The ethical aspects of the current study have been approved by Isfahan university of medical sciences ethics committee with ethics code IR.MUI.REC.1400.008.

Cell culture and treatment

HL-60 and THP-1 cell lines were purchased from Pasteur Institute (Tehran, Iran) and were cultured in Roswell Park Memorial Institute (RPMI) 1640 with 20% heat-inactivated FBS and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) (Bio idea, Iran). THP-1 is of monocytic origin and representative of AML M-5 (https://en.wikipedia.org/wiki/THP-1_cell_line) while HL-60 is representative of promyelocytic leukemia M-2 and M-3 (https://en.wikipedia.org/wiki/HL60). According to previous studies, THP-1 and HL-60 cell lines express PD-L1 which can be overexpressed by PMA stimulation [9, 10]. 1 × 106 of each cell line were seeded in each well of the cell culture plate and stimulated with 50 ng/ml phorbol 12-myristate 13 acetate (PMA, Sigma-Aldrich, St Louis, MO, USA) to increase PD-L1 expression. We divided each cell line into two groups including test and control. Both groups were initially stimulated with PMA to express PD-L1. After 24 h of incubation at 37ºC and 5% CO2, recombinant human PD-1 (CD279)-Fc chimera (carrier-free) (Biolegend, UK) was added to the medium of test group cells in the concentration of 80 ng/ml while the control group cells were not treated. In order to find the effect of PD-L1 stimulation.

Flow cytometry analysis

For detection of PD-L1 expression on the cells before their treatment with recombinant PD-1, Flow cytometric analysis was done 24 h after stimulation with PMA. This analysis was accomplished by a FACS Calibur instrument (Becton Dickinson Bioscience, San Jose, USA). Anti-human CD274 (PD-L1)-PE (Biolegend, UK) was applied to measure the expression of PD-L1 protein on HL-60 and THP-1 cells and after that, the results were analyzed using the Cell Quest Pro software (BD Bioscience, USA).

RNA extraction, cDNA synthesis, and RT-PCR

Total RNA of control and test group cells were extracted after 24, 48 and 72 h following PD-1 treatment using a total RNA extraction kit (Parstus, Inc Iran) according to the manufacturer’s instructions. cDNA synthesis was done immediately after RNA extraction by add Script cDNA synthesis kit (addbio, Korea). The resulting cDNA was applied for real-time quantitative PCR on a Step One Plus™ real-time DNA amplification system (Applied Biosystem, USA) with Ampliqon realQ plus master mix SYBER Green (Denmark) and specific primers. Specific primers for target genes and ACTB, as the housekeeping gene, were designed by Allele ID 7.0 and were ordered to be synthesized by Metabion company (Germany). The primers’ sequences are listed in Table 1.

Table 1 The sequences of specific primers that were used in the studyMetabolic analysisGas chromatography/mass spectrometry

Culture supernatants were harvested at the indicated time points (24, 48, and 72 hours after PD-1 treatment) and gas chromatography (GC) analysis was done using Agilent 7890A GC coupled with an Agilent 5975C mass detector with triple quadrupole mass analyzer and electronic ionization (EI) (Agilent Technology, USA). The gas chromatograph was prepared with an HP‐5 GC capillary column (30 m × 0.25 mm; film thickness 0.25 μm) and the oven temperature was started from 100 ºC, held for 2 min, raised by 12ºC/min up to 210°C, followed by 210–250°C by 3ºC/min with the total run time of 34 min. The carrier gas was helium at a flow rate of 2 ml/min and the MSD ChemStation was used as operating software. Before injection the samples into the device, were washed three times with hexane as a nonpolar detergent in order to extract fatty acids. Afterward, they were mixed with adequate amounts of sodium sulfate to eliminate retained water from samples. After GC was carried out, the samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer.

GSH analysis

In order to analyze NADPH production by the pentose phosphate pathway, the oxidation status of cells was examined by measurement of reduced glutathione (GSH). The HL-60 and THP-1cells were grown under the indicated culture conditions at 4×106 cells per 6ml per well in six-well culture plates.72 hours after PD-1 treatment, the cell groups of control and test were harvested and reduced glutathione (GSH) was measured using GSH kit (Navand Salamat, Iran) according to the manufacturer’s instructions.

Viability analysis (MTT)

For assessing the viability of HL-60 and THP-1 cells before and after treatment with recombinant PD-1, MTT (Methylthiazole tetrazolium) assay was applied. Briefly: 10 µl of a 5 mg/ml MTT (DNA biotech) solution in PBS buffer was added to each well of the 96-well plate and 5 × 104 cells were seeded in each well. After 4-h incubation at 37ºC and 5% CO2, detergent solution (DMSO) was added (100 µl) and Optical density (OD) was measured by a microplate reader (M680 Bio rad, USA) at 570 nm (reference wavelength 690 nm), and the viability of the cells was defined as a percentage compared with untreated (negative) control cells.

Statistical analysis

Statistical analysis was performed using SPSS 26.0 and Graph Pad Prism 9.1.1. The Kolmogorov–Smirnov test was done to assess the assumption of normality. According to that, the parametric methods were used. The two-way ANOVA and t-test were performed to compare the significance of the difference between two or more groups. P < 0.05 was considered to indicate a statistically significant result. Experiment data were presented as mean ± SD. All experiments were performed in triplicates (repeated three times).