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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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Identification of NRP1 as a receptor for C4d. (A) C4d-reactive cells enriched from a BW cell pool expressing a moDCs-cDNA library by multiple rounds of cell sorting. Sorting gates are shown. (B) A single cell clone derived from the C4d-reactive BW cell pool was probed with rh-C4Ad and rh-C4Bd and analyzed via flow cytometry. (C) PCR-amplification of retroviral inserts of a C4d-binding clone. (D) BW cells expressing a 5 kb retroviral insert encoding NRP1 were probed with a NRP1 mAb (monoclonal) or biotinylated rh-C4Ad, rh-C4Bd or ih-C4d (20 μg/ml each; open histograms: reactivity of NRP1 mAb or C4d to BW control cells; gray histograms: reactivity of NRP1 mAb or C4d to BW NRP1 cells). Biotinylation of rh-C4Ad and rh-C4Bd employed the NHS-biotin procedure, except for ih-C4d, which was specifically biotinylated on the thioester carbonyl moiety employing amine-PEG2-biotin reagent. (E) Monocytes and moDCs analyzed for NRP1 expression (open histograms: isotype control; gray histograms: NRP1 mAb). MFI, mean fluorescence intensity.