Igg2a

The ligand-binding domains were compared between CD161/TT and /CC receptors by studying binding of LLT1-Fc to 293-161/TT and /CC transfectants. The cells were incubated with increasing concentrations of LLT1-Fc and binding of the soluble ligand was monitored by staining with PE-conjugated goat anti-human IgG (Jackson Immunoresearch, West Grove, PA, USA). Furthermore, the “epitope landscape” of the ligand-binding loop was compared between CD161/TT and /CC by studying the affinity of the anti-CD161 mAb 191B8 (IgG2a, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Characterization of this mAb and two additional anti-CD161 mAb (DX12, IgG1; B199.2 IgG1, AbD Serotec) revealed that only 191B8 blocks LLT1 binding, suggesting that the 191B8 epitope is part of the ligand-binding domain of CD161. 293-161/TT and /CC transfectants were incubated with an antibody mixture consisting of saturating concentrations (10 μg/ml) of PE-labelled B199.2 and increasing concentrations of unlabelled 191B8. Affinity of 191B8 to this epitope was assessed by studying its capacity to replace/compete with binding of mAb B199.2 which detects an epitope closely related to the 191B8 epitope.

CD14⁺ monocytes were isolated as described before (ten Harkel et al., 2015). Briefly, peripheral blood mononuclear cells were isolated from human buffy coats from healthy donors (Sanquin, The Netherlands) using Ficoll‐Paque density gradient centrifugation. Subsequently, cells were incubated with CD14‐antibody tagged microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted using a manual MACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer's instructions (Davison et al., 2014). The purity of the cells was determined with flow cytometry (FACSverse^™; BD Biosciences, Piscataway). For analysis, cells were incubated with fluoresceine isothiocyanate (FITC) labeled anti‐human CD14 (Miltenyi Biotec) or its equivalent isotype control IgG2a (Miltenyi Biotec), and incubated for 30 min in the dark on ice. After incubation, cells were washed to remove unbound antibodies, recovered in FACS buffer and analyzed (30 s or 100,000 viable events) on a BD Bioscience FACSverse flow cytometer. Purity was confirmed to be at least 80%.

For the initial TRAcP staining and QPCR experiments CD14⁺ monocytes were isolated from the blood from 6 FOP patients and sex- and age- matched controls (20–40 ml blood per donor), for all other experiments human buffy coats (Sanquin, The Netherlands) or blood from healthy donors was used. The CD14+ cells were isolated as described before (23 (link)).
Briefly, peripheral blood mononuclear cells were isolated using Ficoll-Paque density gradient centrifugation. Subsequently cells were incubated with CD14-antibody tagged microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and sorted using a manual MACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer's instructions (24 (link)). The purity of the cells was determined with flow cytometry (FACSverse™, BD Biosciences, Piscataway, USA). For the analysis, cells were incubated with FITC labeled anti-human CD14 (Miltenyi Biotec) or its equivalent isotype control IgG2a (Miltenyi Biotec) for 30 min in the dark on ice. After incubation, cells were washed to remove unbound antibodies, recovered in FACS buffer and analyzed (30 s or 100,000 viable events) on a BD Bioscience FACSverse flow cytometer. Purity was confirmed to be at least 80%.