Chemicals and reagents

CEP (HPLC purity: 99.1%, batch number: 0483686) was purchased from the Cayman Chemical Company. Methylprednisolone (MP) and verapamil (VP) were obtained from Sigma-Aldrich (St. Louis. Mo., USA). CEP, MP, and VP were dissolved in ethanol and stored at 4℃ until use. ConA was purchased from Seikagaku Kogyo Co. Tokyo, Japan. The WST-8 reagent was provided by Dojindo Molecular Technologies, Inc., Tokyo, Japan. The BD Cytometric Bead Array Human Th1/Th2/Th17 Cytokine Kit, FITC mouse anti-human CD4, APC mouse anti-human CD8 and CD25, and Alexa Fluor® 488 mouse anti-human Foxp3 antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-P-glycoprotein antibody was purchased from Kamiya Biomedical Company, Seattle, USA (1 μg/mL, # MC-208). Anti-GR antibody was obtained from Santa Cruz Biotechnology, INC, Texas, USA (G-5, dilution:1:1000, # sc-393232). The anti-β-actin antibody was provided by Proteintech Group, Rosemont, USA (1:5000, # 66009-1-lg). The anti-TATA binding protein (TBP) antibody was purchased from Abcam (Cambridge, UK; dilution:1:1000, # ab818). RPMI-1640 and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). All other reagents used were of the highest quality available from commercial vendors.

Subjects

This study was approved by the Ethical Committee of Tokyo University of Pharmacy and Life Sciences, and written informed consent was obtained from all healthy subjects included in the study. The study included eight healthy subjects (four males and four females with a mean age of 34 years). These subjects had neither a history of immunological disorders nor a history of immunosuppressive drug use.

Isolation of PBMCs and evaluation of drug effects in vitro

PBMCs were isolated from 20 mL of venous blood from healthy subjects as described previously [14,15,16,17]. In brief, heparinized blood was loaded into 4 mL of Ficoll-Hypaque (Nakarai Co., Japan) and centrifuged at 1300 × g for 20 min. PBMCs were collected, washed, and resuspended in RPMI 1640 medium containing 10% FBS, 1000,000 IU/L penicillin, and 100 mg/L streptomycin.

The density of the PBMC suspension was maintained at 1 × 106 cells/mL. The cell suspension (196 μL) with or without the addition of 5 μg/mL ConA was seeded into the wells of a 96-well plate. Then, 2 μL of MP solution was added to obtain final MP concentrations of 0.1, 1, 10, and 100 ng/mL. Subsequently, 2 μL of the CEP solution was added to obtain final CEP concentrations of 0.03, 0.3, 1, and 3 μM. 4 μL of ethanol was added to the control wells. After incubation for 3 days in 5% CO2 at 37℃, 10 μL of WST-8 assay reagent solution was added to each well. The plates were further incubated for 3 hours. Optical density was measured at 450 nm (ref. 650 nm). The percentage of PBMC viability was calculated using the following formula: (Test‐Blank)/(Control‐Blank) × 100 (%). The IC50 values of MP were determined using GraphPad Prism 8 [16, 17]. The combination index was calculated using CompuSyn software, and a combination index < 1, 1, and > 1 represents synergistic, additive, and antagonistic effects, respectively [18].

Human T lymphoblastoid leukemia cell culture

MOLT-4 cells, which express low levels of P-glycoprotein, were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). MOLT-4/DNR cells, which are known to express large amounts of P-glycoprotein, were developed from the MOLT-4 cell line in our laboratory by exposing the parent cells to increasing concentrations of daunorubicin (DNR) over 3 months [18]. The expression of functional P-glycoprotein in these two cell lines was identified in our previous study [18]. MOLT-4 and MOLT-4/DNR cells were maintained in RPMI 1640 medium containing 10% FBS, 1000,000 IU/L penicillin, and 100 mg/L streptomycin [18].

Evaluation of P-glycoprotein function

P-glycoprotein function in PBMCs, MOLT-4 cells, and MOLT-4/DNR cells was detected by rhodamine 123 (Rh123) efflux assay with flow cytometry, as described previously [16,17,18].

Briefly, PBMCs were incubated with 2 μM of Rh123 for 10 min in 5% CO2 at 37℃. After uptake of the dye, the cells were resuspended in Rh123‐free complete media with or without drugs. Subsequently, the cells were incubated for 180 min at 37°C to remove the dye out of cells. VP (5 μM) was used as the positive control. After the efflux period, PBMCs were incubated with FITC mouse anti-human CD4 and APC mouse anti-human CD8 antibodies. The cells were then resuspended in phosphate buffered saline (PBS) and kept on ice in the dark until analysis using FACS Canto TM II, as described previously (BD Biosciences, San Jose, CA, USA) [16, 17]. The percentage of Rh123 accumulation was determined to evaluate P-glycoprotein function of PBMCs.

After the cells were incubated with 2 μM of Rh123 in the presence or absence of drugs, Rh123 accumulation (%) during the dye-uptake period was determined to evaluate the P-glycoprotein function of MOLT-4 and MOLT-4/DNR cells. 1 mL of cell suspension containing 5 × 105 cells was incubated with 2 μM Rh123 in the presence or absence of 0.03, 0.3, and 1 μM CEP for 1 h. After staining, the cells were washed and resuspended in ice-cold PBS. The intracellular Rh123 mean fluorescence intensity was examined using a FACS Canto TM II [18]. Data were analyzed using FlowJo software, version 10.4 [18].

CD4+CD25+Foxp3+ regulatory T cell analysis

After PBMCs were cultured for 3 daysas described above, the culture supernatants were collected and stored at -80°C for measurement of cytokine concentrations (see below). PBMCs were stained with 10 μL FITC mouse anti-human CD4 and 10 μL APC mouse anti-human CD25 antibodies for 20 min at 37°C in the dark. After incubation, the cells were treated with human Foxp3 buffers A and B according to the manufacturer’s instructions. Next, 10 μL of Alexa Fluor® 488 mouse anti-human Foxp3 antibody was added, and the cell suspension was incubated for 30 min at 37°C in the dark. After washing the cells with PBS, 0.4 mL of staining buffer was added to the cell suspension, and then analyzed by flow cytometry. The data were analyzed with FACSCanto ™ II (BD Biosciences) using the BD FACSDiva™ software. CD4+ T cells in the lymphocyte fraction were gated, and the percentages of CD4+CD25+ and CD4+CD25+Foxp3+ cells in the CD4+ cell fraction were calculated as previously described [16].

Evaluation of cytokines with cytometric bead array assay

The culture supernatant obtained as mentioned above was subjected to measure the concentrations of Th1/Th2/Th17 cytokines, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α, by using Cytometric Bead Array assay, followed by flow cytometry, according to the manufacturer’s instructions [15, 16].

Western blot analysis

Cytoplasmic and nuclear proteins were extracted using the Thermo Scientific NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA). Whole cell protein was extracted using radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (#A32961, Thermo Scientific). The protein concentration was quantified using the Pierce BCA Protein Assay Kit (#23227, Thermo Scientific). Western blotting was performed as described previously [17, 18]. 10 μg of whole cell or cytoplasmic protein lysate and 5 μg of nuclear protein lysate were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes respectively. The blots were cut prior to hybridisation with antibodies. The membrane signals were detected using a luminescent image analyzer (Fujifilm; LAS-3000; Fujifilm, Tokyo, Japan). Quantitative densitometry data were evaluated using ImageJ software (version 1.52e, National Institutes of Health, USA; http://imagej.nih.gov/ij) [17, 18].

Statistical analysis

Differences between the values of drug-free controls and those obtained in the presence of serial concentrations of drugs were analyzed using Dunn's multiple comparison test. These analyses were performed using GraphPad PRISM 8.0 (GraphPad Software Inc., San Diego, CA, USA). In each case, a two-sided P value <0.05 was considered significant.