Male NSCLC patients and healthy controls between the age of 40–55 were enrolled in this study. The median age was 46.8 years (range: 40–54) in NSCLC patients (n=13), and 45.3 years (range: 41–52) in healthy controls (n=15). Inclusion criteria for the present study were patients with histological proven NSCLC staging II–IV, who were primarily diagnosed in The Third Affiliated Hospital of Sun Yat-sen University between January 2017 and December 2017. All enrolled patients had no previous treatment with molecular target therapy, chemotherapy or radiotherapy. Exclusion criteria were chronic systemic diseases (including hypertension, diabetes and coronary heart disease) or immune systemic diseases (including HIV, organ transplantation and tumors). All subjects joined voluntarily with informed consents. This present study received ethical approval from the Institutional Review Board of Sun Yat-sen University.

Blood samples (50 ml) were obtained from each subjects. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (GE Healthcare Life Sciences, Little Chalfont, UK) density gradient centrifugation at 1,200 × g and 4°C for 20 min. PBMCs were cultured for 2 h and non-adherent cells were removed. The DC cell culture medium was prepared by adding recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng/ml) and rh interleukin (IL)-4 (100 ng/ml) to RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). PBMCs were incubated for 8 days at 37°C and prepared for subsequent experiments.

Cells were treated with lipopolysaccharide (LPS; 100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml) + LPS (100 ng/ml) for 48 h. According to preliminary experiments in the present study, oxymatrine inhibited the proliferation of dendritic cells, and the effect of inhibiting proliferation was observed at OMT concentration of 0.8 mg/ml. However, the inhibitory effect was not significantly increased with the increase of concentration, so the concentration of OMT in this experiment was chosen to be 1.0 mg/ml (data not shown). The cultured DCs were collected and centrifuged at 1,000 × g for 5 min at 4°C. The supernatant was discarded and the cells were washed three times with PBS. In total, 5 µl CD83 antigen (CD83)-phycoerythrin (PE; 556855; BD Biosciences, San Jose, CA, USA), T-lymphocyte activation antigen CD86 (CD86)-PE (560957; BD Biosciences), CD11 antigen-like family member C (CD11c)-PE (555392; BD Biosciences) and major histocompatibility complex II (MHC II)-PE(555812, BD Biosciences) were added to each tube. Subsequent to gentle mixing, the cells were incubated at room temperature in the dark for 30 min. The cells were washed twice with PBS and resuspended in 100 µl PBS, and the DC surface markers were detected using a flow cytometer (BD Accuri C6; BD Biosciences).

A total of 10 patients with newly diagnosed NSCLC were enrolled to collect 20 ml blood prior to any treatment. Subsequently, PBMCs were isolated using the Ficoll density gradient method at 1,200 × g, 4°C for 20 min. In total, 1×10⁶ cells were used to isolate CD₄⁺ T cells using a magnetic cell separation system. The selected cells were cultured in RPMI-1640 medium containing 10% FBS for subsequent experiments.

The DCs were treated with control, LPS (100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml)+LPS (100 ng/ml) for 48 h. Cells were subsequently centrifuged at 1,000 × g for 5 min at 4°C and the supernatant was discarded. Subsequent to washing three times with PBS, the DCs and peripheral blood-derived CD₄⁺ T cells from patients with NSCLC were implanted into Matrigel-coated Transwell chambers; DCs were seeded into the lower chambers and CD₄⁺ T cells into the upper chambers at 1:5, 1:10 and 1:20 ratios. Cells were cultured in RPMI-1640 medium containing 10% FBS for 48 h; a non-contact cell co-culture system was established.

DC cells and T cells were co-cultured at a ratio of 1:5, 1:10 and 1:20, respectively, as described above. CD₄⁺ T cells were collected and total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. cDNA were synthesized using PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan), strictly following the manufacturer's protocols. The target genes and controls were analyzed by RT-qPCR using SYBR-Green PCR Master Mix (Toyobo Life Science, Osaka, Japan) and the reactions were performed on ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers used in the study were as follows, U6 (housekeeping) primers: 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (forward) and 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (reverse); FOXP3 primers: 5′-CTGACCAAGGCTTCATCTGTG-3′ (forward) and 5′-ACTCTGGGAATGTGCTGTTTC−3′ (reverse). Reagents in the reaction included 0.5 µl of cDNA (1:20), 0.5 µl of forward primer and 0.5 µl of reverse primer, 10 µl of PCR Master Mix and 4.0 µl dH₂O. Thermocycling conditions for PCR were 95°C for 5 min, and 40 cycles of 95°C for 15 sec, 65°C for 15 sec and 72°C for 32 sec. The average value was calculated by the relative quantitative method, and the gene expressions in the treatment group relative to the control group were calculated by the formula 2^−ΔΔCq following Livak's method (16).

The co-cultured CD₄⁺ T cells were collected and centrifuged at 1,000 × g for 5 min at 4°C; the supernatant was discarded. Anti-Human FoxP3 Staining kit with CD4-Fluorescein Isothiocyanate and FOXP3-PE (560132; BD Biosciences) was used in the following experiments. The cells were washed three times with PBS, and CD4-Fluorescein Isothiocyanate (1:20) was added to each tube and incubated for 30 min at room temperature in the dark. For antibodies to enter the intracellular structure, cells were resuspended in fixation/permeabilization buffer (00–5223/00-5123; eBioscience; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Following two washes with PBS, the cells were incubated for 30 min at room temperature in the dark. FOXP3-PE (1:20) was added and incubated for another 30 min in the same condition. Cells were subsequently washed twice and resuspended with the fixation/permeabilization buffer. The ratio of FOXP3⁺/CD₄⁺ cells was detected using a flow cytometer (BD Accuri C6; BD Biosciences).

The DCs were treated with control, LPS (100 ng/ml), OMT (1 mg/ml) and OMT (1 mg/ml) + LPS (100 ng/ml), and mixed with peripheral blood-derived CD⁴⁺ T cells from patients with NSCLC at ratios of 1:5, 1:10 and 1:20. After 48 h, the supernatant of the co-culture system was collected. The concentrations of IL-10, transforming growth factor (TGF)-β, IL-35, interferon (IFN)-γ, IL-2 and IL-12 in the supernatant were detected using ELISA kits: IL-10 (E-EL-H0103c), TGF-β (E-EL-0110c), IL-35 (E-EL-H2443c), IFN-γ (E-EL-0108c), IL-2 (E-EL-H0099c) and IL-12 (E-EL-H0150c; all from R&D Systems, Inc., Minneapolis, MN, USA).

The induction of A549 resistant cells was performed by gradually increasing the concentration of cisplatin from a starting concentration of 1.0 µM, and after 4 weeks, it was increased to 2.0 µM, and serially subcultured for ~100 passages. Then, 6 months later, the A549/DDP resistant cell line was established. The human lung adenocarcinoma cells A549 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China; cat. no. CL-0016). The cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) and 1% penicillin-streptomycin solution (Beyotime Institute of Biotechnology, Haimen, China), and cells were cultured in a humidified atmosphere at 37°C with 5% CO₂. Co-culture systems of A549/DDP plus DC or DC-T were established. OMT (1 mg/ml)-treated DCs and the FOXP3⁺/CD₄⁺ T cells induced by DC-T co-culture (cell ratio, 1:5) were incubated with A549/DDP cells. The cells were seeded for 24 h in the upper and lower chambers of Transwell plates at ratios of 1:1, 1:5, 1:10 and 1:20. At the same time, 22 µM DDP was added to the A549/DDP medium for 24 h. The collected cells were stained using an Annexin V/Propidium Iodide kit (BD Biosciences) according to the manufacturer's protocol, and the apoptotic ratio was analyzed using a flow cytometer (BD Accuri C6; BD Biosciences).

A549/DDP cells were lysed using radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1% NPNP-40, 1% sodium deoxycholate and 0.05% SDS; pH 7.4) following co-culture with OMT-treated DCs/DC-T and treatment with DDP. Total protein was extracted and subsequently quantified using a bicinchoninic acid kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For western blotting, protein loading was set at 40 µg and an 12% SDS-PAGE gel was used for protein separation. The proteins were transferred to a nitrocellulose membrane and incubated with primary antibodies against FOXP3 (1:3,000; cat. no. ab191416), apoptosis regulator BAX, (Bax; 1:1,000; cat. no. ab32503), B cell lymphoma-2 (Bcl-2; 1:1,000; cat. no. ab32124) and β-actin (1:1,000; cat. no. ab8224) overnight at 4°C, and subsequently incubated with Goat Anti-Rabbit (1:1,000; cat. no. ab6702) or Anti-Mouse (1:1,000; cat. no. ab6708) (all from Abcam, Cambridge, UK) secondary antibodies for 1 h at room temperature. Chemiluminescence was used to develop the color of the membrane using an ECL kit according to the manufacturer's instructions (cat. no. FD8030; Guangzhou Fude Biological Technology Co., Ltd., Guangzhou, China). ImageJ 8.0 software (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometric analysis.

Each experiment was repeated at least 3 times. Data were presented as the mean ± standard deviation. Statistical significance was determined by one-way analysis of variance with Bonferroni Correction using SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The monocytes were prepared from the peripheral blood of healthy adult volunteers. To investigate the effect of OMT, the immature DCs were cultured for 8 days containing 1 mg/ml OMT in medium supplemented with rhGM-CSF (100 ng/ml) and rhIL-4 (100 ng/ml). Bacterial LPS is the most common ‘maturation’ signal for DCs (17). With the LPS-treated (100 ng/ml) group (LPS-DCs) as a positive control and mononuclear cells as a negative control, the expression of DC surface markers was detected by flow cytometry analysis, as presented in Fig. 1. Compared with the negative control group, treatment with OMT (1 mg/ml) significantly increased the expression of CD83, CD86, CD11c and MHC II (P<0.001), and the expression level of these markers was higher when LPS and OMT were combined. These results demonstrated that OMT may promote DC maturation.

To examine the response of allogeneic primary CD₄⁺ T cells to DC stimulation pretreated with OMT (1 mg/ml; OMT-DC), the following experiments were performed. CD₄⁺ T cells were isolated from the peripheral blood of patients with NSCLC. DCs were co-cultured with the primary CD₄⁺ T cells for 48 h in Transwell plates at ratios of 1:5, 1:10 and 1:20. The expression levels of FOXP3 in CD₄⁺ T cells were analyzed by flow cytometry, western blotting and qPCR.

As presented in Fig. 2, OMT-DCs stimulated the primary CD₄⁺ T cells to express FOXP3, and the mRNA expression level of FOXP3 decreased as the DCs were more diluted. The FOXP3 protein assay showed that the total amount of FOXP3 was increased when LPS and OMT were used separately or in combination (especially when used in combination). However, in the case of the same treatment, the dilution of DC cells had no apparent effect on FOXP3 protein amount when groups of different DC cell dilution ratios were compared among each other. Whether there are translation level and turnover level factors regulating the expression of FOXP3 protein in this case deserves further investigation. Altogether, these results demonstrated that OMT-pretreated DCs may enhance FOXP3⁺/CD₄⁺ Treg differentiation.

Inflammatory factors serve an important role in the immune response. ELISA was used to quantitatively analyze inflammatory factors in the co-culture supernatants. When OMT-DCs were co-cultured with T cells, a large number of anti-inflammatory factors, including IL-10, TGF-β and IL-35, and pro-inflammatory cytokines, including IFN-γ, IL-12 and IL-2, were secreted. Furthermore, the expression levels of those inflammatory factors decreased as the DCs were more diluted with T cells (Fig. 3). When LPS-DCs were co-cultured with T cells, it additionally promoted the secretion of inflammatory factors, in a similar manner to OMT-DCs. When LPS and OMT were applied simultaneously, the secretion of inflammatory factors reached the highest concentration. These results suggested that OMT-pretreated DCs may promote the primary T cells to polarize into FOXP3⁺/CD⁴⁺ Tregs, and enhance the anti-inflammatory and pro-inflammatory cytokines secretion. Based on these results, the co-culture ratio of DCs to CD⁴⁺ T cells was set at 1:5 for subsequent experiments.

A DDP-resistant cell line, A549/DDP, was established. As presented in Fig. 4A, A549/DDP has a half maximal inhibitory concentration of 22 µM for DDP. In order to investigate the effect of OMT-pretreated DCs and OMT-DCs induced CD⁴⁺ T cells on the sensitivity of A549/DDP to antitumor drugs, OMT-stimulated DCs (OMT-DC) and OMT-DC-stimulated primary CD⁴⁺ T cells (OMT-DCs-T) were co-cultured with the A549/DDP cells at ratios of 1:1, 1:5, 1:10 or 1:20 for 24 h. At the same time, the A549/DDP cells were treated with 22 µM DDP for 24 h. Annexin V/PI staining was used to analyze the apoptosis of the A549/DDP cells.

As presented in Fig. 4B, OMT-DC and OMT-DC-T may increase the percentage of apoptotic cells in A549/DDP, and the apoptotic rate decreased accompanied by the gradual dilution of the OMT-DCs/OMT-DCs-T with A549/DDP.

The western blotting results (Fig. 5) demonstrated that the expression of apoptosis-associated protein Bcl-2 increased as the A549/DDP cell apoptosis decreased; however, the protein expression level of Bax decreased. These results suggested that OMT-pretreated DCs and DC-induced T cells may reverse DDP-resistance in a non-contactable co-culture cell model.