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. 2019 Sep 13;10:2209. doi: 10.3389/fimmu.2019.02209

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Copyright © 2019 Battin, De Sousa Linhares, Paster, Isenman, Wahrmann, Leitner, Zlabinger, Steinberger and Hofer.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Interaction between NRP1 and C4d deposited to surfaces via thioester binding following classical complement activation. (A) C4d biotinylated at its intrinsic thioester carbonyl via amine-PEG2-biotin reagent (ih-C4d-bio) was immobilized to a solid phase either via streptavidin (+SA) or in random orientation without SA (–SA) and detected by ELISA using a monoclonal C4d antibody. (B) Interaction between C4d, immobilized to a solid phase as shown in (A), and rh-NRP1-Ig and rh-ILT4-Ig. Rh-CTLA-4-Ig fusion proteins and goat anti-human-IgG (ctrl) served as negative controls. (C) C4d biotinylated via its intrinsic thioester was tetramerized using streptavidin-APC and probed with BW cells expressing ILT4 or NRP1 and analyzed via flow cytometry. (D) Antibody-coated sheep erythrocytes (EA) bearing C4d were prepared using purified C1, C4, C4 binding protein (C4BP), and factor I (FI) as described in Material and Methods. Deposition of C4d was detected with a monoclonal C4d antibody (upper panel). Interaction between C4d-loaded EA and control EA with immunoglobulin fusion proteins (rh-ILT4-Ig; rh-NRP1-Ig) is shown in the lower panel. (E) Binding of goat anti-human-IgG (ctrl) or rh-NRP1-Ig (20 or 60 μg/ml) to C4d-EA in the presence or absence of an anti-C4d and anti-C4c antibodies. Data shown are representative for two independently performed experiments. gMFI, geometric mean of fluorescence intensity.