Patients
Heparinized peripheral blood from 10 RA patients (age 40 (range 28–50) years, two female, eight male) and 10 HD (age 43 (27–51) years, three female, seven male) was obtained for this study. Patients fulfilled the 1987 criteria of the American College of Rheumatology (ACR) for RA. All the patients, classified as having an early RA (ERA; disease duration 1.7 years (range 5 months to 2 years), were disease-modifying anti-rheumatic drug (DMARD; methotrexate, leflunomide)-naive and had not received prednisone or equivalent for at least 2 weeks before blood collection. Eight out of the 10 patients were anti-citrullinated protein antibody (ACPA)-positive. Increased levels of erythrocyte sedimentation rate (30.4 ± 15.7 mm/h) and C-reactive protein (0.69 ± 1.2 mg/dl) were also found in all patients. The study was approved by the Ethical Committee of the University Hospital in Palermo where the patients were recruited. Informed consent was signed by all participants.
γδ T cell identification
Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation using Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden).
Fc receptor blocking was performed with human immunoglobulin (Sigma; 3 μg/ml final concentration) followed by surface staining with different fluorochrome-conjugated antibodies to study the phenotype and the cytokine production by Vγ9Vδ2 T cells.
The following fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PE-Cy5-, PE-Cy7-, allophycocyanin (APC)-, and APC-Cy7-conjugated anti-human monoclonal antibodies (mAbs) were used to characterise the Vγ9Vδ2 T-cell population: live/dead-FITC, anti-CD45-APCH7 (clone2D1), anti-CD3-PECy7 (clone SK7), anti-TCRVδ2-PE (clone B6), anti-CD27-APC (clone MT271), and anti-CD45RA peridinin chlorophyll protein (PerCP)-Cy5.5 (clone HI100). Expression of surface markers was determined by flow cytometry on a FACSCanto II Flow Cytometer with the use of FlowJo software (BD Biosciences).
For the intracellular cytokine assay, PBMC (106/ml) were stimulated with ionomycin (Sigma, St. Louis, MO, USA; 1 μg/ml final concentration) and phorbolmyristate acetate (PMA; Sigma; 150 ng/ml final concentration). Cells were cultured in a humidified incubator at 37 °C with 5% CO2 for 6 h in the presence of 5 μg/ml Brefeldin A (Sigma, St. Louis, MO, USA). Following incubation, PBMC were harvested, washed in phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS) and 0.1% sodium azide, and then stained as follows: live/dead-FITC, anti-CD45-APCH7 (clone 2D1), anti-CD3-PECy7 (clone SK7), anti-TCRVδ2-PE (clone B6), in incubation buffer (PBS, 1% FCS, 0.1% sodium azide) for 30 min at 4 °C.
Subsequently, PBMC were washed, fixed, and permeabilized (Cytofix/Cytoperm Kit, BD Pharmingen) according to the manufacturer’s instructions and stained for intracellular cytokines with conjugated anti-IFN-γ-APC (clone 25723.11), anti-IL-8-APC (cloneE8N1), and anti-IL-6-APC (clone MQ2-13A5) mAbs. Isotype-matched control mAbs were used. All mAbs were obtained from BD (San Josè, CA, USA) except IL-8 (from Biolegend, San Diego, CA, USA). Cells were washed, fixed in 1% paraformaldehyde, and at least 1 × 106 lymphocytes were acquired using a FACSCanto II Flow Cytometer (BD Biosciences) after gating by forward (FSC) and side scatter (SSC) plots. FACS plots were analysed using FlowJo software (version 6.1.1; Tree Star, Ashland, OR, USA). Negative controls were obtained by staining PBMC in the absence of any stimulation. Cut-off values for a positive response were pre-determined to be in excess of 0.01% responsive cells. Results below this value were considered negative and set to zero [11]. Values found using isotype control mAbs were subtracted in all the samples analysed. The gating strategy used for the phenotype distribution of Vγ9Vδ2 T cells and for the evaluation of the intracellular cytokine content was made starting with the initial lymphocyte gate (SSC vs FSC), followed by gating on single cells, live/dead cells vs CD45, CD3 vs Vγ9Vδ2 T cells, followed by further surface or intracellular molecules.
Generation of γδ T-cell lines
Polyclonal Vγ9Vδ2 T-cell lines were generated by first enriching PBMC using a γδ T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by sorting single Vγ9Vδ2 T cells through a FACSAria I Cell Sorter (BD Biosciences) with specific mAbs. Sorted cells at the concentration of 2 × 104 were then cultured into each well of round-bottomed plates, containing 2 × 104 irradiated (40 Gy from a caesium source) allogeneic PBMC, plus zoledronic acid (2 μM) and 200 U/ml recombinant IL-2 (Proleukin, Novartis Pharma) [12]. Growing T-cell lines were expanded in 200 U/ml IL-2 and re-stimulated every 3 days. Cells were collected after 2 weeks and sorted according to their phenotype into four different subsets: naive (Tnaive; CD45RA+CD27+), central memory (TCM; CD45RA−CD27+), effector memory (TEM; CD45RA−CD27−), and terminally differentiated effector memory (TEMRA; CD45RA+CD27−).
RNA purification and miRNA expression analysis
For analysis of miRNA17–92 among total Vγ9Vδ2 T cells and the different cell subsets, total RNA containing miRNA was purified using an miRNeasy mini-kit (Qiagen). miRNA labelling, hybridization, scanning, and expression profiling was performed using miRCURY LNA microarray service (Exiqon).
Real-time polymerase chain reaction (RT-PCR) analysis of the whole population of Vγ9Vδ2 T cells
Total RNA was extracted from γδ T-cell lines derived from RA patients and HD with the miRNeasy Mini Kit (Qiagen) isolation kit according to the manufacturer’s instructions. The quality of RNA was accessed with a NanoDrop 1000 Spectrophotometer V3.7 (Thermo Scientific). The obtained RNA was subsequently used as a template for cDNA generation. For this purpose, a reverse transcription reaction was performed with miScript II RT Kit (Qiagen; 300 ng of RNA per reaction) following the manufacturer’s protocol. The resulting cDNA was used to conduct an RT-PCR reaction with miScript SYBR Green PCR Kit (Qiagen) applying primers specific for hsa-miR-21a-5p, hsa-miRNA-hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, and hsa-miR-106a-5p (commercially available from QIAGEN) in a Rotor-Gene Q system. The expression level of RNU6 was used as an endogenous control.
RT-PCR was also performed to evaluate IL-8, IL-6, and programmed cell death 4 (PDCD4) mRNA using the commercially available Illustra RNAspin Mini Isolation Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions. For quantitative TaqMan RT-PCR, master mix and TaqMan gene expression assays for GAPDH (glyceraldehyde 3-phosphate dehydrogenase, Hs99999905_m1) control and target genes were obtained from Applied Biosystems. Samples were run in duplicate using the Step-One Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Relative changes in gene expression between paired patients before and after treatment were determined using the ΔΔCt method. Levels of the target transcript were normalized to a GAPDH endogenous control constantly expressed in both groups (ΔCt). For ΔΔCt values, additional subtractions were performed between untreated and treated samples ΔCt values. Final values were expressed as fold of induction (FOI).
Statistical analysis
miRNA microarray data were analysed by miRCURY LNA microarray service (Exiqon). Data were normalized using the non-parametric regression method, LOESS. Unsupervised two-way clustering of miRNAs and samples was performed on log2 (Hy3/Hy5) ratios (with each sample versus the common reference pool) to produce a heat map. Heat map expression data were displayed using Gene-E software developed by Joshua Gould (http://www.broadinstitute.org/cancer/software/GENE-E). Hierarchical clustering using one minus Pearson’s correlation was applied to samples and genes/miRNAs. Global or relative map colours were applied using the minimum and maximum values in the data. Network analysis to identify miRNA targets using gene and miRNA expression data was performed using MIR@NT@N [13].
Obtained Ct values were used to calculate expression levels of tested miRNAs with the 2ΔΔCt method in two groups, each composed of HD and RA patients. To assess the statistical significance of observed differences, independent student t tests and Mann Whitney tests were performed on all groups and p values *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 were considered as significant, very significant, and extremely significant, respectively.
The normal distribution of the data was assessed by a Shapiro-Wilk normality test. Analysis of variance (ANOVA) was performed as part of the data analysis, and these data are reported as a heat map.