Supplementary MaterialsS1 Fig: Technique for analysis of cytokine-producing B cells. B cells creating TNF-, IL-6, or IL-10. Mononuclear cells from healthful donors (HD; N = 12) and sufferers with relapsing-remitting multiple sclerosis (RRMS; N = 13) had been either still left unstimulated (-stim), or activated with entire MBP every day and night (+MBP) or with MBP every day and night and PMA + ionomycin going back 4 hours of incubation (+MBP+PMAiono). Cells had been stained intracellularly with antibodies against (A) TNF-, (B) IL-6 and (C) IL-10 before evaluation by movement cytometry. The organic data matching to Fig 1 are proven as Tanshinone I median, interquartile range (container) and range (whiskers). allele, was genotyped by TaqMan allelic discrimination PCR assay (Lifestyle Technologies European countries BV, Denmark) using predesigned primers and probes as previously referred to . Antibodies and Antigens Entire individual MBP was purchased from HyTest Rabbit Polyclonal to BAZ2A Ltd. (Turku, Finland). The monoclonal antibody MK16, which identifies MBP85-99 within the framework of HLA-DRB1*15:01, was utilized as probe for antigen display . Tanshinone I The MK16 IgG1 antibody was affinity-purified by protein A from the supernatant of MK16-expressing Chinese hamster ovary cells produced in HAMS F-12 media (GIBCO) supplemented with 10% fetal calf serum (FCS; Biological Industries) and 0.8 mg/ml geneticin (Invitrogen, Carlsbad, CA). Antibodies used for flow cytometry were: PE-Cy7-streptavidin, PerCP-Cy5.5-anti-human CD19 (clone HIB19), PE-anti-human CD3 (clone UCHT1), APC-anti-human CD3 (clone UCHT1), PE-anti-human TNF- (clone MAb11), FITC-anti-human IL-6 (clone AS12) (all from BD Biosciences) and APC-anti-human IL-10 (clone JES3-19F1)(Biolegend, San Diego, CA). Tanshinone I Assessment of MBP presentation and intracellular cytokine staining 0.5×106 PBMCs were incubated for 18 h at 37C under 5% CO2 in RPMI-1640 containing 30% (v/v) serum from healthy blood group AB donors in a final volume of 200 l with either: no stimulating antigen, 30 g/ml MBP, or 30 g/ml MBP plus cell stimulation cocktail containing PMA and ionomycin (500x diluted from stock; PMA 40.5uM and 670 M ionomycin)(eBioscience, San Diego, CA). The cocktail was added during the last 4 h of culture. To block secretion of cytokines, 1 l/ml of 1 1:5 diluted brefeldin A (1000x #555029 BD Biosciences), was added to all cultures during the last 4 h. Next, the cells were incubated with IgG for intravenous use (IVIg; CSL Behring, Bern, Switzerland) at a concentration of 6 mg/ml with 2% mouse serum (Statens Serum Institut, Copenhagen, Denmark) to block unspecific binding. Subsequently, MK16 was incubated at a concentration of 50 ng/ml for 30 min at 4C in 2% FCS; antibodies against cell-surface markers were included in the same step. Following two washes, streptavidin-PE-Cy7 was incubated with the samples for 30 min at 4C. For intracellular staining of cytokines, Cytofix/Cytoperm? answer (BD Biosciences) was used according to the manufacturers instructions. The LIVE/DEAD? Fixable Near-IR Dead Cell Stain Kit from Molecular Probes? (Molecular Probes, Eugene, OR, USA) was used to discriminate between live and lifeless cells. First a live/lifeless cell gate was used to discriminate living cells from lifeless cells. Next, doublets were excluded based on FSC-A and FSC-W. Finally, B cells were identified as CD19 positive cells within the lymphocyte gate. Cells were analyzed on a FACS Canto flow cytometer (BD Biosciences), and data was analyzed using FlowJo v.X, (TreeStar, Inc, Ashland, OR). Statistics Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software, La Jolla, CA). Comparisons between RRMS patients and healthy donors were performed using the two-tailed Mann Whitney U-test. Comparisons between non-stimulated and MBP-stimulated B cells were done using the Wilcoxon matched-pairs signed-rank test. Column statistics were calculated using the Wilcoxon signed-rank test. The non-parametric Spearmans correlation test was used to analyze the association between cytokine positive B cells and EDSS or MSSS. Results MBP-induced cytokine-producing B cells To study the ability of an MS-relevant self-antigen to stimulate cytokine production by B cells derived from RRMS patients and those derived from healthy donors, we decided the frequencies of B cells producing TNF-, IL-6 or IL-10 before and after stimulation of PMBCs from these groups with MBP. The flow cytometric gating strategy is shown in S1 Fig. Stimulation with MBP elevated the percentage of TNF–producing B cells as well as the percentage of IL-6 creating B cells from RRMS sufferers, while only minimal changes had been observed in the proportions of TNF– or IL-6-creating B cells from healthful donors (Fig 1A and 1B). MBP induced just few IL-10-creating B cells both in groupings (Fig 1C). Organic values for everyone cytokine data are shown in S2 Fig. Open up in another home window Fig 1 MBP-induced cytokine creation by B cells.Mononuclear cells from 12 healthful donors (HD) and 13 individuals with relapsing-remitting multiple sclerosis (RRMS) were activated with entire myelin simple protein (MBP) every Tanshinone I day and night and stained intracellularly for (A) TNF-, (B) IL-6, and (C) IL-10.