NK cell isolation

Healthy volunteers were recruited through the NCI-Frederick Research Donor Program (http://ncifrederick.cancer.gov/programs/science/rdp/default.aspx). The KIR genotype of each donor was determined as previously described (Martin and Carrington 2008). Donors possessing only the KIR2DL1*003 allele were chosen to match the allele present in the YTS cell line. NK cells were separated from the peripheral blood of healthy donors by Histopaque (Sigma-Aldrich) gradient centrifugation using the RosetteSep Human NK Cell Enrichment Cocktail (STEMCELL Technologies).

Cell culture

YTS cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Gibco) at 37 °C and 5% CO2. Cells were passaged every 3–4 days and plated at 3 × 105 cells/ml in 75 cm2 flasks. The KIR+ cell line was derived by treating parental YTS with 5-azacytidine transiently for 72 h followed by washing and staining with anti-KIR2DL1 antibody. The KIR2DL1-expressing cells were purified by three rounds of FACS sorting until a stable line was generated.

CRISPR/Cas 9 deletion of the KIR2DL1 intermediate promoter

The following oligonucleotide sequences were used as CRISPR guides to delete a 281 bp region containing the KIR2DL1 intermediate promoter/enhancer region:

3′ Guide-For5′-CACCGTTAGGCATCTCGTGTTCGGG-3′

3′ Guide-Rev5′-AAACCCCGAACACGAGATGCCTAAC-3′

5′ Guide-For5′-CACCGAAACTCCAGAATTTACAGGT-3′

5′ Guide-Rev5′-AAACACCTGTAAATTCTGGAGTTTC-3′

Bold bases indicate sequence added for cloning into vectors. The 3′ guide was inserted into pSpCas9(BB)-2A-GFP (PX458) and the 5′ guide was inserted into pU6-(BbsI)-CBh-Cas9-T2A-mCherry, both obtained from Addgene. Plasmids (1 µg each) were digested with BbsI (New England Biolabs) for 30 min at 37 °C. The digested plasmids were purified using ChargeSwitch PCR Clean-Up Kit (Invitrogen). Each pair of target sequence oligos (10 µM final) was phosphorylated with T4 Polynucleotide Kinase (New England Biolabs) at 37 °C for 30 min, denatured at 95 °C for 5 min, and then slowly annealed down to 25 °C at 5 °C per minute in a thermocycler. Phosphorylated and annealed target sequence oligos were diluted 1:200 and ligated into BbsI digested plasmids with T4 DNA Ligase (New England Biolabs). Plasmids were transformed into One Shot™ Top10 Chemically Competent E. coli (Invitrogen) and purified using the ZymoPURE™ II Plasmid Midiprep kit (Zymo Research). 1 × 106 YTS cells were transfected with GFP and mCherry plasmids containing guide oligos using an Amaxa Nucleofector II transfection system. Cells were pelleted at 200 × g and resuspended in 100 µl of Amaxa transfection buffer with 5 µg of both plasmids. Cells were electroporated using program X-005 and subsequently were cultured for 48 h prior to single-cell sorting of GFP and mCherry double-positive clones into 96 well plates.

Single-cell sorting for GFP/mCherry+ cells

KIR2DL1 intermediate promoter edit clones were sorted using either a Symphony S6 or FACS Aria-II cell sorter (BD Biosciences) to deposit single GFP/mCherry double-positive cells into individual wells of 96 well plates. YTS cells that had been transfected were counted, washed twice, resuspended in sorting buffer (1 × PBS pH7.4, 1% BSA, 0.5 mM EDTA, and 25 mM HEPES), and passed through 30-μm mesh (CellTrics, Sysmex) prior to sorting to remove doublets and aggregated cells. Stringent signal pulse width gates were applied by the sorting software for further assurance of clonality. Sorted single cells were collected in 96 well plates filled with 100 µl/well of RPMI 1640 supplemented with 10% FBS and glutamine/pen/strep. Plates were monitored for growth, and clones were subsequently expanded.

Screening of YTS intermediate promoter edit clones

Genomic DNA was isolated from the YTS intermediate edit clones with the Quick-DNA™ Mini Prep kit (Zymo Research). Thirty nanograms of DNA from each clone was screened by PCR using 2DL1 distal-For (5′-GGAGAGAGACAGACGGAAAAC-3′) and 2DL1 intermediate-Rev (5′-CGCTAGAATTTGACACCTAGTG-3′) primers to identify clones with homozygous or heterozygous deletions and unedited (wild-type) clones.

Cell transfection and luciferase reporter assay

5 × 105 YTS cells were seeded per well in a 24-well plate the day before transfection; 4.5 µg of reporter construct DNA, 400 ng of Renilla DNA, and 6 µl of jetOPTIMUS transfection reagent (Genesee Scientific) were diluted in 150 µl buffer and incubated at room temperature for 10 min before addition to each well. The DNA mixture containing plasmid DNA was then added to each well and incubated at 37 °C in 5% CO2 for 48 h before luciferase activity analysis. Luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Briefly, the culture medium was removed 48 h post-transfection, and cells were washed with 0.5 ml of phosphate-buffered saline (PBS, pH 7.4). Cells were then lysed with passive lysis buffer. The suspensions were centrifuged at 14,000 rpm for 1 min. A total of 20 μl of cell lysate supernatant was mixed with 100 μl of luciferase substrate, and the light units were measured on a luminometer. Measurements of firefly luciferase activity from promoter constructs were normalized relative to the activity of the Renilla luciferase produced by the pRLSV40 control vector, and each construct was tested in duplicate in at least three independent experiments.

RNA isolation

Total RNA was isolated from YTS cells using the RNeasy® Mini Kit (Qiagen). Cells were lysed in buffer RLT per manufacturer’s recommendations, and lysates were loaded on RNeasy Mini Spin Columns and washed with buffer RW1 and spun by centrifugation at 10,000 × g for 1 min. On-column DNase I digestion was performed using the RNase-Free DNase Set (Qiagen). Digested samples were washed with buffer RW1 followed by two washes with buffer RPE spinning by centrifugation at 10,000 × g after each wash. RNA samples were eluted in 25 µl of elution buffer, and RNA concentrations were determined by using a NanoDrop 2000 Spectrophotometer (Fisher Scientific).

Poly(A)+ mRNA was isolated using the NEBNext® High Input Poly(A) mRNA Isolation Module (New England Biolabs). Fifty micrograms of purified total RNA was the input for each poly(A)+ mRNA sample in a 200 µl reaction. Poly(A)+ mRNA was captured and washed on oligo(T)25 paramagnetic beads provided by the manufacturer, eluted with 17 µl of Tris buffer, and quantified using a NanoDrop 2000 Spectrophotometer (Fisher Scientific). Typical yields ranged from 400 to 600 ng of poly(A)+ mRNA per sample.

cDNA synthesis

cDNA samples were synthesized using TaqMan® Reverse Transcription Reagents (Applied Biosystems). Each cDNA synthesis reaction was performed in a 20 µl volume with either 1 µg of total RNA or 100 ng of poly(A)+ RNA, 1.75 mM MgCl2, 2 mM dNTPs mix, 2.5 µM random hexamers, 1 U RNase Inhibitor, and 1 U of MultiScribe™ RT. The cDNA was synthesized using a 2720 Thermal Cycler (Applied Biosystems) with a single cycle of 25 °C for 10 min, 37 °C for 30 min, and 95 °C for 5 min, followed by a 4 °C hold.

PCR of KIR2DL1 transcripts

PCR amplifications of KIR transcripts were performed in 40 µl reactions with ZymoTaq premix. Each PCR reaction utilized 2 µl of cDNA template, 1 µM of forward and reverse primers, and 20 µl of 2X ZymoTaq premix. Initial denaturation was performed at 95 °C for 10 min per the manufacturer’s recommendation, 30–35 cycles of 95 °C for 30 s, 30 s annealing at a primer-specific temperature, and extension at 72 °C for 0.5–1.5 min for transcripts up to 1 kb in length. A final extension was performed at 72 °C for 7 min.

PCR primers

2DL1 exon 1-For5′-CGGCAGCACCATGTCGCTCT-3′

2DL1 exon 3-Rev5′-AGGTCCCTGCCAGGTCTTGCG-3′

2DL1 intermediate-For5′GAGTTGGTCATAGTGAAGGACACTAG-3′

2DL1 distal-For5′-GGAGAGAGACAGACGGAAAAC-3′

B-actin-For5′-CCTGGCACCCAGCACAATG-3′

B-actin-Rev5′-GGGCCGGACTCGTCATACT-3′

KIR2DL1 detection by flow cytometry

1 × 106 YTS cells were washed twice in staining buffer (1 × PBS pH 7.4 without Ca/Mg, 1% BSA, 0.5 mM EDTA, and 0.1% NaN3 sodium azide) by centrifugation at 100 × g for 10 min in 5-ml polystyrene round-bottom tubes (Falcon 352,052). YTS cell pellets were incubated with 1 µg PE-conjugated CD158/KIR2DL1 antibody (clone REA284, Miltenyi Biotec) for 30 min on ice, washed twice with staining buffer, gently resuspended in Cytofix™ buffer (4.2% formaldehyde, BD Biosciences), and then stored at 4 °C until analysis. Data was collected using a FACS Symphony A5 cytometer (BD Biosciences) and analyzed with FlowJo v10.8.1 (BD). Antibody binding data were expressed as % positive compared to unstained control cells; PE median fluorescence was also assessed to indicate receptor density.

Bulk cell sorting for KIR2DL1+ cells

KIR2DL1+ cells were sorted to uniform high purity using either a Symphony S6 or FACS Aria-II cell sorter (BD Biosciences). YTS cells were counted, washed twice, resuspended in sorting buffer (1 × PBS pH7.4, 1% BSA, 0.5 mM EDTA, and 25 mM HEPES), and incubated with 10 µl PE-conjugated CD158/KIR2DL1 antibody (clone REA284, Miltenyi Biotec) for 30 min on ice. Labeled cells were washed twice with sorting buffer, resuspended in 0.5–15 ml sorting buffer, passed through 30-μm mesh (CellTrics, Sysmex), and maintained at 4 °C during sorting. Sorted KIR2DL1+ cells were collected in RPMI 1640 supplemented with 10% FBS and glutamine/pen/strep. Sorted cells were monitored for growth and subsequently expanded.

Statistical analysis

Mann–Whitney U and two-tailed t-tests were performed using Prism version 9 for Mac OS; p < 0.05 was regarded to be statistically significant.