Cell lines and medium
The human leukemia cell line K562 was kindly provided by Dr. C. H. June[15] and the non-small-cell lung cancer cell line A549 was kindly provided by Dr. K. Suzuki[16]. All cells were maintained in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% FBS, 50 mM b-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA), 10 mM Hepes (Thermo Fisher Scientific), and penicillin-streptomycin (Thermo Fisher Scientific).
Flow cytometry
Cells were stained with the following antibodies: anti-TCR Va24-FITC (clone C15) and anti-TCR Vb11-PE (clone C21) from Beckman Coulter (Brea, CA); anti-CD56-FITC (clone NCAM16) from BD Biosciences (Franklin Lakes, NJ); anti-PD-1-Pacific Blue (clone EH12.EH7) and anti-CD3-APC/Cy7 (clone HIT CD3a) from Biolegend (San Diego, CA). Data were collected by a FACSCanto II flow cytometer (BD Biosciences), and analyzed using FlowJo software (FlowJo LLC, Ashland, OR).
Cell preparation and culture
PBMCs were isolated from peripheral blood of healthy donors by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL). For NKT cell expansion, PBMCs were cultured in RPMI 1640 medium in the presence of 200 ng/mL a-GalCer (REGiMMUNE, Tokyo, Japan) and 100 JRU/mL recombinant human IL-2 (Shionogi, Osaka, Japan) for 6 days. Nivolumab or ultra-LEAF Purified Human IgG4 recombinant antibody (Biolegend) were added to the culture on days 0, 3, and 5. Nivolumab was provided by Ono Pharmaceutical Co. Ltd. (Osaka, Japan). After 6 days of culture, NKT cells were labeled with the anti-TCR Va24-FITC antibody, followed by positive selection using anti-FITC MicroBeads and an autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, purified NKT cells (>90% purity) were rested in RPMI 1640 medium in the presence of 100 JRU/mL IL-2 overnight and used in further experiments.
Cytotoxicity assay of NKT cells
The K562 and A549 cancer cell lines were labeled with an Invitrogen CellTrace Violet Cell Proliferation Kit (CTV) (Thermo Fisher Scientific), in accordance with the manufacturer’s protocol. NKT cells preincubated with 10 μg/mL nivolumab or 10 μg/mL hIgG4 isotype control for 30 min were co-cultured with the cancer cell lines at E/T ratios of 2.5:1, 5:1, and 10:1 in 96-well plates for 4-6 h. The cells were then stained with AnnexinV-FITC and PI using an Invitrogen Dead Cell Apoptosis Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. CTV+AnnexinV+ cells were considered apoptotic cancer cells. Cytotoxicity was calculated by the following equation: Cytotoxicity (%) = CTV+ [% AnnexinV+ target cells – % AnnexinV+ spontaneous cells].
To assess the expression levels of effector molecules, NKT cells and K562 cells were co-cultured at an E/T ratio of 10:1, harvested for RNA isolation, and subjected to real-time PCR.
Cytotoxicity assay of NK cells co-cultured with NKT cells
NK cells were negatively selected from PBMCs using an NK Cell Isolation Kit (Miltenyi Biotec) and the autoMACS Pro Separator. NKT cells cultured with nivolumab or the isotype control were seeded in the upper compartment of transwells separated by a 0.4-µm pore membrane with K562 or A549 cells at an E/T ratio of 10:1. At 4 h after NKT cell seeding, NK cells were seeded in the lower compartment with CTV-labeled K562 cells at an E/T ratio of 1:1 or CTV-labeled A549 cells at an E/T ratio of 2:1. The lower compartments containing cells were harvested after 4 h and the cytotoxicity of NK cells was assessed as described above.
RNA isolation and quantitative real-time PCR
Total RNA was isolated using Invitrogen TRIzol Reagent (Thermo Fisher Scientific). cDNA was synthesized using Invitrogen SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Real-time PCR was performed using Applied Biosystems TaqMan™ Fast Universal PCR Master Mix (Thermo Fisher Scientific) with an Applied Biosystems Step One Plus Real-time PCR system (Thermo Fisher Scientific). Primers and probes were purchased from Roche (Basel, Switzerland). Relative gene expression was calculated by the DDCt method using HPRT1 as a reference gene for normalization. The primer sequences were: Granzyme B: 5′-AGATGCAACCAATCCTGCTT-3′ and 3′-CATGTCCCCCGATGATCT-5′; Perforin: 5′-CACTCACAGGCAGCCAACT-3′ and 3′-GGGAGTGTGTACCACATGGA-5′; TNF-a: 5′-CAGCCTCTTCTCCTTCCTGAT-3′ and 3′-GCCAGAGGGCTGATTAGAGA-5′; TRAIL: 5′-TCACAGTGCTCCTGCAGTCT-3′ and 3′-GCCACTTTTGGAGTACTTGTCC-5′; FASL: 5′-AGCTGGCAGAACTCCGAGAG-3′ and 3′-GGCATGGACCTTGAGTTGGA-5′.
Statistical analysis
Statistical analysis was performed using GraphPad PRISM software version 6.0b (GraphPad Software, San Diego, CA). Data were presented as mean ± standard deviation. Values of P < 0.05 were considered statistically significant.