Cell lines and cell culture conditions
SNK-6, SNT-8, and NK-92 cell lines were purchased from BeNa Culture Collection (BNCC), NK-YS cell line was provided by Dr. Wenqi Jiang (Sun Yat-sen University Cancer Center), EL4 and 293 T cell lines were obtained from the American Type Culture Collection (ATCC, USA). All cell lines were validated by short-tandem-repeat (STR). NK-YS, SNK-6, and SNT-8 cells were cultured in RPMI-1640 and IL-2 (100 IU/ml, for NK-YS, SNK-6, and SNT-8 cells) and 293 T cells were grown in DMEM medium containing 10% fetal bovine serum. EL4 cells were cultured in DMEM with 10% fetal equine serum. NK-92 cells were grown in 75% alpha-MEM supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 2 mM l-glutamine, 10 mL/L penicillin-streptomycin, 10 ng/mL recombinant human IL-2 (rIL-2), and 5 mg/mL plasmocin to prevent mycoplasma contamination. All cells were maintained at 37 °C in a humidified 5% CO2 incubator [25, 26].
Cell viability assays
NK-YS, SNK-6, and SNT-8 cells were treated with 0, 100 and 500 ng/ml Recombinant Human GM-CSF Protein (R&D Systems, USA) for 0, 24, 48, 72 h. For Fedratinib treatment assays, NK-YS, SNK-6, and SNT-8 cells were treated with DMSO or Fedratinib (MedChemExpress, MCE)) at a concentration of 3 nM for 12 h. Cell viability was determined by CCK-8 (Yeasen Biotech, China) [27].
Western blot analysis
Cells were treated with the indicated concentrations as shown in the figures and washed twice with cold PBS. Whole-cell extracts were collected in RIPA lysis buffer (Santa Cruz Biotechnology, Germany), and protein concentration of the lysates was measured using a BCA Protein Assay Kit (Pierce Biotechnology, USA). The protein samples were electrophoresed through a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking, membranes were probed with primary antibodies (1:1000) followed by washing and incubation with a secondary antibody (1:5000) conjugated to horseradish peroxidase (Amersham GE Healthcare, USA). Protein bands were visualized by applying a chemiluminescent reagent (Pierce ECL kit, Thermo Fisher Scientific, USA) [28]. The antibody against Phospho-Jak2 (Tyr1007/1008), JAK2, Phospho-Stat5 (Tyr694), STAT5, PD-L1 were purchased from Cell Signaling Technology (USA). The antibody against Phospho-STAT5A (Y694), STAT5A, Phospho-STAT5B (S731), STAT5B were purchased from Abcam (USA).
Dual-luciferase reporter assay
The template of PD-L1 (Accession: NM_001267706) promoter fragment was purchased from GeneCopoeia Inc. (# HPRM40139) and was amplified into the promoter-less plasmid pGL3-Basic vector by PCR (Promega, USA). The STAT5A and STAT5B fragments were amplified using the relevant primers by PCR and inserted into the pReceiver-M12 vector (GeneCopoeia Inc., USA). When reaching an approximately 80% confluence, 4 × 105 293 T cells each were co-transfected with 3.8 μg/well of pGL3 luciferase construct (empty vector or pGL3-PD-L1 promoter) and 0.2 μg/well pRL-TK (Promega, USA). The relative luciferase activity was examined by Dual Luciferase Assay Kit (Promega, USA) following the manufacturer’s protocols [29].
RNA extraction and quantitative real-time PCR
Total cellular RNA was isolated using Trizol (Life Technologies, USA) according to the manufacturer’s protocol. For first-strand cDNA synthesis, 5μg of total RNA was reverse-transcribed using the GoScript™ Reverse Transcription System kit (Promega, USA) followed by quantitative polymerase chain reaction (qPCR) with GoTaq qPCR Master Mix (Promega, USA), according to the manufacturer’s instructions. Real-time PCR analyses were conducted using the Biorad CFX96 system with SYBR green (Bio-Rad, USA) and the appropriate primers to estimate the mRNA expression levels of PD-L1. The primers are as follows: PDL1 forward: 5′-TATGGTGGTGCCGACTACAA-3′; reverse: 5′-TGCTTGTCCAGATGACTTCG-3′; GAPDH forward: 5′-CTCCTCCTGTTCGACAGTCAGC-3′; reverse: 5′-CCCAATACGACCAAATCCGTT-3′. Data were normalized to GAPDH levels. Experiments were performed in triplicates [30].
Transfection of shRNAs and plasmid DNAs
STAT5A and STAT5B shRNAs and an shRNA scramble control (Open Biosystems GE Healthcare Dharmacon Inc., USA) were transiently transfected along with a pSIH-H1-puro Lentivector Packaging Kit (System Biosciences, USA). Transfections were carried out in 293 T cells reaching ∼80% confluency in 10 cm dishes using Lipofectamine 2000 transfection reagent (Life Technologies, USA) and following the manufacturer’s instructions. Transfection medium was replaced with fresh growth medium 5 h after transfection. At 48 h following the initial transfection, viral supernatant was collected and filtered through a 0.45 nm filter (System Biosciences, USA). SNK-6 and SNT-8 cells were infected and incubated with the viral particles overnight at 37 °C. At 48 h after transfection, cells were placed under puromycin selection by supplementing the growth medium with puromycin (3 μg/ml for SNK-6, and 5μg/ml for SNT-8). Growth medium was replaced every 48 h for 2–3 weeks until isolated colonies (∼2 mm diameter) were apparent on the plate. At this point, individual clones were transferred to 12-well dishes and expanded in 1 μg/ml puromycin for further analysis. Individual clones were verified by Western blot and RT-PCR [31].
In vivo mouse studies
C57BL/6 mouse was purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and kept in a specific pathogen-free (SPF) barrier facility at the Animal Center of Sun Yat-sen University Cancer Center (n = 80). 8–12 weeks old female mice were used for all animal experiments. Experiments were approved by the institutional committee of Sun Yat-sen University Cancer Center (No. L102042018120I), and conducted following protocols approved by the Guangdong Provincial Animal Care and Use Committee.
EL4 cells or B16-F10 cells (5 × 105 cells in 200 μL growth medium) were subcutaneously injected into the right flank of immunocompetent C57BL/6 mouse. Tumor sizes were measured with calipers every 2 days and tumor volumes were calculated by applying the following formula: 1/2 (length×width2). When tumor sizes reached approximately 100 mm3, mice were randomized into control or experimental groups. The terminal event was defined as tumors reaching a size of 2000 mm3 in the control group, at which point animals were euthanized [32, 33].
Mice were intraperitoneally injected with murine GM-CSF (315–03-20; Peprotech; USA) or anti-mouse PD-1 (BE0273; Bio X cell, USA) alone, the combination of murine GM-CSF and anti-mouse PD-1, or saline and rat IgG2a isotype control (BE0089; Bio X cell, USA) (each group, n = 8). Murine GM-CSF (9 μg/kg), anti-PD-1 antibody therapy (10 mg/kg), or saline was administered intraperitoneally from day 7, every 3 days, after tumor implantation [34,35,36,37]. Survival analysis was performed using Kaplan-Meier analysis and log-rank test.
Patients and tissue specimens
Clinical data and/or samples of 109 ENKTL patients, 4 peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) patients, and 4 angioimmunoblastic T-cell lymphoma (AITL) from September 1999 to March 2019 were collected from Sun Yat-sen University Cancer Center (Figs. 6B, 7B), including medical records, tumor samples, peripheral blood, and buccal swabs samples. The clinical characteristics were summarized in Additional file: Table S2. Clinical data were collected from pathology reports and unprocessed medical files. The study was conducted with the permission of the Ethics Committee of the Sun Yat-sen University Cancer Institutional Board, and all patients involved provided informed written consent.
Histology and immunohistochemistry (IHC)
For IHC staining of the xenografts, tumor tissues were fixed, embedded, and sectioned (3 μm thick). Immunohistochemistry staining for mouse tissues was performed following standard procedures [39]. Two qualified professional pathologists performed a subsequent blinded evaluation. PBS, instead of the primary antibody, was used as a negative control, and specimens were scored according to the intensity of the dye color and the number of positive cells. A staining index (values 0–12), obtained as the product of the intensity of mouse PD-L1, CD3, CD8, granzyme B, and human STAT5, JAK2, PD-L1-positive staining. The intensity of the dye color was graded as 0 (no color), 1 (light yellow), 2 (light brown), or 3 (brown), and the number of positive cells was graded as 0 (< 5%), 1 (5–25%), 2 (25–50%), 3 (51–75%), or 4 (> 75%). The intensity was graded as -, negative staining (0); +, mild expression (1–4); ++, moderate expression (5–8); +++, strong expression (9–12). The following antibodies were used for ENKTL tissues: Anti-Jak2 (phospho Y1007 + Y1008, Abcam, USA), Anti-STAT5 (phosphor Y694, Abcam, USA). The antibody against human PD-L1 was purchased from Cell Signaling Technology (USA). The following antibodies were used for mouse tissues: Anti-CD3 antibody (Abcam, USA), Anti-CD8 antibody (Abcam, USA), Anti-Granzyme B antibody (Abcam, USA). The antibody against mouse PD-L1 was purchased from Cell Signaling Technology (USA) [40].
Tumor digestion
Tumors were extracted and finely minced. Tumor tissue was additionally blended with Collagenase type IV (Absin, China) for 2 h, 37 °C. Tumors were extracted and re-suspension in PBS buffer containing 2% FBS for flow cytometric analysis.
Flow cytometry
Tumors were extracted and processed as described above before re-suspension in PBS buffer containing 2% FBS and PBS for flow cytometric analysis. Zombie NIR™ Fixable Viability Kit (Biolegend, USA) was applied to cells for 30 min on ice in the dark. Cells were washed and incubated with fluorochrome-conjugated antibody (anti-mouse CD45 Birliant violet 605, BioLegend; anti-mouse CD3 APC, BioLegend; anti-mouse CD8 FITC, BioLegend) at the manufacturer’s recommended dilution for 30 min on ice in the dark. For samples requiring intracellular staining, cells were fixed with Fixation/Permeablization Diluent (eBioscience cat. 00–5223-56) for 30 min at room temperature, washed twice with Permeablization Buffer (eBioscience cat. 00–8333-56), and incubated with antibody (anti-mouse Granzyme B PE, BioLegend; anti-mouse Ki-67 Alexa Fluor 700, BioLegend) in permeabilization buffer for 30 min at room temperature in the dark. Following staining, cells were washed again with permeabilizaton buffer, subsequently washed with PBS, and re-suspended in PBS buffer for flow cytometric analysis on the CytoFLEX LX Flow Cytometer. 50,000–100,000 cells were analyzed per sample per mouse using Beckman CytExpert Software.
Next-generation sequencing
Formalin-fixed paraffin-embedded (FFPE) tumor samples and matched peripheral blood or buccal swabs samples of fourteen patients diagnosed with ENKTL were used to extract DNA and detect mutations by targeted next-generation sequencing (NGS) with a panel of the coding sequence of 102 ENKTL-relevant genes (Additional file: Table S1) (GeneseeqOne, Nanjing Geneseeq Technology Inc., China). Sequencing was performed on the Illumina HiSeq4000 platform followed by data analysis as previously described [41].
Generation and expression of STAT5A/B constructs
Sanger sequencing was used to identify the STAT5A/B mutations in ENKTL cell lines, including NK-YS, SNK-6, and SNT-8. Next-generation sequencing (NGS) was used to identify the STAT5A/B mutations in ENKTL tumor samples. Wild-type STAT5A (STAT5AWT) and wild-type STAT5B (STAT5BWT) were amplified with PrimeSTAR MAX DNA Polymerase (TaKaRa Bio Inc., code no. R450A) using NK-92 cell line cDNA as the template inserted into the pReceiver-M12 vector. STAT5A/B mutations were generated from STAT5A/BWT constructs and confirmed by Sanger sequencing. Eukaryotic expression vectors harboring STAT5A/BWT or STAT5A/B mutations were introduced into NK-92 cells.
Statistical analysis
Statistical analysis was carried out using IBM SPSS statistics software or GraphPad Prism using Student’s t-test or one-way ANOVA or Dunnett’s test. All experiments were repeated in triplicate. Data are expressed as mean ± standard deviation (SD). Statistical significance was defined as P < 0.05.