Name: two flow cytometric approaches of nkg2d ligand surface detection to distinguish stem cells from bulk subpopulations in acute myeloid leukemia
Uploaded: 2021-02-21T13:00:00
Description: Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

This study was supported by grants from the Swiss National Science Foundation (179239), the Foundation for Fight Against Cancer (Zuerich),&#160;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&#8217;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No. 765104. We thank the Flow Cytometry Facility in Basel for support.

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&#8217;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 &#176;...

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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...

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 &#8212;such as natural cytotoxicity receptors (NCRs), NKG2D and CD16&#8212; 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, absence of self-recognition and increased binding of activating receptors to their ligands on the target cells trigger the release of cytotoxic granules leading to NK cell-mediated cytotoxicity1. Finally, NK cells can exert antibody-dependent cellular cytotoxicity (ADCC) by binding of the activating receptor CD16 to targets expressing the Fc portion of Ig (FcR)2. Apart from direct cytotoxicity, NK cells can also trigger cytokine release bridging the innate with the adaptive immune system3.
NKG2D is a major activating receptor expressed on NK, NKT, &#947;&#948; T, and na&#239;ve CD8+ T cells4 that enables such cytotoxic immune cells to recognize and lyse NKG2D ligand (NKG2DL) expressing target cells. Healthy cells commonly do not express NKG2DL. Instead, NKG2DL expression is upregulated on malignant or virus-infected cells to make these amenable to immune clearance5.
The human NKG2DL family comprises eight known molecules among which the two MHC I chain-related molecules A and B (MICA and MICB6) and the cytomegalovirus UL16-binding proteins 1&#8211;6 (ULBP1-67). The expression of NKG2DL is regulated on the transcriptional, post-transcriptional as well as the post-translational levels8. As such, while NKG2DL expression is commonly not detectable on the surface of healthy cells, NKG2DL mRNA9 and intracellular protein expression were reported in healthy tissues. The functional relevance of such expression and the mechanisms underlying such discrepant expression patterns remain to be defined10.
The mechanistic regulation of NKG2DL expression in the cancer cells is a fascinating area of investigation. Pathways known to be involved in either cellular stress, e.g., the heat shock stress pathway9, or DNA damage-associated pathways, such as the ataxia telangiectasia mutated (ATM) and Rad3 related (ATR) pathway11, as well as viral or bacterial infections have been directly linked to the induction of NKG2DL expression12. However, even if surface expression of NKG2DL has been effectively induced, this expression can be lost again through proteolytic-mediated shedding, a mechanism associated with immune escape and poor clinical prognosis in some cancers13.
The absence of cell surface NKG2DL may also play important roles in patients with AML. Here, treatment with intensive chemotherapy often induces remission, but relapse often occurs from leukemic stem cells (LSC), which selectively survive chemotherapies and evade immune response. As we recently showed, LSCs, for example, escape NK cell lysis by suppressing NKG2DL surface expression14.
Inversely, the absence of surface NKG2DL expression can be used as a method to identify and viably isolate putative stem-like subpopulations of cells from bulk counterpart leukemic subpopulations. Here, we present two flow cytometric approaches that can be used to detect NKG2DL surface expression and thereby identify NKG2DL negative stem cells in leukemia and perhaps also in other cancers: A method for pan-ligand surface recognition and a method involving staining with single or pooled antibodies recognizing individual known NKG2DL proteins.

<table>
	<tbody>
		<tr>
			<td>7-amminoactinomycin D (7-AAD)</td>
			<td>Invitrogen</td>
			<td>A1310</td>
			<td>Viability dye</td>
		</tr>
		<tr>
			<td>96 well plate U bottom</td>
			<td>Sarstedt</td>
			<td>833925500</td>
			<td>96 Well plate for our Flow cytometer</td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human cd34</td>
			<td>BD</td>
			<td>555824</td>
			<td>Antibody detecting CD34 
			RRID: AB_398614</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>PanReac AppliChem</td>
			<td>A1391,0050</td>
			<td>Component of the staining buffer</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Roth</td>
			<td>8043.1</td>
			<td>Component of the staining buffer</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>BioConcept</td>
			<td>2-01F10-I</td>
			<td>Component of the supplemented RPMI medium</td>
		</tr>
		<tr>
			<td>FlowJo 10.2</td>
			<td>BD</td>
			<td>/</td>
			<td>Software enabling data analysis for flow cytometry experiment</td>
		</tr>
		<tr>
			<td>Goat- anti-Rabbit IgG (H+L) Alexa Fluor 488</td>
			<td>Thermo Scientific</td>
			<td>A21222</td>
			<td>Secondary antibody detecting the primary antibodies for MICA and MICB 
			RRID: AB_1037853</td>
		</tr>
		<tr>
			<td>Human NKG2D Fc Chimera Protein, CF</td>
			<td>R&#38;D</td>
			<td>1299-NK-050</td>
			<td>Fusion Protein detecting all NKG2DLs 
			RRID:</td>
		</tr>
		<tr>
			<td>Human ULBP-1 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1380</td>
			<td>Antibody detecting ULBP1 
			RRID: AB_354765</td>
		</tr>
		<tr>
			<td>Human ULBP-2/5/6 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1298</td>
			<td>Antibody detecting ULBP2/5/6 
			RRID: AB_354725</td>
		</tr>
		<tr>
			<td>Human ULBP-3 Antibody</td>
			<td>R&#38;D</td>
			<td>AF1517</td>
			<td>Antibody detecting ULBP3 
			RRID: AB_354835</td>
		</tr>
		<tr>
			<td>MICA Polyclonal Antibody</td>
			<td>Thermo Scientific</td>
			<td>PA5-35346</td>
			<td>Antibody detecting MICA 
			RRID: AB_2552656</td>
		</tr>
		<tr>
			<td>MICB Polyclonal Antibody</td>
			<td>Thermo Scientific</td>
			<td>PA5-66698</td>
			<td>Antibody detecting MICB 
			RRID: AB_2663413</td>
		</tr>
		<tr>
			<td>One-step Antibody Biotinylation Kit 1 strip, for 8 reactions</td>
			<td>Miltenyibiotec</td>
			<td>130-093-385</td>
			<td>Biotinylation kit for the NKG2DL fusion protein</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537-500ML</td>
			<td>Component of the staining buffer</td>
		</tr>
		<tr>
			<td>Rabbit anti-Goat IgG (H+L) Alexa Fluor 488</td>
			<td>Thermo Scientific</td>
			<td>A11034</td>
			<td>Secondary antibody detecting the primary antibodies for the ULBPs 
			RRID: AB_2576217</td>
		</tr>
		<tr>
			<td>Rainbow Calibration Particles (8-peaks) 3.0 um</td>
			<td>Spherotech Inc.</td>
			<td>RCP-30-20A</td>
			<td>Beads used for flow cytometry device maintainance</td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Sigma Aldrich</td>
			<td>R8758-500ML</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>RayBright Universal Compensation Beads</td>
			<td>Raybiotech</td>
			<td>137-00013-100</td>
			<td>Beads used to create the compensation matrix</td>
		</tr>
		<tr>
			<td>Streptavidin, R-Phycoerythrin Conjugate (SAPE) - 1 mg/mL</td>
			<td>Invitrogen</td>
			<td>S866</td>
			<td>Seondary step for the biotinylated NKG2DL fusion protein detection</td>
		</tr>
	</tbody>
</table>

two flow cytometric approaches of nkg2d ligand surface detection to distinguish stem cells from bulk subpopulations in acute myeloid leukemia

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.

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 ...

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.

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

This protocol shows two distinct ways of detecting NKG2DLs on the surface of AML cells. The technique provides a fast, user-friendly staining method to detect all known and possibly unknown NKG2DLs. This method allows for separation of leukemic stem cells from bulk AML cells, which makes it possible to further characterize these cells.Begin by thawing the biotin and the NKG2D fusion protein tubes at room temperature. Quickly spin down the NKG2D FC powder, then add 500 microliters of PBS to reconstitute the powder and mix thoroughly using a P-1000 micropipette. Add 100 microliters of the NKG2DL fusion protein to a biotin tube to obtain a final concentration of 10 micrograms per milliliter, and mix the solution thoroughly with a P-100 micropipette.To thaw the AML cells, remove the cryovial containing primary AML from the liquid nitrogen storage, and immediately place it in a 37 degrees Celsius water bath. Gently move the tube back and forth in the water, allowing the contents of the vial to thaw until there is only a small ice crystal left. Immediately transfer the thawed cells into the medium containing tube and rinse the vial using one milliliter of medium.Centrifuge the cells at 300 times G for 10 minutes and discard the supernatant without disturbing the pellet. Wash the cells with five milliliters of RPMI medium containing 10%FCS and repeat the centrifugation. Resuspend the cell pellet with staining buffer to a final concentration of 0.5 times 10 to the seventh cells per milliliter.Then transfer 100 microliters of the cell suspension to a cell culture 96 well U-bottom plate and centrifuge the plate at 300 times G for 10 minutes. Discard the supernatant without disturbing the pellet. Prepare a master mix of biotinylated NKG2D fusion protein so that the cells are resuspended in a final volume of 50 microliters per well with a final NKG2D concentration of 10 micrograms per milliliter per well.Add the prepared master mix and resuspend the cell pellets with a 200 microliter pipette. Incubate and centrifuge the plate as described in the text manuscript. Prepare a master mix using streptavidin PE so that the cells are resuspended in a final volume of 50 microliters.Add the master mix to the cells and resuspend the pellets with a 300 microliter multichannel pipette. After centrifuging the plate and discarding the supernatant, use a 300 microliter multi-channel micropipette to resuspend the cell pellets in 200 microliters of staining buffer plus 7-AAD. Then analyze the cells using a flow cytometry device.The analyzed AML samples are positive for CD34 and NKG2DL, but negative subpopulations also exist, with four different populations in total. The typical gating strategy starts with the selection of the main population of cells via their FSC and SSC. Doublets and dead cells are excluded in the downstream analysis.The gates are adjusted using FMO controls to ensure proper identification of positive cells. The fluorescence intensities of cells positive for CD34 versus NKG2DL are highlighted here. AML cells that are positive for CD34 show a lower surface expression of NKG2DL, indicating that NKG2DL expression is associated with lack of stemness.Three primary AML samples demonstrated 19.8, 49.8, and 89.4%positive events for the fusion protein staining versus 20.4, 50.4, and 90.6%for the pooled anti-NKG2DL antibody staining. On the other hand, single ligand staining showed a range of positive events up to 92%with the percentages varying based on the ligand. When attempting this method, it is very important to be quick when thawing the primary AML cells, because they are fragile and can die easily during this step.After performing this protocol, a cell sort based on the NKG2DL signal can be performed to further investigate the populations.

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