Human synovial tissue samples were collected from RA patients (n=7, 4 females, 30-68 years old, mean age of 54) during knee joint arthroscopic synovectomy procedures. The diagnosis conformed to the revised criteria of the American College of Rheumatology. The RA patients were medicated with nonsteroidal anti-inflammatory drugs to help reduce the pain and swelling of the joints and to decrease stiffness. Patients were enrolled between June 2017 and October 2018 at Shandong Provincial Qianfoshan Hospital. All of the patients signed written informed consent statements for participation in the study. The study protocol was approved by the Medical Ethical Committee of Shandong Provincial Qianfoshan Hospital at Jinan (Approve number：20170306), China.
Induced amplification of Treg cells
Healthy human (n=7) peripheral blood was aseptically collected. The volunteer signed written informed consent for participation in the study. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation and were then plated in a culture flask coated with 5 µg/ml anti-human-CD3 antibody (Sungene, China). Treg cell differentiation and amplification were induced in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, USA) containing TGF-β (PeproTech, USA), recombinant human IL-2 (T&L, China), anti-human CD28 antibody (Sungene, China) and 10% fetal bovine serum (FBS, Gibco, USA). Fresh growth medium was added every 2 days, and the first round of amplification was completed after 6 days. The cells were transferred to a culture flask without the antibody and were further cultured for 2 days in DMEM containing recombinant human IL-2 and 10% FBS. After 2 days, the cells were transferred to a culture flask coated with anti-human CD3 antibody and were amplified following the same method used for the first amplification cycle. The entire process required 14 days.
Assessment of Treg cell population expansion by flow cytometry
One-hundred microliter samples of the unamplified PBMCs and the Treg cell suspension following 14 days of amplification were collected, and anti-human CD25-fluorescein isothiocyanate (FITC, BioLegend, USA) and anti-human CD4-PerCP (BioLegend, USA) antibodies were added. The samples were then incubated in the dark for 30 min at 4°C. The cells were washed twice with phosphate-buffered saline (PBS) and then resuspended in PBS to form single-cell suspensions. Flow cytometry was used for detection (ACEA, USA) and, after phenotypic characterization, 5 Treg cell samples were pooled in equal proportions for the subsequent experiments.
Primary culture of RASFs
RA synovial tissue samples were aseptically disrupted, and DMEM containing 4% type II collagenase (Solarbio, China) was added, and the samples were then incubated in a 5% CO2 at 37°C for 4 h until the tissue pieces were dispersed into a cell suspension. The cell suspension was filtered through a 70-μm cell strainer and then centrifuged at 1200 rpm for 5 min. The cells were resuspended in DMEM containing 10% FBS and incubated at 37°C in a 5% CO2 incubator to obtain primary RASFs. Cells passaged for 3-8 generations were used in the subsequent experiments.
Assessment of RASF amplification via the cell counting kit-8 (CCK-8) assay
A single-cell suspension of RASFs was seeded into 96-well plates. Induced mature Treg cells were washed with PBS and seeded into the 96-well plates at a 1:1 ratio of RASFs to Treg cells. The cells were cocultured for 24-72 h. At the end of the coculturing, the suspended Treg cells were washed away with PBS, and CCK-8 (Dojindo, Japan) solution was added to the plates, which were then incubated for 3 h. The OD450 values were measured using a microplate reader. The data were statistically analyzed using an independent samples t-test (BioTek, USA).
Assessment of RASF apoptosis using annexin V-propidium iodide (PI)/FITC
A single-cell suspension of RASFs was seeded into 6-well plates. Induced mature Treg cells were seeded into the 6-well plates at a 1:1 ratio of Treg cells to RASFs, and the cells were cocultured for 48 h. At the end of the coculturing, the suspended Treg cells were washed away with PBS, and a single-cell suspension of RASFs was collected and resuspended in binding buffer. FITC-conjugated antibody and PI-conjugated antibody (BioLegend, USA) were added, and the samples were then mixed well and incubated at room temperature for 15 min in the dark. Apoptosis was assessed by flow cytometry. The data were statistically analyzed using an independent samples t-test.
Assessment of the effects of coculture on cytokine levels via flow cytometry
A single-cell suspension of RASFs was seeded into 6-well plates. Induced mature Treg cells were seeded into the 6-well plates at a 1:1 ratio of Treg cells, and the cells were cocultured for 48 h. After the coculturing, the supernatant was collected, centrifuged at 1500 rpm for 10 min, and assayed for changes in cytokine levels using a human Th1/Th2 cytokine assay kit (Cell-Genebio, China). The data were statistically analyzed using one-way analysis of variance (ANOVA).
Treg cell treatment of CIA rats
A total of 30 rats (six week old) were purchased from Shandong Laboratory Animal Center (Jinan，China) and were randomly divided into a normal control (NC) group, a CIA group and a Treg treatment group (n=10 rats per group). The CIA rat model was established in all of the groups except for the NC group. Bovine type II collagen (Chondrex, USA) was mixed with complete Freund's adjuvant (Sigma, USA) in equal amounts and the mixture was fully emulsified. The initial immunization with 0.2 ml of emulsion per rat was performed by intracutaneous injection into the tail root. One week later, bovine type II collagen was mixed with incomplete Freund's adjuvant (Sigma, USA) in equal amounts and the mixture was fully emulsified. A booster immunization was performed via intracutaneous injection of 0.2 ml of emulsion per rat into the tail root. The rats in the Treg treatment group were injected with Treg cells (107 Treg cells/kg) via the tail vein simultaneously with the booster immunization, and the injection was repeated again one week later. The NC and CIA groups were injected with the same dose of PBS at the same time points. All mice were humanely euthanized by a lethal dose of ketamine and xylazine。
The breeding and operation of the experimental animals were carried out in accordance with the Helsinki Convention on Animal Protection and the Regulations of the People's Republic of China on the Administration of Experimental Animals. The study protocol was approved by the Medical Ethical Committee of Shandong Provincial Qianfoshan Hospital at Jinan (Approve number：20170306), China.
Evaluation of the general CIA conditions
After the start of treatment, the degree of ankle joint swelling was measured with a Vernier caliper, and the joints were photographed every 3 days. At the end of the experiment, an inflammation curve was plotted based on the degree of joint swelling over time. The data were statistically analyzed by repeated measurements ANOVA.
The experiment was terminated on the 20th day after the booster immunization, following the start of treatment. Tissue samples located 1 cm above and below the right knee joint were collected, fixed in 4% paraformaldehyde (Solarbio, China) for 48 h, decalcified with EDTA and embedded in paraffin. Pathological changes in the joint tissue samples were observed under light microscopy after hematoxylin-eosin (HE) staining.
Assessment of the lymphocyte subsets in the peripheral blood and spleen of rats
The experiment was terminated on the 20th day after treatment. The animals were anesthetized via intraperitoneal injection of 30 mg/kg 3% sodium pentobarbital. For sample collection, each rat was fixed on its back, the abdominal cavity opened, and a blood sample was collected from the inferior vena cava. The spleen was removed and placed in 2 ml PBS where it was then cut into pieces with ophthalmic scissors and filtered through a strainer to obtain a single-cell suspension. Samples of both the peripheral blood and the spleen single-cell suspensions were collected after red blood cell lysis using red blood cell lysis buffer. Next, anti-rat CD4-FITC (BioLegend, USA) and anti-rat CD25-phycoerythrin (PE, BioLegend, USA) were added for the detection of Treg cells, anti-rat CD3-allophycocyanin (APC, BioLegend, USA) and anti-rat CD161-FITC (BioLegend, USA) were added for the detection of NK cells, and anti-rat CD3-APC (BioLegend, USA) and anti-rat CD45RA-PE (BioLegend, USA) were added for the detection of B cells. The samples were incubated at 4°C for 30 min in the dark, washed twice with 500 μl PBS, and examined after resuspending in PBS. The data were statistically analyzed using one-way ANOVA.
Assessment of cytokine concentrations in rats
The obtained blood samples were coagulated at room temperature for at least 30 min and then centrifuged at 3000 rpm for 20 min at 4°C to obtain serum. Changes in cytokine expression levels in the peripheral blood were detected by flow cytometry using a rat Th1/Th2 cytokine assay kit (BioLegend, USA). Th1 cells are characterized by interferon gamma (IFN-γ) secretion, and Th2 cells mainly secrete IL-4. The Th1/Th2 ratio was calculated based on the average fluorescence intensities detected for IFN-γ and IL-4. The data were statistically analyzed using one-way ANOVA.
Normal and variance homogeneity tests were performed using SPSS 17.0 software, and data that met the test criteria are represented as x s. Independent and paired sample t-tests were used to determine significant differences between two groups. Repeated measurements and one-way ANOVA were used to determine significant differences between multiple groups. The least significant difference (LSD) method or the Tamhane method was used for pairwise comparisons. P<0.05 was considered statistically significant.