Endothelial Nectin-2 is the major Nectin-3 counter receptor.

<p><b>A</b>: Direct binding of soluble Nectin-3 (Nec3-Fc) (10^-7 M) to indicated immobilized ligands (10^-7 M). Interactions were measured by ELISA. <b>B</b>: Binding of Nec3-Fc (1.5 µM) to HUVECs pre-treated with the indicated mAbs (20 µg/ml). HUVECs were either untreated or pre-treated with anti-Nectin-1 (R1.302), anti-Nectin-2 (R2.477) or anti-PVR (PV.404) mAbs alone or in combinations. Nec3-Fc and DNAM1-Fc binding to HUVECs was then analyzed by FACS.</p

Nectin-3 is expressed on T-lymphocytes and binds to a junctional ligand on ECs.

<p><b>A</b>: Fresh PBMCs were gated on lymphocytes on the basis of both size and granularity. Cells were analyzed by two color-immunofluorescence and cytometry with anti-CD3 in combination with anti-Nectin-2 (R2.477), anti-Nectin-3 (N3.12) and anti-DNAM-1 (FS123) mAbs. DNAM-1, previously described to be expressed on T-cells, was taken as a positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077424#B51" target="_blank">51</a>]. <b>B</b>: Binding of soluble Nectins and DNAM-1 to HUVECs was analyzed by FACS as indicated. <b>C</b>: Binding of soluble Nectins and DNAM-1 to HUVECs was analyzed by one color-immunofluorescence as indicated. Bold arrows show junctional stainings. Bar, 50 µm.</p

Nectin-2 interacts with Nectin-3 during lymphocyte transmigration.

<p><b>A</b>: Lymphocyte transmigration through HUVEC monolayers was performed in the presence of anti-Nectin-2 (R2.477 or L14) blocking mAbs on ECs only, anti-Nectin-3 (N3.12) blocking mAbs on ECs only (EC), lymphocytes only (PBLs) or ECs and lymphocytes (ECs + PBLs). Anti-CD99 blocking mAbs were used as a positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077424#B35" target="_blank">35</a>]. <b>B</b>: Monocyte transmigration was performed in the presence of anti-Nectin-2 (R2.477 or L.14) or anti-Nectin-3 (N3.12 or N3.2) blocking mAbs. In all experiments, either anti-Nectin-4 (N4.40) or anti-CD34 (Immu133) mAbs were used as isotype matched irrelevant antibodies. The value 100% corresponds to the number of lymphocytes or lymphocytes that transmigrate in the presence of the anti-CD34 mAbs. Each measurement was performed in triplicate. The results were obtained from three independent experiments. Values are mean ± SEM (error bars; N≥3); ***, P < 0.001; **, P < 0.01; *, P < 0.05.</p

Nectins are expressed on primary ECs.

<p>HUVEC confluent monolayers were analyzed by FACS (<b>A</b>) and immunofluorescence (<b>B</b>) with anti-Nectin-1 (R1.302), anti-Nectin-2 (R2.477), anti-Nectin-3 (N3.12), anti-PVR (PV.404) and (<b>C</b>) with anti-AF-6 and anti-β-catenin mAbs. Bar, 50 µm.</p

Nectin-2 is expressed on HEVs.

<p>Frozen sections of human placenta, skin and lymph nodes were analysed by immunohistochemistry with anti-Nectin-1 (R1.302), anti-Nectin-2 (R2.477), anti-Nectin-3 (N3.12), anti-PVR (PV.404) or anti-AF-6 mAbs. Representative images are shown. Bold arrows show positive specific HEV staining and arrows show negative HEV staining. Bars, 20 µm.</p