreptavidin PE

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved