Isolation of NK cells and Cell culture
Human NK cells were isolated from peripheral blood mononuclear cells of healthy donors with the Dynabeads® Untouched™ Human NK Cell-Kit according to the manufacturer`s instructions (Invitrogen). The study was approved by the Ethics Committee of the Leibniz Research Center (#213) and all blood donors gave informed consent. For experiments with resting NK cells, isolated NK cells were rested in medium overnight and then used for experiments. To generate pre-activated NK cells, isolated NK cells were seeded in 96-well round-bottom plates with irradiated feeder cells (K562-mbIL15-41BBL) with 200 U/mL IL-2 (National Institutes of Health Cytokine Repositor) and 100 ng/mL IL-21 (Miltenyi Biotec). On day 8, NK cells were re-stimulated with fresh feeder cells and then cultured with 100 U/mL IL-2. On day 14, 2.5 ng/mL recombinant IL-15 (PAN Biotech) was added and after three weeks NK cells were used for experiments. In case of long-term treatment, Glupin (100 nM) or DMSO (0.1%) were added to the culture every 72 h. For inhibition of CPT1 long-term treated NK cells were treated with 1 µM of the CPT1-inhibitor Etomoxir for 24 h and analyzed for degranulation or IFN-γ secretion as described below. For cultivation with short-chain fatty acids, expanded NK cells were cultivated with 0.1% DMSO, Pentanoate (2.5 mM, 5 mM, 7.5 mM) or Butyrate (0.5 µM, 0.75 µM, 1 µM) for 72 h, followed by a degranulation-assay against K562 targets. K562 cells were cultured in DMEM (Gibco) with 10% FCS and 1% penicillin/streptomycin. MCF7 cells were cultured in DMEM (Gibco) with 10% FCS and 1% penicillin/streptomycin, 1% Sodium Pyruvat, 0,1% NeAA, 0,1% Insulin. HepG2 cells were cultured in DMEM (Gibco) with 10% FCS and 1% penicillin/streptomycin.
RTCA analysis
3x104 MCF7 cells or HepG2 cells were seeded in e-plates and placed into the xCELLigence device. After 16 h of measurement, 3.75x103 NK cells in combination with DMSO (0.1%) or Glupin (100 nM) were added. Cell index was measured every 10 minutes for 72 hours.
51 Chromium-release-assay
Cytotoxicity was analyzed using a standard 4 h chromium release assay as already described (26). In short, target cells were labeled for 1 h at 37°C and 5% CO2 on a Rotator with 51Cr. Afterwards, target cells were washed twice and added to the NK cells in a 96 v-well plate. NK cells were serial diluted (1:2) starting at an E:T 2:1. After 4 h incubation time, supernatant was collected and analyzed with the Wizard2 (Perkin Elmer) gamma counter. The percentage of specific lysis was determined as follows:
\(\frac{experimental release-spontaneous release}{maximal release-spontaneous release}\) ∗100%
Flow cytometry
To measure degranulation, resting or pre-activated NK cells were treated with Glupin (100 nM) or DMSO (0.1%) for 30 minutes and then stimulated via plate-bound CD16-mAb (2 µg/mL), NKp30 (2 µg/mL), NKG2D + 2B4 (2 µg/mL) or control IgG (2 µg/mL) in the presence of anti-CD107a PE-Cy5 for 2 h. CD107a- expression was then measured by flow cytometry. For intracellular staining NK cells were stained for live/dead (Zombie NIR; BioLegend) and CD56 using anti-CD56 BV421 (Clone:NCAM16.2; BD Biosciences) for 15 min at RT. Intracellular staining was performed using 2% paraformaldehyde for fixation, Permeabilizing Solution 2 (BD Bioscience) for permeabilization and anti-GLUT1 (PE; Clone:202915; R&D Systems), anti-pmTOR (PE-Cy7; Clone:MRRBY; Thermo Fisher Scientific) and anti-HK1 (AF647; Clone: EPR10134(B); abcam) or Granzyme B (AF647; Clone:GB11; BioLegend) and Perforin (FITC; Clone:dG9; BioLegend). Surface staining was performed using two different panels. Panel 1: Zombie NIR (BioLegend), CD56 (BUV805; Clone:B159; BD Biosciences), CD3 (BUV563; Clone:UCHT1; BD Biosciences), NKp46 (BV421; Clone:9E2; BioLegend), 2B4 (FITC; Clone:C1.7; BioLegend), NKp44 (PerCP-Cy5.5; Clone:P44-8; BioLegend), DNAM-1 (AF647; Clone:DX11; BD Biosciences), NKp30 (APC-Fire750; Clone:P30-15; BioLegend), CD16 (PE-Dazzle; Clone:3G8; BioLegend) and NKG2D (AF700; Clone:FAB139N; R&D Systems). Panel 2: CD38 (BUV395; Clone:HB7; BD Biosciences), NKG2C (BUV496; Clone:134591; BD Biosciences), CD3 (BUV563; Clone:UCHT1; BD Biosciences), KLRB1 (BUV661; Clone:NKR-3G10; BD Biosciences), CD56 (BUV805; Clone:B159; BD Biosciences), 4-1BB (BV421; Clone:4B4-1; BioLegend), KLRG1 (BV510; Clone:2F1; BioLegend), HLA-DR (BV605; Clone:L243; BioLegend), FasL (BV650; Clone:NOK-1, BD Biosciences), TIGIT (BV786; Clone:741182; BD Biosciences), CD11a (AF488; Clone:HI111; BioLegend), CD8 (AF532; Clone:RPA-T8; Thermo Fisher Scientific), CD27 (PerCP-Cy5.5; Clone:M-T271; BD Biosciences), NKG2A (PE; Clone:Z199; Beckmann Coulter), CTLA-4 (PE-Dazzle; Clone:BNI3; BioLegend), TRAIL (PE-Cy7; Clone:N2B2; BioLegend), PD-1 (AF647; Clone:EH12.1; BD Biosciences), CD18 (AF700; Clone:TS1/18; BioLegend), Tim-3 (APC-Fire 750; Clone:F38-2E2; BioLegend) and Zombie NIR (BioLegend). Cells were measured on a Cytek Aurora spectral flow cytometer and analysed with FlowJo.
SCENITH
Assays were performed as described before (27). In brief, NK cells were incubated in the presence or absence of Oligomycin (1 µM), 2-DG (100 nM) or Oligomycin + 2-DG for 30 min. Next, Puromycin (10 µg/mL) was added for 20 min followed by a wash using ice cold PBS. After a Fc-Block for 5 min surface staining was performed using zombie NIR (BioLegend), anti-CD56 BV421 (Clone:NCAM16.2; BD Biosciences), anti-CD16 PE-Dazzle (Clone:3G8; BioLegend). Cells were then washed, fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (InvitrogenTM) followed by intracellular staining using anti-Puromycin AF488 (Clone:12D10; Sigma-Aldrich) and anti-pmTOR PE-Cy7 (Clone:MRRBY; Thermo Fisher Scientific) and analysis using a Cytek Aurora flow cytometer. Glycolytic dependency, mitochondrial dependency, glycolytic capacity and FAO (fatty acid oxidation) and AAO (amino acid oxidation) capacity was calculated using the formula as already described by Argüello et al (14).
ELISA
Resting or pre-activated NK cells were treated with Glupin (100 nM) or DMSO (0.1%) for 30 minutes followed by stimulation via plate-bound CD16-mAb (2 µg/ml), NKp30 (2 µg/ml), NKG2D + 2B4 (2 µg/ml) or control IgG (2 µg/ml) or via IL-12 (0.25 ng/µl) + IL-18 (1.25 ng/µl) as previously described (28). After 16 h supernatants were collected and IFN-γ was analyzed using the ELISA MAX™ Deluxe Set Human (BioLegend).
Glucose-uptake Assay
Glucose uptake-Glo™ Assay (Promega) was performed according to the manufacturer’s instructions. Luminescence was recorded using a GloMaxR instrument (Promega).
NAD/NADH-Assay
NK cells were analyzed for NAD+/NADH concentrations using NAD/NADH-Glo™ Assay (Promega) according to the manufacturer’s instructions. Measurement was done with a GloMaxR instrument.
Serial-killing-assays
Assays were performed as described before (15). In brief, microchips were seeded with MCF7 cells to obtain 60–80 tumor cells/well. After attachment of tumor cells medium containing the dead cell stain SYTOX Blue (1:1000) was added and the microchip was placed into the incubation chamber of the ApoTome System with Axio Observer 7 microscope (Zeiss) equipped with a 20×/0.8 Plan-Apochromat objective and an incubation chamber with environmental control (37°C, 5% CO2, humidity device PM S1). 10–20 NK cells per well were added and time-lapse microscopy was started. During 16h a picture was taken every 3 minutes using an Axiocam 506 mono camera. SYTOX Blue was excited using the Colibri 7 LED-module 475 (filter set 38 HE LED) and brightfield was acquired using the TL LED module.
Seahorse-analysis
Energetic phenotype of pre-activated NK cells was analyzed using the glycolysis stress test kit (Agilent). In brief, culture plates were coated with poly-l-lysine, followed by the addition of pre-activated NK cells. Injection ports were loaded with 10 x concentrated substance (final concentration after injection: Port A: 100 nM Glupin / 0.1% DMSO / medium; Port B: 10 mM Glucose; Port C: Oligomycin; Port D: 50 mM 2-DG) and the experiment was performed according to the manufacturer`s instructions (Agilent).
Metabolomics
For metabolomics, 10x106 long-term treated NK cells were lysed analyzed by the Metabolomics Innovation Centre (TMIC Canada) using a combination of direct injection mass spectrometry with a reverse-phase LC-MS/MS custom assay for the targeted identification and quantification of up to 143 different endogenous metabolites including amino acids, acylcarnitines, biogenic amines & derivatives, glycerophospholipids, sphingolipids and sugars. Isotope-labeled internal standards and other internal standards were used for metabolite quantification. Data analysis was done using Analyst 1.6.2.
RNA-Seq
10x106 long-term treated NK cells were used for RNA-isolation using RNAeasy Mini Kit (Qiagen) according to the manufacturer`s instructions with an additional step of DNAse treatment. RNA-sequencing were performed at the genomics & transcriptomics facility (GTF) at the university hospital Essen. In brief, QuantSeq 3’ mRNA-Seq Library Prep Kit FWD was used for library preparation. Sequencing was performed on the Illumina NextSeq500. Reads were processed with standard Illumina base calling. Trimmed with Trim Galore v0.6.0 and standard settings. Alignment was performed with hisat2 v2.2.1 to grch38 and standard settings. Raw counts were summed up with summarize Overlaps from R-Package Genomic Alignments v1.8.4. RNA-Seq data were stored in the Gene expression omnibus archive (accession: GSE216156).
Statistics
Statistical analyses were performed using GraphPad Prism version 9.