Details on primary cell seeding and differentiation
Freshly isolated monocytes were initially seeded at appropriate cell densities in 6, 12 or 96 well plates for flow cytometry/Western Blot, RT-PCR or cell viability/apoptosis assays with 1.2, 0.5 or 0.04 x 106 cells per well. For initial analysis cells were cultured in serum-free XVivo10 media (Lonza AG) and compared to RPMI 1640 media (PAN Biotech) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Life TechnologiesTM).
RNA handling, primer design, selection of reference gene and RT-PCR setup
Initial RNA yield and quality were ascertained on a NanoDrop (PEQLAB Biotechnologie GmbH) and 2% Agarose gel (Life TechnologiesTM) to account for protein and DNA contamination as well as for crude digestion via the 28S:18S ratio. RNA was stored at -80°C until further needed. Primers were designed to exclusively target the transcript variant(s) of interest (Tab.S1). Multiple reference genes (β-actin, G6PDH, GAPDH, PDBG and RPII) from literature were assessed a priori for specificity and lack of regulation by various culture conditions. Based on these criteria, β–actin was selected (Ref#S2). RT-PCR experiments were performed using the LightCycler 480II and SYBR Green I kit (Roche). Relative expression was calculated by the ∆∆cp-Method, normalizing the target ∆cp to the ∆cp value from an average of 4-9 donors of either monocytes, normal skin or M(IL-4) + IgG, depending on the specific assay requirements.
DNA handling and sequencing strategy
Similar to RNA – DNA yield and quality, e.g. contamination with RNA/protein was roughly estimated via NanoDrop. Single-nucleotide polymorphism (SNP) sequencing and further editing were carried out double-stranded by SequiServe GmbH and ambiguous sequence reads were reproduced with the same or novel sets of primers.
Surface plasmon resonance spectroscopy
The experiment was performed at 37°C on a Biacore T200 instrument and evaluated with the corresponding software 2.0, both from GE Healthcare as described (Ref#S3). Analyzed human recombinant IL-4 protein was concentration-adjusted with running buffer, supplemented with 0.1% BSA and used in seven increasing concentrations of IL-4 (c= 0.046-300 nM, dilution factor 3) before being injected at a flow rate of 50 µL/min for a 180s association time and a variable dissociation time of 600 or 3600s for multi-cycle kinetics analysis. Soluble IL-4Rα was covalently coupled to the chip surface at a concentration of 5 or 10 µg/mL by using 10 mM sodium-acetate at pH 4.5 and regeneration without major loss of activity was achieved with 10 mM Glycine at pH 3.0.
Cynomolgus monkey (Maccacafascicularis of Mauritius origin) study
A thirteen week toxicity study was conducted and RNA isolated from lung, liver and colon tissue after repeated i.v. doses of 0 or 60 mg/kg given once weekly for thirteen weeks to male and female cynomolgus (n=3 per dose group and per sex). This study was conducted in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Office of Animal Welfare. Raw read data was aligned against the cyno transcriptome using Bowtie2. Read mappings were then counted on the gene level using in-house tools and again using in-house software, RPKM values were computed as described.
Ref#S1 Allantaz F, Cheng DT, Bergauer T, Ravindran P, Rossier MF, Ebeling M, et al. Expression profiling of human immune cell
subsets identifies miRNA-mRNA regulatory relationships correlated with cell type specific expression. PLoS One
2012;7:e29979
Ref#S2. Lin L, Goldberg YP, Ganz T. Competitive regulation of hepcidin mRNA by soluble and cell-associated hemojuvelin.Blood
2005;106:2884-9
Ref#S3 Castoldi R, Jucknischke U, Pradel LP, Arnold E, Klein C, Scheiblich S, et al. Molecular characterization of novel trispecific
ErbB-cMet-IGF1R antibodies and their antigen-binding properties. Protein Eng DesSel 2012;25(10):551-9