Vpu-mediated control of IFN-I production by pDCs involves engagement and activation of ILT7 by BST2.

<p><b>(A-C)</b> Vpu-mediated BST2 antagonism enhances activation of ILT7. ILT7+ NFAT-GFP reporter cells were co-cultured with HEK293T (mock) or BST2-expressing HEK293T cells mock-transfected or transfected with the indicated pNL4.3 constructs (WT or dU). <b>(A)</b> Representative example of ILT7 activation as determined by the percentage of NFAT-GFP positive cells measured by flow cytometry. <b>(B)</b> Percentage of ILT7 activation after co-culture with the indicated HIV/BST2 expressing HEK 293T cells relative to WT HIV-producing cells (100%) (n = 4). <b>(C)</b> Percentage of BST2 surface expression in HEK293T cells after co-transfection of BST2 with the indicated HIV provirus relative to dU HIV-producing cells (100%) (n = 4). <b>(D-F)</b> Effect of ILT7 depletion on IFN-I production by pDCs. <b>(D)</b> Non-pDC fraction (BDCA-2-) and siRNA-treated enriched pDCs (CD14-/BDCA-2+) were stained using anti-ILT7 Abs as indicated. A representative example of absolute levels <b>(E)</b> or relative percentages <b>(F)</b> of IFN-I produced after co-culture of control pDCs (pDC-siCTRL) or ILT7-depleted pDCs (pDC-siILT7) with the indicated infected MT4 cells are shown. The amount of IFN-I released by pDC siCTRL in contact with dU HIV-infected cells was set at 100% (n = 4). <b>(G-I)</b> Effect of recombinant soluble ILT7 on IFN-I production by pDCs. <b>(G)</b> Expression of a HA-tagged soluble ILT7 (soILT7-HA) in HEK 293T cells. Cells and supernatants (sup) were analyzed by Western blot (WB) using anti-HA Abs. Purity of secreted soILT7-HA was confirmed by Coomassie staining (sup Coom). A representative example of <b>(H)</b> absolute levels or <b>(I)</b> relative percentages of IFN-I production after co-culture of PBMCs with the indicated infected MT4 cells pre-treated with control (CTRL) or soILT7-HA-containing supernatants are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of CTRL supernatant was set at 100% (n = 6). Two-tailed paired <i>t</i>-test was used in B-C while repeated measures ANOVA with Bonferroni’s multiple comparison test was used in F and I (*** p<0.001, ** p<0.01, ns not significant (p>0.05)). Error bars represent SD.