Vpu-mediated control of IFN-I production by pDCs requires the presence of BST2 on infected donor cells.

(A-C) Control (MT4-shNT) or BST2-depleted (MT4-shBST2) MT4 cells were mock-infected, or infected with GFP-marked NL4.3 WT or dU viruses for 48 h. (A) Surface expression of BST2 on GFP-positive MT4 cells infected with WT (dashed grey histogram) or dU (solid black histogram) was evaluated by flow cytometry. Mean fluorescence intensity (MFI) values are indicated for each sample (staining using pre-immune rabbit serum, PI, shaded grey histograms). (B-C) The indicated MT4 donor cells were co-cultured with PBMCs. After 24 h, levels of bioactive IFN-I were measured in supernatants. A representative example of absolute levels (B) or relative percentages (C) of IFN-I produced after co-culture of the indicated infected MT4 cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected MT4-shNT cells was set at 100% (n = 12). (D-E) MT4-shNT (BST2 +) or MT4-shBST2 (BST2 -) cells were infected with GFP-marked NL4.3 WT or T/F CH077 viruses for 48 h. Similar number of p24+ infected cells were then co-cultured with PBMCs. After 24 h, levels of bioactive IFN-I were measured in supernatants. A representative example of absolute levels (D) or relative percentages (E) of IFN-I produced after co-culture of the indicated infected MT4 cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with infected MT4-shBST2 cells was set at 100% (n = 6). Repeated measures ANOVA with Bonferroni’s multiple comparison test was used. (*** p<0.001, ns not significant (p>0.05)). Error bars represent SD.