Il 10rα

Monocytes were cultured at 2 × 10⁶ cells/well on a 24-well plate for 24 h in RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum (CSL Biosciences, Parkville, VIC, Australia), 2 mm GlutaMax-1 (Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were then treated with the following cytokines alone or in combination for 24 h and collected for phenotype detection. For the IL-10 blockade assay, monocytes were pretreated with blocking Abs against IL-10Rα (R&D Systems) and IL-10Rβ (R&D Systems) or isotype control (R&D Systems) for 1 h. After washing, cells were cultured in the presence of IL-10 or supernatant from lymphoma cells.

DARPins have been described previously [19 (link), 21 ]. Two rounds of cell-based affinity selections were each performed on 10⁷ activated expanded Treg cells originating from seven donors using a DARPin phage display library containing up to 10⁹ binding members, as described previously [17 (link), 18 (link)]. The following recombinant human proteins were included as de-selection antigens: TCR α and β chains, CD5 (Cambridge Biosciences); CD69, CD3ε, and ITGB2 (Sino Biological); CD2 (Abcam); CD132, CD122, CD39, IL-10Rα, sCD4, CD109, IL-1R, IL-6Rα, IL-14Rα, IFN-γR1, CD45, CD38, TNFRI, 4-1BB, CD30, GITR, PD-1, B7-H1, CD44, CD25, CD27, and CD28 (R&DSystems). DARPins isolated from two rounds of selections were reformatted as either human IgG₁ Fc domain fusions or mouse IgG_2a Fc domain fusions and expressed in HEK293 cells (ATCC).