sHLA-G1 binds to the CD160 receptor expressed by endothelial cells. (A, left) anti-CD160 mAb (open profiles) stains Jurkat-CD160 but not untransfected Jurkat (filled profiles, isotype control). Flow cytometry analysis. (right) sHLA-G1 tetramer binds to HUVECs and Jurkat-CD160 control transfectant (open profiles) but not to untransfected Jurkat cells (filled profiles, control staining with streptavidin-PE). Flow cytometry analysis. (B) Recombinant sHLA-G1 (sG1) blocks CD160 mAb binding to HUVECs (filled profile, isotype control). Flow cytometry analysis. Results are representative of 3 independent experiments. (C) Soluble CL1-R2 anti-CD160 mAb triggers inhibition of in vitro angiogenesis. HUVECs were seeded on Matrigel in the presence or absence of FGF2 (10 ng/mL) and sHLA-G1 (sG1, 1 μg/mL) or CD160 mAb (+++, 10 μg/mL; +, 1 μg/mL) or IgG1-isotype control (10 μg/mL). Photographs of each well were taken after 24 hours and angiogenesis quantified. Results are mean ± SD of triplicate wells and are representative of 5 independent experiments. ^***P < .001. ^*P < .005. ns indicates not significant (ANOVA) compared with FGF2-treated cells. (D) Soluble CL1-R2 anti-CD160 mAb induces HUVEC but not fibroblast apoptosis. HUVECs were treated with sG1 (1 μg/mL), CD160 mAb (10 μg/mL), or control IgG1 (10 μg/mL) or were untreated (UT) for 50 hours in the presence of VEGF (50 ng/mL). Apoptotic cells were detected by flow cytometry using annexin V/PI double staining. (Left) Results of representative experiment. (Right) Histograms. Data are shown as mean ± SEM percentage of annexin V-positive cells. ^*P ≤ .02, Student t test.