Soluble PD-1 aggravates progression of collagen-induced arthritis through Th1 and Th17 pathways

A total of 83 patients with RA were included in the study (Table 1). All patients fulfilled the American College of Rheumatology criteria for RA. This group included 61 females and 22 males with mean disease duration of 12.1 ± 8.0 years. The mean age of the patients was 58.30 ± 13.01 years. They were recruited from inpatient and outpatient clinics at the rheumatology departments of the First and Third Affiliated Hospitals of Soochow University. Disease history was recorded for all patients, including presenting symptoms, affected joint counts, and medication history. The activity of disease was evaluated by calculation of 28-joint Disease Activity Score (DAS28) [34]. The level of RA disease activity can be interpreted as low (Lo-RA; 2.6 ≤ DAS28 ≤ 3.2), moderate (Mo-RA; 3.2 < DAS28 ≤ 5.1), or high (Hi-RA; DAS28 > 5.1), and a DAS28 < 2.6 can be considered as remission (Re-RA), according to the European League against Rheumatism criteria. According to extraarticular involvement, the subjects were divided into patients with RA with limited joint manifestations and those with extraarticular manifestations. Eight of the patients received methotrexate (MTX) therapy (10 mg/week for 20 weeks by oral administration, including follow-up periods of 16 and 32 weeks). None of the patients had received steroid or immunosuppressive drugs within 1 year before the study period. Complete sets of paired SF and peripheral blood were obtained from 15 of the 83 patients for paired analyses. Additional sets of SF and paired serum specimens (no cells) derived from the remaining 68 patients with RA were used only for analyses of protein concentrations of sPD-1 by enzyme-linked immunosorbent assay (ELISA). Complete sets of paired SF and peripheral blood samples from a total of 67 patients with osteoarthritis (OA) were also included in the study. Control PBMCs and sera were obtained from a group of 88 healthy individuals who were matched for sex ratio and mean age with the patient group from the same hospitals and who had not received immunosuppressive or immunomodulatory drugs for various reasons for at least 2 months before the time of sample collection. Informed consent was obtained from all subjects before sample collection. The study protocol and consent form were approved by the Institutional Medical Ethics Review Board of Soochow University. SF was centrifuged at 350 × g for 3 minutes, and supernatants were collected and immediately stored at −80 °C until use. Mononuclear cells were prepared by Ficoll-Hypaque separation (GE Healthcare Life Sciences, Little Chalfont, UK) in all cases from blood specimens of patients with RA and controls using the standard protocol.

PBMCs were isolated from patients with RA or controls. Cells were washed by centrifugation in RPMI 1640 medium and subsequently resuspended in cold phosphate-buffered saline (PBS) containing 2.5 % fetal bovine serum (FBS) at a cell density of 1 × 10⁷/ml. CD4⁺ T cells were prepared from freshly isolated PBMCs by depleting cells expressing CD8, CD14, CD16, CD19, CD36, CD56, CD123, γ/δ-T-cell receptors, and glycophorin A using No-Touch T-cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD4⁺ T cells was 95–98 % as determined by flow cytometry using specific antibodies.

Purified T-cell preparations derived from peripheral blood of patients with RA or controls were cultured at 1 × 10⁶/ml in RPMI 1640 medium containing 10 % FBS in the presence of 1 μg/ml anti-CD3 monoclonal antibody (mAb) (clone OKT-3; eBioscience, San Diego, CA, USA) and 0.05 μg/ml anti-CD28 mAb (clone CD28.2; eBioscience). Cells were maintained at 37 °C in a 5 % CO₂ atmosphere for 48 h and were then harvested for RNA extraction before real-time polymerase chain reaction (PCR) analysis.

PBMCs from healthy individuals were cultured in 24-well plates at 1 × 10⁶/ml in RPMI 1640 medium containing 10 % FBS in the presence or absence of a panel of recombinant human cytokines [interferon (IFN)-γ, interleukin (IL)-17A, or tumor necrosis factor (TNF)-α; R&D Systems, Minneapolis, MN, USA), respectively, at the indicated concentrations. Cells were kept in culture at 37 °C in a 5 % CO₂ atmosphere for 12 h or 24 h and were then harvested for isolation of CD4⁺ T cells before real-time PCR analysis.

Concentrations of sPD-1 were measured quantitatively in SF and sera using ELISA according to our established protocol. For sPD-1, Costar ELISA 96-well plates (Fisher Scientific, Pittsburgh, PA, USA) were precoated with the capture anti-PD-1 mAb (4B9) [31, 34] at 3 μg/ml in 0.05 M carbonate buffer solution (pH 9.6) overnight at 4 °C. The coating solution was aspirated off, and unoccupied binding sites on the plates were blocked with 2 % bovine serum albumin in PBS at 37 °C for 1 h. After being washed three times with PBS containing 0.2 % Tween-20, samples and standards (PD-1 fusion proteins; R&D Systems) were added to the wells for 2 h at 37 °C in duplicate. The specific binding protein was detected with biotinylated anti-PD-1 mAb (bio-9H1, 1 μg/ml) [31, 34] for 1 h at 37 °C, followed by streptavidin-horseradish peroxidase at 1:2000 for 1 h at 37 °C, and then revealed with the substrate 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped with 2 M H₂SO₄, and the plates were analyzed at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The plates were washed five times with PBS containing 0.2 % Tween-20 after each step. The serial twofold dilutions of the soluble CD28-Fc, PD-L1-Fc, and PD-L2-Fc proteins starting from 100 ng/ml were detected by the ELISA to assess the specificity of the established system. For the detection of serum expression of TNF-α, IFN-γ, and IL-17A, high-sensitivity ELISA kits for soluble cytokines were obtained from eBioscience and used according to the manufacturer’s instructions.

The effects of PD-1 crosslinking during T-cell activation were determined according to the method described by Bertsias et al. [20]. CD4⁺ T cells (1 × 10⁵/well) stimulated in 96-well plates with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (250 ng/ml) were incubated with a pool of L929-PD-L1 cells (PD-L1 transgenic cell line) or with various concentrations of plate-bound PD-L1-Fc (0.5 μg/ml; R&D Systems), a chimeric protein containing the extracellular part of human PD-L1 linked to the Fc fragment of human immunoglobulin G1 (IgG1). Then the above CD4⁺ T cells were incubated in the presence or absence of the recombinant fusion protein PD-1-Fc for 4 days. After 3 days of coculture supernatants were collected for cytokine measurements, and after 4 days cells were pulsed with the reagent of Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) for another 4 h to measure proliferation.

Male DBA/1 mice were purchased from the Chinese Academy of Sciences (Beijing, China) and maintained in a specific pathogen-free animal facility at Soochow University. All animal procedures were approved by the Institutional Animal Care and Use Committee of Soochow University for the use of laboratory animals. CIA was induced according to the standard protocol. Briefly, an emulsion was formed by dissolving 2 mg/ml chick type II collagen (CII; Sigma-Aldrich) overnight at 4 °C in 10 mM acetic acid and combining it with an equal volume of complete Freund’s adjuvant containing 5 mg/ml heat-killed Mycobacterium tuberculosis (Difco H37Ra; BD Diagnostics, Sparks, MD, USA). Eight-week-old mice were injected intradermally at two sites in the base of the tail with a total of 100 μl of emulsion. This was repeated as a booster injection 21 days later. In some experiments, DBA/1 mice received intraperitoneal injections of a high dose of PD-1-Fc (0.15 mg/mouse, n = 7) or a low dose of PD-1-Fc (0.05 mg/mouse, n = 7) on days 1, 3, and 5 postimmunization. PD-1-Fc protein consists of the extracellular domains of murine PD-1 linked to the Fc fragment of mouse IgG1. Animals were assessed for redness and swelling of all four limbs, and a clinical score ranging from 0 (no inflammation) to 4 (extensive swelling and erythema of the entire paw) was assigned to each mouse two or three times per week for up to 42 days. After the mice were killed, their rear paws were removed, fixed, decalcified, and embedded in paraffin. Frontal sections of the paw tissue (5 mm) were stained with hematoxylin and eosin and evaluated according to the presence or absence of inflammatory cell infiltrates (defined as focal accumulations of leukocytes).

Five days or ten weeks after the second immunization, the spleen was removed. Single-cell suspensions of erythrocyte-depleted splenocytes were prepared in RPMI 1640 medium supplemented with 10 % FBS, 2 mM glutamine, 1 mM sodium pyruvate, and antibiotics. Whole splenocytes were seeded into 96-well flat-bottom microtiter plates and cultured in the presence or absence of the indicated amounts of denatured (60 °C, 30 minutes) bovine CII for 72 h. PD-1-Fc protein was added at the start of the assay. CCK-8 was added (10 μl/well), and incubation was continued for 2 h, followed by measurement of absorbance at 450 nm. Supernatants from similar cultures were collected after 96 h for assessment of cytokine production using a cytometric bead array (CBA; BD Biosciences, San Jose, CA, USA).

Cytokine concentrations in supernatants were determined using a mouse Th1/Th2/Th17 CBA kit (BD Biosciences), which allowed for the simultaneous detection of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A. Aliquoted samples were thawed, and CBA analysis was performed according to the manufacturer’s protocol. Briefly, beads coated with capture antibodies were mixed, and 50 μl of the capture bead mixture was added to 50 μl of sample. To these sample bead complexes, 50 μl of phycoerythrin (PE)-conjugated detection antibody was added, and this mixture was incubated for 3 h in the dark at room temperature. Samples were washed with 1 ml of wash buffer at 1100 rpm for 5 minutes, and the pellets were resuspended in 300 ml of wash buffer. Cytokine standards were serially diluted to facilitate the construction of calibration curves necessary for determining protein concentrations of test samples. Flow cytometric analysis was performed on a BD FACSCanto II (BD Immunocytometry Systems, Erembodegem, Belgium) with BD FACSDIVA version 6 software, and data were analyzed with FCS Express version 3 software (De Novo Software, Glendale, CA, USA).

Human cells were stained with the following antibodies: fluorescein isothiocyanate (FITC)-anti-CD4, PE-cyanine 7 (Cy7)-conjugated anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend, San Diego, CA, USA). Mouse cells were stained with FITC-conjugated anti-CD4, PE-anti-chemokine (C-X-C) motif receptor 5, PE-Cy7-conjugated anti-inducible costimulatory molecule, anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend).

Intracellular staining was performed as follows. PBMCs or splenocytes were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate, 750 ng/ml ionomycin (both from Sigma-Aldrich), and 1 μl/ml GolgiStop (BD Biosciences) for 5 h at 37 °C. Surface staining was performed for 20 minutes with FITC anti-human/anti-mouse CD4 antibody on ice. Cells were washed and resuspended in fixation/permeabilization solution (BD Cytofix/Cytoperm kit; BD Biosciences) and stained with PE-Cy7-conjugated anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend) for flow cytometric analysis. PE-Cy7-conjugated IgG1 and FITC-conjugated IgG1 (BD Biosciences) were used as isotype controls. All data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Micro–computed tomography was performed using a cone beam scanner (μCT 20; SCANCO Medical, Brüttisellen, Switzerland) with a fixed x-ray fan beam of 7-μm spot size at 50 kVp and 160 mA. Integration time was 140 milliseconds, and slices were scanned at high resolution (1024 × 1024–pixel matrix per slice) and size of 25 microvoxels.

CIA mice were killed at day 35 or day 60. Anteroposterior radiographs of the four limbs were obtained with a cabinet soft x-ray apparatus (CMB-2; Softex, Tokyo, Japan). The hind paws were then removed, fixed in formalin, decalcified in 10 % ethylenediaminetetraacetic acid, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

All statistical analyses were performed using IBM SPSS 20.0 for Windows software (IBM, Armonk, NY, USA). All the quantitative data are presented as mean ± standard deviation. Student’s t test was used to analyze differences between the groups. A Mann–Whitney U test based on nonparametric analysis was performed for independent samples. A paired-samples t test or nonparametric Wilcoxon signed-rank test was performed for paired samples. For multiple comparisons, one-way analysis of variance or the Kruskal–Wallis test was initially performed to determine whether an overall statistically significant change existed before using the paired or unpaired Student’s t test. For correlation analyses, a Spearman’s r value derived from Pearson’s r was calculated. A p value less than 0.05 was considered statistically significant.