Patients and healthy donors (HD)
Blood samples of 4 volunteers (37 ± 9 years) with autoimmune arthritis (2 patients with rheumatoid arthritis (RA), 1 patient with spondylarthritis (SA) and 1 patient with systemic lupus erythematosus (SLE) and significant polyarthritis and 4 age-matched HD were taken at the Department of Internal Medicine II, Rheumatology and Clinical Immunology University Hospital Würzburg (Table 1). The study was approved by the local ethics committee of the Medical Faculty of the University Hospital of Würzburg (No. 123/14) and performed in accordance with the Declaration of Helsinki 2013 and the Medical Research Involving Human Subjects Act (WMO).
Table 1
|
Patient 1
|
Patient 2
|
Patient 3
|
Patient 4
|
Age (y)
|
60.8
|
34.4
|
23.7
|
29.8
|
Sex (m/f)
|
f
|
f
|
f
|
m
|
Diagnosis
|
Seropositive RA
|
Seropositive RA
|
SLE Polyarthritis
|
AS
|
Disease duration (y)
|
31
|
15
|
5
|
7
|
DAS28
|
3.50
|
3.35
|
-
|
-
|
BASDAI
|
-
|
-
|
-
|
2.3
|
BASMI
|
-
|
-
|
-
|
2
|
SLEDAI
|
-
|
-
|
12
|
-
|
ESR (1h)
|
30mm
|
4mm
|
54mm
|
4mm
|
CRP (mg/dl)
|
0.25
|
0.01
|
0.04
|
0.31
|
Leukocytes (n*1000/µl)
|
9.9
|
6.7
|
6.4
|
12.8
|
Remission (yes/no)
|
no
|
no
|
no
|
yes
|
Methotrexate
|
10mg 1x/week
|
-
|
-
|
-
|
Leflunomide
|
20mg 1-0-0
|
-
|
-
|
-
|
Prednisolone
|
40mg 1-0-0
|
7.5mg 1-0-0
|
40mg 1-0-0
|
-
|
Infliximab
|
-
|
-
|
-
|
400mg 1x/8 weeks
|
Mycophenolate mofetil
|
-
|
-
|
500mg 2-0-2
|
-
|
Etanercept
|
-
|
50mg 1x/week
|
-
|
-
|
Table shows age in years (y); sex - male/female (m/f); Diagnosis (RA = rheumatoid arthritis, SLE = systemic lupus erythematosus, AS = ankylosing spondylitis); Years since initial diagnosis; Disease Activity Score 28 (DAS28); Bath Ankylosing Spondylitis Disease Activity Index (BASDAI); Bath Ankylosing Spondylitis Metrology Index (BASMI); Systemic Lupus Erythematosus Disease Activity Index (SLEDAI); Erythrocyte sedimentation rate (ESR) after one hour (1h); C-reactive protein (CRP) in mg/dl; Leukocytes in n*1000/µl; Medication at the time of blood sample collection (MTX = methotrexate, LEF = leflunomide, PRE = prednisolone, ETA = etanercept, MYC = mycophenolate mofetil, INF = infliximab); Remission yes/no (patient in remission phase yes or no). A DAS28 < 2.6 was defined as remission for patients 1 and 2. Patient 3 showed an increased disease activity corresponding to a total of 12 points in the SLEDAI. In patient 4, a remission of the underlying disease could be assumed on the basis of low numerical values in the BASDAI and BASMI.
Exclusion criteria were malignancies, congenital anomalies, syndromes, immunodeficiencies, clinically relevant infections in the last eight weeks and vaccinations in the last four weeks. Written informed consent was given from patients, HD and MSC donors.
Mesenchymal stem cells (MSC)
MSCs were harvested from spongy bone of the acetabulum during orthopedic hip surgery for the treatment of osteoarthritis of 2 otherwise HD and expanded in vitro 34. MSCs of both donors were in passage 1 at the beginning of the co-culture. Patients with malignancies, femoral head necrosis, femoral head trauma, congenital anomalies, syndromes, immune defects, clinically relevant infections in the last eight weeks and autoimmune inflammatory rheumatic diseases were excluded from the study (Table 2).
Table 2
MSC healthy donor characteristics
|
MSC 1
|
MSC 2
|
Age (y)
|
48.9
|
72.2
|
Sex (m/f)
|
m
|
m
|
Diagnosis
|
coxarthrosis
|
coxarthrosis
|
Origin MSC
|
acetabulum
|
acetabulum
|
CRP (mg/dl)
|
1.6
|
0.2
|
Leukocytes (n*1000/µl)
|
7.7
|
6.5
|
Dabigatran
|
110mg 0-0-2-0
|
-
|
Pantoprazol
|
20mg 1-0-0-0
|
-
|
Paracetamol
|
500mg 2-2-2-0
|
-
|
Diclofenac
|
75mg 1-0-1-0
|
-
|
Levetiracetam
|
-
|
500mg 1-0-1-0
|
Lorazepam
|
-
|
1mg 0-0-1-0
|
Simvastatin
|
-
|
10mg 0-1-0-0
|
Tromcardin® forte
|
-
|
0-2-0-0
|
Perindopril
|
-
|
4mg 0-0-1/2 − 0
|
Table shows age in years (y); sex - male/female (m/f); diagnosis; origin of MSCs; c-reactive protein (CRP) in mg/dl; leukocytes in n*1000/µl; medication (in mg) at the time of harvesting MSCs.
Naive and non-naive cell sorting
CD4+ cells were isolated from peripheral blood mononuclear cells (PBMCs) from HD and patients using the CD4+ magnetic isolation kit according to the manufacturers’ instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Naive (depleted) and non-naive CD4+ (retained) fractions were obtained using a naive isolation kit according to the manufacturer’s instructions (Miltenyi). A purity > 90% of the positive fractions was evaluated by flow cytometry. Non-CD4+ T-cells were irradiated (30Gy) and used as autologous antigen presenting cells (feeder cells).
Co-culture MSC and T-cells
For co-culturing MSCs and T-cell subpopulations, adherent MSCs were removed from the cell culture surfaces by using trypsin / EDTA (1x) solution (PAA Laboratories GmbH, Pasching, Austria) following treatment with cell culture media containing 10% FCS to stop the reaction. To examine the effect of the soluble factors in the modulation of the T-cell response by MSCs, naive or non-naive CD4+ T-cells (2.5x105 cells/well) together with feeder cells (5x104) were seeded in the upper part of the Transwell system (Greiner Bio-one GmbH, Frickenhausen, Germany), while MSCs (0.25x105 cells/well) were seeded on the bottom. Wells with direct cell-to-cell contact between MSCs and T-cells were not included in the study. For Th17-inducing cytokine conditions, wells were stimulated under a Th17 cytokine cocktail consisting of IL-1β (10ng/mL), IL-6 (20ng/mL), TGF-β (5ng/mL), IL-23 (100ng/mL), anti-CD3/anti-CD28 (1µg/mL) for 6 days at 37°C. Wells containing no MSCs were used as control.
Flow cytometry analysis
The cytokine production profile on CD4+ naive and non-naive T-cells treated in the presence or absence of MSCs was determined upon stimulation with phorbol 12-myristate 13-acetate (PMA) (15ng/ml), ionomycin (1µg/ml) and brefeldin A (5µg/ml) for 3h at 37°C. After incubation, expression of CD45RA and CD27 on naive and non-naive cells were determined by using monoclonal antibodies labeled with brilliant violet 421 (BV421, BioLegend, San Diego, USA) and CD27, labeled with phycoerythrin-cynanine7 (PE-Cy7, BioLegend, San Diego, USA) respectively. According to the expression of CD45RA and CD27, subpopulations were characterized as terminal effector memory cell re-expressing RA (TEMRA) (CD45RA+CD27-), memory (CD45RA-CD27+), effector (CD45RA-CD27-) and naive cells (CD45RA+CD27+) 35.
Intracellular cytokine production of IL-9 and IL-17 was determined after treatment with fixation and permeabilization buffer (Biolegend, San Diego, USA) using IL-9 (PE, BD Biosciences, Franklin Lakes, USA) and IL-17 (Alexa Fluor 700, BioLegend, San Diego, USA) monoclonal fluorescent labeled antibodies. Surface marker and cytokine production was evaluated by flow cytometry using FACSCanto II, BD. Data analysis was performed using FACSDiva software V6 (BD, San Jose, CA).
Statistics
Statistical evaluation was performed non-parametrically using the Mann-Whitney U test. For calculation of the data sets IBM SPSS Statistics Version 22 (Armonk, New York, USA) was used. A p ≤ 0.05 was considered statistically significant.