Patients and samples
Two cohorts of patients were collected: a first cohort consisted of a total of 29 patients treated with abatacept. Patients had a mean age of 55(18–74), 79% female (n=23), 65% ACPA-positive (n=19). PBMCs and serum from RA patients were collected at month 0, on initiation of abatacept therapy, and after 3 months and 6 months of therapy. Patients were treated with abatacept by intravenous infusion according to baseline weight (< 60 kg, 500 mg; 60–100 kg, 750 mg; and > 100 kg, 1000 mg) on days 1, 15, 29, and then every 4 weeks. Clinical assessment of the patients was performed after 3 and 6 months and the clinical response was evaluated using the European League Against Rheumatism (EULAR) response criteria, based on the disease activity score using the 28 joint count (DAS28) and erythrocyte sedimentation rate. The second cohort consisted of 16 RA and 6 JIA patients, from which Synovial fluid and serum was obtained. (Mean age 51(23–86), 77% female (n=17), 50% ACPA-positive (n=11)). All patients attended the Rheumatology Clinic at Karolinska University Hospital. All RA patients fulfilled the ACR criteria for RA .
Peripheral blood and synovial fluid samples prepared by ficoll (Ficoll-Paque Plus, GE Healthcare, Uppsala, Sweden) separation and cryopreserved until use. Serum samples were stored at −70°C until use.
The ethics review board of the Karolinska University Hospital approved this study, and all study subjects gave informed consent according to the declaration of Helsinki.
Anti-CCP assay (ACPA)
Serum samples from 33 patients were used to determine anti-citrullinated cyclic peptides (anti-CCP) levels using the anti-CCP-2 ELISA kit (Immunoscan RA Mark 2; Euro-Diagnostica, Arnhem, The Netherlands) according to the manufacturer’s instructions.
Intracellular cytokine staining
Cells from 19 patients collected at 0 and 6 months after abatacept treatment were cultured for 6h or 5 days in complete media (RPMI, HEPES, L-Glutamine, Penicillin, Streptomycin) containing 5% human serum and stimulated with plate-bound α-CD3 monoclonal antibody (2.5 μg/ml, clone OKT-3). All cultures were incubated at 37°C, 5% CO2. Supernatants were collected on day 5 and stored at −80°C until use.
In order to prevent produced cytokines from being secreted, 10 μg/ml Brefeldin A (Sigma-Aldrich, Steinheim, Germany) was added to the cultures 4 h prior to harvesting. Extracellular and intracellular cytokine staining was performed using Cytofix/Cytoperm Kit (BD) according to the manufacturer’s instructions. Antibodies: α-CD28 PE (clone: L293, BD), α-CD14 APC Cy7 (clone: MphiP9, BD), α-CD4 PeCy7 (clone: SK3, BD), α-CD3 PB (clone: UCHT1, BD and Biolegend), α-IFN-γ FITC (clone: B27, BD), α-TNF PerCP Cy5.5 (clone: Mab11, Biolegend), α-IL17 Alexa 647 (clone: BL168, Biolegend).
Beriglobin was added in order to prevent unspecific staining and LIVE/DEAD Aqua Dead Cell Stain (Invitrogen) was used to exclude dead cells. The PBMCs were run on a Beckman Coulter CyAn. Analyses were performed with FlowJo software, version 8.1.0 or higher (Treestar Inc.).
Luminex analysis of cell culture supernatants
Cytokine analysis was performed on the collected supernatant samples from the 5 day cultures using the multiplex assay LEGENDplex (Biolegend, San Diego, CA, USA) and read on a Luminex100™ platform (Bio-Rad, Hercules, CA, USA) with Bio-Rad software. The cytokines analyzed were; interleukin IL-1β (1.9), IL-2 (0.3), IL-3 (4.1), IL-4 (0.6), IL-7 (1.5), IL-9 (0.1), IL-10 (0.2), IL-13 (0.2), IL-17A (0.7), IL-17F (1.6), IL-21 (0.2), IL-22 (4.4), IL-23 (1.2), TNF (0.6) and IFN-γ (0.2). Detection limits for these cytokines/chemokines are indicated in brackets in pg/ml.
Phenotypic characterization of Tregs
PBMCs taken from 12 patients at 0 and 3 months post-treatment were utilized for phenotypic analyses by flow cytometry. Intranuclear staining of FOXP3 and Helios was performed using FOXP3/Transcription factor staining kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Antibodies: α-CD3 Alexa700 (clone: UCHT1, Biolegend), α-CD3 FITC (clone: UCHT1, BD), α-CD4 PE (clone: RPA-T4, BD), α-CD39 FITC (clone: A1, Biolegend), α-CD45RA ECD (clone: 2H4LDH11LDB9, Beckman Coulter), α-Helios Alexa647 (clone: 22F6, Biolegend), α-FOXP3 PB (clone: 206D, Biolegend), α-CTLA4 PE (clone: BNI-3, BD). LIVE/DEAD Near-IR Dead Cell Stain (Invitrogen) was used to exclude dead cells. The PBMCs were run on a Beckman Coulter Gallios (Beckman Coulter, Brea, CA, USA). Analyses were performed with FlowJo software, version 8.1.0 or higher (Treestar Inc., Ashland, OR; USA).
In vitro study of SFMC in the presence of abatacept
SFMC (n=16) were thawed and stimulated with either plate-bound α-CD3 (1 μg/ml, clone: OKT-3) for 72 hours or with influenza vaccine (Fluvirin vaccine 2001/2002, Evans Vaccines Limited, Liverpool, UK) for 6 days. For Treg suppression assays, SFMC (n=6) were thawed and sorted by flow cytometry into CD3-APC, CD3+4+25- effector T cells and CD3+4+25++ Tregs and co-cultured with plate-bound α-CD3 (0.5 μg/ml, clone OKT-3) for 6 days  in presence or absence of 10 μg/ml abatacept. For total SFMC Abatacept or a control compound (Chimeric L6 Bd2.1 IgG) was added at 10 μg/ml (both provided by Bristol-Myers Squibb, Princeton, NJ, USA), which represents the physiological concentration in the blood of patients post-treatment . During the last 15–18 hours of the incubation 3H-labelled thymidine (1uCi/well, Perkin Elmer, Boston, MA, USA) was added to the wells and the counts per minute were measured, indicating cell division. Cytokines were measured in supernatants with the CBA Inflammation Kit (Becton Dickinson (BD), San Jose, CA, USA) according to the manufacturer’s instructions and were acquired and analyzed with a FACS calibur (BD).
A nonparametric Wilcoxon signed-rank test was used to compare control and abatacept-treated SFMC cultures, Treg co-cultures, Treg frequencies, and differences between baseline and post-treatment cytokine secretion and cytokine levels. P values less than 0.05 were considered statistically significant. Spearman’s rank correlation coefficient was used to analyze the ratio between different combinations of cytokines.
All statistical analyses were performed using Prism 5.0 (GraphPad Software, La Jolla, CA, USA).