Patients

This study was conducted in accordance with the ethical guidelines stipulated in the 2013 Declaration of Helsinki. The Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University (No.2023192) approved the histochemical analysis of samples from the patients. Written informed consent for the sample collection and use was obtained from all patients. All authors accessed, reviewed, and approved the data and contents of the manuscript. Cancer tissue and paired para-cancerous tissue of 93 TNBC patients from 2017 to 2018 were collected by the department of oncology of the Second Affiliated Hospital of Xi’an Jiaotong University to investigate the expression of IDO-1 in TNBC patients. The diagnosis of TNBC was histologically confirmed in 93 patients who were at least 18 years old as described previously [16]. The inclusion requirements included a minimum 3 month survival rate, adequate bone marrow, hepatic, and renal function, and signed informed permission. Incomplete case information, post-operative death from complications, and the presence of other cancers were all considered exclusion criteria.

Cell culture and reagents

HEK293T cells and human TNBC cell lines (MDA-MB-231 and HCC-1937) were obtained from American Type Culture Collection (Manassas, USA). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher, Waltham, USA) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher, Waltham, USA) at 37 °C in a humidified 5% CO2 incubator.

Plasmid construction and lentivirus infection

Short hairpin RNAs (shRNAs) targeting IDO-1 and its flanking control sequence were cloned in pLKO.1-EGFP-puro vector. The target sequence was as follows: shIDO-1#1: 5ʹ-CCATCTGCAAATCGTGACTAA-3ʹ, shIDO-1#2: 5ʹ-CGTAAGGTCTTGCCAAGAAAT-3ʹ, shIDO-1#3: 5ʹ-CGCTGTTGGAAATAGCTTCTT-3ʹ, shNC: 5ʹ-GGTTCTCCGAACGTGTCACGT-3ʹ. The qualified plasmids were then transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) into HEK293T cells for packaging. At 48 or 72 h following transfection, virus-containing supernatant was harvested and centrifuged to remove cell debris, and then stored at – 80 ℃ until use. MDA-MB-231 cells in the logarithmic growth phase were washed with phosphate-buffered saline (PBS), adjusted to a cell density of 5 × 105 cells/mL, and re-inoculated into a 6-well dish. The recombinant lentiviruses were used to infect the cells for 48 h, followed by selection of infected cells using puromycin.

Western blot

Cells were lysed in lysis buffer (50 mM NaCl, 50 mM EDTA, and 1% Triton X-100) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were then blocked with 5% nonfat milk diluted in PBS for 2 h at room temperature and incubated with the following primary antibodies: anti-IDO-1 (Cat.No.86630S, Cell Signaling Technology, Boston, USA); anti-HLA-G (Cat.No.66447-1-Ig, Proteintech, Wuhan, China), and anti-GAPDH (Cat.No.60004-1-Ig, Proteintech, Wuhan, China). Then, membranes were washed with PBS containing 0.05% Tween and incubated with secondary antibodies conjugated with HRP for 1 h at room temperature. The bands were developed using a chemiluminescence reagent (Thermo Fisher, Waltham, USA).

Flow cytometry

Primary NK cells were analyzed via FCM using antibodies as follows: APC-labeled anti-human CD56 antibody (Cat.No.362503, Biolegend, California, USA) and FITC labeled anti human CD3-antibody (Cat.No.300405, Biolegend, California, USA). Briefly, 5 × 105 cells were washed and resuspended in PBS buffer containing 2% bovine serum albumin and 0.2% sodium azide and incubated with either directly labeled antibody or unconjugated antibody (followed by directly labeled secondary antibody) for 30 min at 4 °C. After incubation, the cells were washed three times and analyzed using FCM (Becton, Dickinson and Company, New Jersey, USA).

In vitro NK cell expansion

All experiment protocols are approved by the Ethics Committee of the Fourth Military Medical University. NK cells were isolated from the peripheral blood of healthy adult donors. Written informed consent was obtained from all participants. PBMCs were isolated by Ficoll density gradient centrifugation, and NK cells were purified by negative magnetic selection using an NK Cell Isolation Kit (Cat.No.130-092-657, Miltenyi, Teterow, Germany) following the manufacturer’s instructions. NK cells were expanded in the RPMI 1640 medium (Thermo Fisher, Waltham, USA) supplemented with 10% FBS (Thermo Fisher, Waltham, USA) and 10 ng/ml anti-CD3 antibody (Cat.No.GTX79905, GeneTex, California, USA), and 15 ng/ml IL-2 (Cat.No.200-02, PeproTech, New Jersey, USA) and 50 ng/ml IL-15 (Cat.No.200-15, PeproTech, New Jersey, USA) were added every other day. Cells from expansion days 14–21 with purity > 90% were used for further experiments.

siRNA synthesis and transfection

The small interfering RNAs (siRNAs) targeting IDO-1 were synthesized by GenePharma (Shanghai, China). The siRNA was transfected into TNBC cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. The siRNA sequence used is as follows: siIDO-1 #1, 5′-CAAAGUAAUUCCUACUGUATT-3′; siIDO-1 #2, 5′-GUAUGAAGGGUUCUGGGAATT-3′; and negative control, 5′-UUCUCCGAACGUGUCACGUTT-3′. Cells were collected for analysis 48 h after transfection with indicated siRNA.

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Thermo Fisher, Waltham, USA) following the manufacturer’s instruction. RNA was reverse transcribed using the Prime-Script RT Reagent Kit (TaKaRa, Kusatsu, Japan). Quantitative PCR was conducted using a CFX96TM Real-Time PCR system (Bio-Rad, Hercules, USA) with SYBR Green Reagents (TaKaRa, Kusatsu, Japan). Gene expression levels were normalized to that of GAPDH. The primers used for real-time PCR were as follows: IDO-1-F:5ʹ-GCCAGCTTCGAGAAAGAGTTG-3ʹ, IDO-1-R:5ʹ-ATCCCAGAACTAGACGTGCAA-3ʹ; HLA-G-F: 5ʹ-CGTCCTGGGTCTGGTCCT-3ʹ, HLA-G-R:5ʹ-GTGGCTCCACAGATACCTG-3ʹ; GAPDH-F:5ʹ-AGGTCCACCACTGACACGTT-3ʹ, GAPDH-R:5ʹ-GCCTCAAGATCATCAGCAAT-3ʹ.

ELISA

Human IL-2 (Cat.No.1110202, Dakewe, Shenzhen, China), human TNF-α (Cat.No.1117202, Dakewe, Shenzhen, China) and human IFN-γ (Cat.No.1110002, Dakewe, Shenzhen, China) in the tissue supernatants or cell supernatants were quantified using commercially available ELISA Kits following the manufacturer’s instructions. Briefly, the supernatants were added to an ELISA Kit plate that was precoated with a specific antibody. A biotinylated secondary antibody was then added and the plate was incubated at room temperature for 2 h. The color development catalyzed by HRP was terminated with 2.5 mol sulfuric acid, and the absorption was measured at 450 nm. The protein concentration was normalized to the relative absorbance rate of the respective standard and expressed as the mean ± SD.

CCK8 assays

NK cell activity and the proliferation of IDO-1 knockdown TNBC cells or IDO-1 inhibitors (1-L-MT, Cat.No.447439, Sigma-Aldrich, Shanghai, China) treated TNBC cells were determined by CCK8 (Yeasen, Shanghai, China) according to the manufacturer’s instructions. Briefly, indicated cells in 96-well plates were incubated in 100 μl CCK-8 working solution at 37 °C for 2 h. The OD value was then measured by microplate reader (Thermo Fisher, Waltham, USA), at a wavelength of 450 nm. The killing rate of NK cells were estimated using the formula: [1 − (OD value of effector-target cell)/OD value of target cell] × 100%.

Histochemical analysis

Paraffin-embedded tissue sections were subjected to IHC staining as described previously with minor modifications [17]. Briefly, the slides were deparaffinized in xylene and rehydrated in a graded alcohol series, and the endogenous peroxidase activity was blocked with 3% H2O2. Nonspecific binding sites were also blocked using pre-immune rabbit serum before incubation of the sections overnight in a humidity chamber at 4 °C with the following primary antibodies: anti-IDO-1 (Cat. No. ab156787, Abcam, Cambridge, UK). Slides were then washed three times with PBS, followed by incubation with a biotinylated secondary antibody for 30 min at room temperature. Signal was visualized by incubation with 3,3ʹ-diaminobenzidine chromogens for 2–3 min. The IHC results were quantified according to the staining intensity and extent as previously described [18]. Briefly, the staining intensity was scored from 0 to 3 points (0 for no staining, 1 for weak immunoreactivity, 2 for moderate immunoreactivity and 3 for strong immunoreactivity). The score for positive percentage was determined as follows: 0 for all negative cells, 1 for < 25% positive cells, 2 for 25–50% positive cells, 3 for 50–75% positive cells, and 4 for more than 75% positive cells. The final score was obtained by multiplying the intensity score and positive proportion score, and ranged from 0 to 12. For immunofluorescent staining of tumor tissues, the sections were deparaffinized and blocked as aforementioned, and were incubated at 4 °C overnight with primary antibodies against IDO-1 (Cat. No. ab156787, Abcam, Cambridge, UK), CD69 (Cat. No. PA5-102562, Invitrogen, Carlsbad, USA) and NKG2D (Cat. No. PA5-97904, Invitrogen, Carlsbad, USA). Subsequently, incubation with goat anti-mouse IgG H&L (Alexa Fluor® 488) (Cat. No. ab150113, Abcam, Cambridge, UK) and goat anti-rabbit IgG H&L (Alexa Fluor® 594) (Cat. No. ab150080, Abcam, Cambridge, UK) was performed for 1 h at 37 °C in the dark. Nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole. Fluorescent images were acquired by a confocal laser-scanning microscope (Ti2-E-A1, Nikon, Tokyo, Japan).

Analysis of in vivo tumor growth

Animal experiments were approved by and conducted in full compliance with the regulations of the Institutional Animal Care and the Ethics Committee of the Fourth Military Medical University (No. 20220830 and KY20224215). Nude mice (6 weeks old, 18 ± 0.39 g weight, female) were purchased from Hunan Slater Jingda Experimental Animal Co., Ltd. All mice were fed autoclaved food and water. The right groin of mice was injected with 5 × 106 control cells or IDO-1 knockdown MDA-MB-231 cells for the development of TNBC xenografts. Mice were randomly assigned into three groups receiving treatment with PBS, NK cells alone, or NK cells combined with an HLA-G-blocking antibody (1 mg/kg, Cat. No. MAB11223, Abnova, Taiwan, China) (n = 6 in each group). When tumor volume reached 200 mm3 (defined as day 0), NK cells and the antibody were administered via tail vein on days 0, 3 and 6. Tumor diameter was measured using a Vernier caliper every 5 days from day 5 until day 23. In order to analyze the anti-tumor efficiency of NK cells and HLA-G blocking antibody, mice were sacrificed on day 23. Tumor size was estimated using the formula: tumor volume = length × width2/2, where the length and width represented the longest and shortest tumor diameters.

Statistics

SPSS 20.0 software was used for data processing and GraphPad Prism 8.0 for data analysis and graphing. Using the t test (unpaired), the data from the two groups were compared. For three groups and more than three groups, one-way ANOVA was utilized. Data were provided as mean ± standard deviation, and P < 0.05 denoted a significantly difference in the outcomes.