Study population and clinical information
Ninety-eight RA patients and 73 healthy individuals were enrolled and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were isolated on a Ficoll-Hypaque (GE Healthcare, USA) gradient. All RA patients met the 2010 classification criteria of American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) (20). Laboratory investigations included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (1), anti-citrullinated protein antibody (ACPA). Disease activity was evaluated by measuring tender joint count (TJC; TJC 68 and TJC 28), swollen joint count (SJC; SJC 68 and SJC 28), ESR and CRP in each patient. The 28 Joints disease activity score (DAS28-ESR and DAS28-CRP) was available in 94 RA patients. Remission status was defined by ACR/EULAR Boolean criteria (21). Clinical characteristics and treatments for the enrolled patients are listed in Table 1.
This study was approved by the institutional review board of Seoul National University Hospital, and all samples were collected after obtaining written informed consent.
Isolation of mononuclear cells from synovial tissue
Synovial tissue was obtained from knee joint replacement surgery. Tissue was chopped, treated with Type 2 collagenase (Worthington biochemical, USA) and incubated for more than 2 hours at 37℃. After incubation, culture media (RPMI-1640 (Welgene, Korea), 10% fetal bovine serum (Biowest, France), 1% penicillin/streptomycin (Gibco, USA)) was added, and cells were filtered and separated by Ficoll-Hypaque gradient (GE Healthcare).
Immuno-phenotyping and cell sorting
For cell surface phenotyping, Fc receptors on PBMCs (1 × 107 cells/mL) were blocked with purified mouse anti-human IgG (BD Biosciences) and the cells were stained with adequate fluorescent antibodies. Detailed flow cytometry gating strategy was described in Supplementary Figure 1. To measure the absolute number of CD4+ or CD8+ Tscm cells, Accucheck counting beads (Thermo Fisher, USA) were used.
Following antibodies were obtained from BD Biosciences (San Jose, USA): CD3 (clone SK7), CD4 (SK3), CD8 (SK1), CD45RO (UCHL1), CD62L (DREG-56), CD45RA (HI100), CD27 (M-T271), CXCR5 (RF8B2), ICOS (DX29), PD-1 (EH12.1)
Anti-CD28 (CD28.2) antibody was obtained from eBioscience (San Diego, USA) and antibodies specific for CD127 (A019D5), CD122 (Tu27), CD95 (DX2), CCR7 (G043H7) were obtained from BioLegend (San Diego, USA).
Stained cells were analyzed using a LSR Fortessa flow cytometer (BD Biosciences) and were sorted using a FACSAria instrument (BD Biosciences). All data were analyzed using FlowJo software (Treestar, USA).
Differentiation capacity of Tscm cells
Sorted Tscm cells were stimulated for 6 days with anti-CD3/CD28-coated beads (Dynabeads, Thermo Fisher) at a 1:1 ratio. Differentiation capacity into other daughter T cell subsets (Tcm, Tem, and Temra and Tfh cells) were examined by staining CD3, CD4, CD45RO, CCR7, CD45RA, CXCR5, ICOS and PD-1.
Intracellular transcription factor staining
Transcription factors associated with helper T subsets were stained. Fc receptors were blocked and cells were stained for CD3, CD4, CCR7, CD45RA. After surface-staining, cells were permeabilized with fixation/permeabilization solution (eBiosciences) and stained for transcription factors: T-bet (4B10), GATA3 (16E10A23) (Biolegend), RORγt (Q21-559) (BD Biosciences), FoxP3 (PCH101) (eBiosciences).
IL-6 ELISA analysis
Plasma from RA patients and HCs were collected and stored at below -80℃. IL-6 levels in plasma were measured using the Quantikine high sensitivity human IL-6 ELISA kit (enzyme-linked immunosorbent assay) (R&D systems, USA). All procedures were followed according to the instructions of the manufacturer. Standards were measured in triplicate and samples were measured in duplicate.
Assays for activation markers and cytokines (Cytometric Beads Assay)
Sorted Tscm cells (4 x 104 cells) were stimulated for overnight with 50 ng/mL of recombinant human IL-6 protein (Peprotech, USA) and additionally stimulated with anti-CD3/CD28-coated beads (Thermo Fisher) if applicable. Next day, culture supernatants were stored for the cytokine assay and cells were stained for activation markers including CD69, CD25 (IL-2 receptor alpha) and CD154 (CD40 ligand).
Cytokine assay was proceeded with Cytometric Beads Assay (CBA). IFN-γ, IL-4, IL-17A, and TNF cytokine levels were measured according to the manufacturer’s protocol (LEGENDplex (Biolegend)) using LSR-Ⅱ flow cytometer (BD biosciences).
RNA was extracted from FACS-sorted Tscm cells using RNeasy mini kit or RNeasy Plus Micro kit (both Qiagen, Germany). RNA-seq libraries were produced using the NEXTflex Rapid Directional mRNA-Seq Kit Bundles (Bioo Scientific, USA) and sequenced on the Illumina HiSeq 2500 platform (San Diego, USA). The sequence reads were analyzed by alignment to the Human Ensembl Archive Release 90 using STAR (22) with ENCODE options and quantification using RSEM (23).
Data are expressed as the mean ± SEM. For continuous variables, Student’s t-test (unpaired, 2-tailed) was used when the sample size was > 30, while the Mann-Whitney U test was used when the sample size was < 30. All graphs were depicted using the Prism software (GraphPad software, Inc., USA). For RNA transcriptome analysis, differentially expressed genes were identified by DESeq2 (24) with Wald test and Likelihood ratio test corrected by Benjamini-Hochberg procedure (FDR < 0.05) and one-way ANOVA (P < 0.01) corrected by FDR < 0.05. The enrichment analysis of Gene Ontology terms and pathway were performed using Metascape (25) and volcano plots were drawn by R package EnhancedVolcano (Ver. 1.01). All statistical analyses were performed using SPSS (IBM, USA), otherwise stated.