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

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, 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, γδ T, and naïve 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 1–6 (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.

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 °C.

1. Thaw both the biotin, as well as the NKG2D fusion protein tubes at room temperature (RT).
   NOTE: If the cells need to be sterile for further experimental use (injection in mice, colony forming unit assays, etc.), reconstitute the fusion protein under a laminar flow hood to avoid contamination. Otherwise, each step can be performed on a bench.
2. Quickly spin down the 50µg of lyophilized NKG2D fusion protein. Add 500 µL of phosphate-buffered saline (PBS) to reconstitute the powder and mix thoroughly using a P1000 micropipette.
3. Add 100 µL of the NKG2DL fusion protein to a biotin tube to obtain a final stock concentration of 100 µg/mL.
4. Mix the solution thoroughly by continuous resuspending with a P100 micropipette.
5. Incubate the fusion protein/biotin solution at a controlled RT for 24 h. The fusion protein is now ready to use.

2. Thawing of primary AML cells

NOTE: Approximately 5,000,000 frozen leukemic cells per patient stored in liquid nitrogen were thawed and then used for the assays right away. Leukemic cells were obtained and frozen as previously described¹⁵. Briefly, peripheral blood samples collected from patients with AML and high blast cell percentages (>90% of blasts among mononuclear cells) were processed with a density gradient separation to obtain mononuclear cells and subsequently frozen in fetal calf serum (FCS) containing 10% dimethyl sulfoxide solution (DMSO). Cell numbers highly varied between patients in dependence on the leukocyte concentration in the patient (range: 1,000,000 to 30,000,000 leukemic cells per mL blood).

1. Pre-warm RPMI medium containing 10% of FCS at 37 °C using a water bath. For each vial of AML cells (up to 30,000,000 cells in 1 mL FCS + 10% DMSO), add 10 mL of pre-warmed medium to a 15 mL reaction tube.
2. Remove the cryovial containing primary AML from the liquid nitrogen storage and immediately thaw the cells using a 37 °C 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.
3. Immediately transfer the thawed cells into the medium-containing tube and rinse the vial using 1 mL of medium.
4. Centrifuge the cells at 300 x g for 10 min and discard the supernatant without disturbing the pellet.
5. Wash the cells with 5 mL of RPMI medium containing 10% of FCS and centrifuge at 300 x g for 10 min.

3. Cell counting

1. Resuspend the cell pellet in a known volume of RPMI medium containing 10% of FCS.
2. Transfer a small volume of the cell suspension to a 1.5 mL microcentrifuge tube and dilute at a known ratio with trypan blue or any other alternative dye that allows live cell/dead cell discrimination.
3. Use any device to count the cells.
4. Centrifuge the cells at 300 x g for 10 min and discard the supernatant without disturbing the pellet.

4. Staining of primary AML cells using the biotinylated NKG2D fusion protein

1. Prepare the staining buffer by supplementing 500 mL PBS with bovine serum albumin (BSA) and Ethylenediaminetetraacetic acid (EDTA) to a final concentration of 0.07 mM and 2 mM, respectively. Adjust the pH (7.2–7.4) if necessary.
2. Resuspend the cell pellet with staining buffer to a final concentration of 0.5 x 10⁷ cells/mL.
   NOTE: Optionally, prior to staining, the cells can be incubated with a blocking agent (e.g. hIgG (1µg/ml) for 30 min at RT or FcR-blocking agent)..
3. Transfer 100 µL of the cell suspension to a cell culture 96-well U-bottom plate and centrifuge the plate at 300 x g for 10 min. Discard the supernatant without disturbing the pellet.
4. Prepare a master mix of biotinylated NKG2D fusion protein so that cells are resuspended in a final volume of 50 µL per well with a final concentration of 10 µg/mL per well.
5. Add the master mix prepared in step 4.4 using a 100 µL pipette and resuspend the cell pellets with a 300 µL multichannel pipette.
   NOTE: Scale down the final volume according to the number of cells to reduce the amount of fusion protein necessary for the staining.
6. Incubate the cell suspension for 25 min at RT or 50 min at 4 °C.
7. Wash the cells by adding 200 µL of staining buffer per well with a 300 µL multichannel pipette.
8. Centrifuge the plate at 300 x g for 10 min and discard the supernatant without disturbing the pellet.
9. Repeat steps 4.7 and 4.8.
   NOTE: The staining described here is performed in a cell culture 96-well U-bottom plate. Therefore, washing steps are performed twice because of the small volume capacity of such plates. The staining can also be performed in other tubes and washing steps might then be performed only once using a larger volume.
10. Prepare a master mix using Streptavidin-PE (see Table 1 for dilutions) so that cells are resuspended in a final volume of 50 µL.
    NOTE: Add selection antibodies for your cell type of interest as e.g. CD33, CD34…. for AML.
11. Add the master mix prepared in step 4.10 using a 100 µL pipette and resuspend the cell pellets with a 300 µL multichannel pipette.
12. Incubate the cell suspensions for 15 min at RT or 30 min at 4 °C in the dark.
13. Wash the cells by adding 200 µL of staining buffer per well with a 300 µL multichannel pipette.
14. Centrifuge the plate at 300 x g for 10 min and discard the supernatant without disturbing the pellet.
15. Repeat steps 4.13 and 4.14.
16. Prepare a solution of staining buffer including 7-AAD (7-Aminoactinomycin D, 1:1000) or any reagent to distinguish between live and dead cells.
17. Resuspend cell pellets in 200 µL staining buffer + 7-AAD prepared in step 4.16 in using a 300 µL multichannel micropipette.
18. Analyze the cells using a flow cytometry device.

5. Staining of single or pooled single anti-NKG2DL antibodies

1. Use the same cell suspension as in step 4.2.
2. Transfer 100 µL of the cell suspension to a cell culture 96-well U-bottom plate using a 300 µL multichannel pipette and centrifuge the plate at 300 x g for 10 min. Discard the supernatant without disturbing the pellet.
3. Prepare an antibody master mix for each primary antibody or pooled antibodies (see Table 2 for dilutions) so that the cells are resuspended in a final volume of 50 µL.
4. Add the master mix prepared in step 5.3 using a 100 µL pipette and resuspend the cell pellets with a 300 µL multichannel pipette.
5. Incubate the cells for 25 min at RT.
6. Wash the cells by adding 200 µL of staining buffer per well using a 300 µL multichannel pipette.
7. Centrifuge the cell culture 96-well U-bottom plate at 300 x g for 10 min and discard the supernatant without disturbing the pellet.
8. Repeat steps 5.6 and 5.7.
9. Prepare an antibody master mix by adding the secondary antibody (see Table 2 for dilutions) so that the cells are resuspended in a final volume of 50 µL per well.
10. Add the master mix prepared in step 5.9 using a 100 µL pipette and resuspend the cell pellets with a 300 µL multichannel pipette.
11. Incubate for 15 min at RT or 30 min at 4 °C in the dark.
12. Wash by adding 200 µL of staining buffer per well using a 300 µL multichannel pipette.
13. Centrifuge the cell suspension at 300 x g for 10 min and discard the supernatant without disturbing the pellet.
14. Repeat steps 5.12 and 5.13.
15. Resuspend cell pellets in 200 µL staining buffer containing 7-AAD prepared in step 4.16.
16. Analyze the cells using a flow cytometry device as described in step 6.

6. Data acquisition

NOTE: Make sure that weekly quality controls of the flow cytometer are performed to ensure that lasers are functioning properly. For the protocol presented here, a weekly Cytometer and Tracking (CTS) process is performed with relative beads to check laser performances on all channels. After the CTS, 10,000 events are recorded using the 8-peak beads as an internal control. All peaks should be in the same position inside their gates and well separated from each other.

1. Create the matrix compensation to subtract spectral overlap between detectors with beads or with cells¹⁶.
2. Record 10,000 events for the unstained cells and the single stained beads. For the fluorescence minus one (FMO) controls, record 50,000 events and for the full-stained samples record up to 100,000 events.
   NOTE: Alternatively, single stained samples can be performed on the cells. If so, make sure that the cells have a positive signal for the desired marker.
3. Acquire a single stained cell or bead sample for each fluorophore used in the experiment to reveal the amount of spectral overlap. Use the compensation creator of the flow cytometry device to calculate spill-over values and apply the compensation matrix to all the measured samples.
   NOTE: Secondary antibodies cannot be used for compensation creation using beads if the compensation is calculated with beads. Depending on the type of compensation beads, secondary antibodies cannot be used for the compensation with beads.
4. For the FMO controls and the full stained samples with the fusion protein, firstly, select the cells based on their forward scatter (FSC) and side scatter (SSC). After the exclusion of doublets, gate the cells based on their negativity for 7-AAD (PercP-Cy5.5) signal to exclude the dead cells (see step 5.15). The final plot shows the expression of CD34 (Y-axis) and NKG2DL (X-axis) on living singlets.
5. For the single anti-NKG2DL-antibodies, apply the same strategy to the samples but note that ligand positivity lies in Alexa Fluor 488 and not the PE channel.
6. Adjust gates according to the FMO controls for gating strategy from step 6.4 and 6.5 (see Table 3) for appropriate FMO controls to perform for this experiment: In the analysis software, draw a gate on the negative fraction. While recording the full-stained sample, the cells above the previously created gate are positive for the analyzed marker.

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...

Materials

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