One-hundred and fifty-five patients with ankylosing spondylitis (AS) that fulfilled the modified New York criteria for AS  were included in the Backbone study in northern Sweden, investigating severity and comorbidities in AS. The mean age of the AS patents in Backbone was 55.5 ± 11.4 years and 107 (69.0%) was men. The patients with AS underwent clinical examination and answered questionnaires regarding medication and disease-related data. Mobility was measured by the Bath Ankylosing Spondylitis Metrology Index (BASMI). Disease activity and physical function were assessed using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), the Ankylosing Spondylitis Disease Activity Score based on C-reactive protein (ASDAS-CRP) and the Bath Ankylosing Spondylitis Function Index (BASFI) . Spinal radiographic alterations were graded according to the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) . The overall scoring scale ranges from 0 to 72, with 72 representing complete ankyloses. An mSASSS score ≥ 2 at a vertebral corner was considered as having a syndesmophyte.
Blood samples were drawn in the morning after overnight fasting and erythrocyte sedimentation rate (ESR), high sensitivity C-reactive protein (hsCRP) were analyzed consecutively. HLA-B27 were analyzed using standard laboratory techniques. Plasma was stored and frozen in -80°C. Plasma levels of IL-17A, IL-22 and IL-10 were measured on the Meso Scale Discovery U-PLEX platform according to the manufactory instructions (Meso Scale Discovery®, Rockville, USA). According to the manufacturer the lower level of detection (LLOD) for IL-17A was 2.6 pg/mL, for IL-22 0.13 pg/mL and for IL-10 0.14 pg/mL. IL-17A was below the LLOD in 90% of the patients and is not further presented.
At inclusion in Backbone, 106 (68.4%) patients donated samples for storing and freezing PBMCs in -80ºC according to the protocol described below. Based on results from previous studies  a sample size of 50 patients and controls was calculated to be sufficient for this project (i.e. power 80%). The control group consisted of blood donors fulfilling the Swedish criteria for blood donation .The upper age limit for blood donors was 65 years and thus AS patients > 65 years of age were excluded, leaving 81 remaining patients. We also excluded HLA-B27 negative patients (n=3) resulting in 78 patients. In order to keep similar sex-distribution as in Backbone nineteen men and nine women were further excluded, evenly distributed according to age. Finally, 50 patients with AS, mean age 52.1±9.0 years, 33/50, 66% men and 17/50, 34% women and 50 blood donor controls matched on age (mean age 54.6±9.1 years) and sex (33/50, 66% men, 17/50, 34% women) were included in this study. No significant difference in age (p=0.1) or sex (p=1) was found between cases and controls. PBMCs from the controls were handled according to the same protocol as the AS patients. The study was approved by the Ethics Review Board at Umeå University, Umeå, Sweden (Dnr 2016/208-31, Forsblad-d’Elia, PI), and carried out in accordance with the Helsinki declaration. Written consent was obtained from all the participants.
Sample collection and freezing
Blood from AS patients and controls was collected in cell preparation tubes, BD Vacutainer® CPTTM (Becton Dickinson, NJ, USA). The tubes were centrifuged for 15 min at 1500 x g and 50% of the plasma was removed, followed by mixing of buffy coat and remaining plasma. The cell suspension was transferred to a 50 mL tube and kept on ice. Roswell Park Memorial Institute (RPMI) -1640 medium (Gibco, Thermo Fisher Scientific, USA)) was added to a total volume of 40 mL. Cell viability and concentration were assessed. The cell suspension was then centrifuged at 250 x g for 10 min followed by removal of supernatant. The cell pellet was resuspended in fetal bovine serum (FBS) to a concentration of 10 x 106 cells/mL FBS. Freeze medium (90% FBS/10% DMSO) was carefully added, drop by drop, in the same amount as FBS. Cell samples were stored at -80ºC.
Frozen cells were thawed lightly in 37°C, put in ice-cold Gibco™ PBS 1x pH 7.4 (Thermo Fisher scientific, Waltham, MA, USA), and centrifuged at 300xg for 10 min in 4°C. Cells were washed in PBS and 0.5-2 x 106 cells were transferred to tubes and stimulated 5h in 37°C in an 5% CO2 incubator in RPMI-1640, l-glutamine, PenStrep (Gibco, Thermo Fisher scientific, Waltham, MA, USA )with PMA 50ng/ml, Ionomycin(both from Sigma-Aldrich, Saint Louis, MO, USA) 1µg/ml) in the presence of Golgiplug (diluted 1/1000) (BD Biosciences). After a PBS wash, cells were stained in BD Horizon™ Fixable Viability Stain 510 according to manufacturer’s instruction. After washing with FACS buffer (Gibco™ PBS 1x pH 7.4 containing 3% Gibco™ FBS (Thermo Fisher scientific) and 0.01% sodium azide (Sigma-Aldrich)), Brilliant Stain Buffer (BD Biosciences, San Jose, CA, USA) and surface stained with a mix of the following antibodies (all from BD Biosciencesunless otherwise noted): anti-CD4 (RPA-T4, BV605), anti-CD3 (APA1/1, FITC), anti-CD8 (SK1, APC-H7) and intracellularly stained using BD Perm/Wash™, anti-IL-17A (SCPL1362, PE), anti-IL-22 (IL22JOP, APC) (eBioscience™) according to manufacturer’s instruction.
Stained samples were resuspended in FACS buffer andanalyzed in an LSRII FACS machine (BD Biosciences). Data was analyzed using FACSDiva Software (BD Biosciences) or FlowJo v10.4.2 (FlowJo, LLC, Ashland, OR, USA) software. In the analysis doublet discrimination were applied, i.e. events in the flow cytometry that potentially could be the result of cells appearing in pairs and not as single cells were excluded from the analysis. Gates were set using fluorescent minus one (FMO) controls or on discrete populations.
Descriptive statistics are presented as median with interquartile range (IQR) or absolute number with percentages. Wilcoxon signed ranks samples test with paired samples was applied when comparing cases and controls and when comparing groups with AS, Mann-Whitney U test was used for continuous variables. Correlation analysis was performed using Spearman’s rank correlation test. All tests were two-tailed and p ≤ 0.05 was considered statistically significant. All data were analyzed using IBM SPSS Statistics 25 (IBM, Armonk, NY, USA).