Role of pDCs in mTRAIL expression and IFN-α production by CD4⁺ cells. (a) Isolated CD4⁺ cells were depleted of pDCs and cultured with HIV-1_MN or rIFN-α. After 24 h, mTRAIL expression was detected on CD4⁺ T cell by flow cytometry. Undepleted CD4⁺ cells cultured with HIV-1_MN were used as positive control for mTRAIL expression. (b) Isolated CD4⁺ cells depleted or not of pDCs were cultured for 24 h with AT-2 HIV_MN and supernatants were tested for their IFN-α content. (c) Isolated pDCs were cultured with AT-2 HIV-1_MN or AT-2 HIV_Ada in presence or absence of anti-IFN-α/β Abs for 24 h and culture supernatants (SN) were used to stimulate CD4⁺ cells depleted of pDCs. After 24 h mTRAIL expression on CD4⁺ T cells was determined by flow cytometry. Data in a and c are representative of four independent experiments. Data in b are mean values with SEs of four independent experiments for each condition tested. **, P < 0.01, determined by a two-tailed Student t test

Regulation of TRAIL expression on CD4⁺ T cells. (a) Detection of mTRAIL by flow cytometry on CD4⁺ T cells after a 24-h incubation with IFN-α or rIFN-β.(b) HIV-1_MN-induced mTRAIL expression on CD4⁺ T cells is inhibited by anti-IFN-α/β- or anti-IFNAR2-blocking Abs. (c) STAT1 and STAT2 expression determined by Western blot in CD4⁺ cells after a 24-h culture with AT-2 HIV-1_MN, AT-2 HIV-1_Ada, rIFN-α, AT-2 HIV-1_MN plus anti-IFN-α/β Abs, or AT-2 HIV-1_Ada plus anti-IFN-α/β Abs. β-Actin was used as a loading control. (d) Intracellular staining of phosphorylated STAT1 (STAT1-P) in CD4⁺ T cells after a 24-h culture with HIV-1_MN or AT-2 HIV-1_MN. Control is CD4⁺ T cells cultured with microvesicles or media. (e) Inhibition of STAT1 and STAT2 protein in CD4⁺ T cells. Anti-STAT1, anti-STAT2, and isotype Abs were delivered into CD4⁺ T cells by using a Chariot kit. Cells were cultured for 24 h with AT-2 HIV_MN plus isotype Ab, AT-2 HIV_MN plus anti-STAT1, or -STAT2 Abs. Chariot was also used without Abs as negative control (mock). Cells were stained for mTRAIL and acquired and analyzed by flow cytometry. Data are representative of four experiments

Role of CD4-gp120 interaction in IFN-α/β production by pDCs. (a) Isolated pDCs were culture 24 h with AT-2 HIV-1MN in presence of sCD4 or AMD at 5 μg/ml. Culture supernatants (SN) were used to culture CD4⁺ cells depleted of pDCs for an additional 24 h. mTRAIL expression on CD4⁺ T cells was determined by flow cytometry. (b) (Left) IFN-α levels in SN of pDCs cultured with AT-2 HIV-1 and viral entry inhibitors were quantified by ELISA. Control (open bar); sCD4 (solid bar); AMD (horizontal lined bar); RANTES (vertical lined bar). (Right) pDCs cultured with or without AMD or RANTES and were stained with anti-CXCR4 or anti-CCR5 Abs and analyzed by flow cytometry. Isotype control (Iso) was used as negative control. (c) Titration of AT-2 HIV-1 for inducing INF-α production by isolated pDCs. (d) pDCs were culture for 24 h with gp120_SF (open bar), gp120_MN (blue bar), gp160_MN (gray bar), or Tat (diagonal bar) at several concentrations (0-5,000 ng/ml). IFN-α levels were quantified by ELISA. (e) PBMC from 13 patients' blood were cultured for 2 d in medium supplemented with 50% autologous plasma in the absence or presence of anti-IFN-α/β Abs at 5 μg/ml. Cells were stained for CD3, CD4, TRAIL, and Annexin V and analyzed by flow cytometry. Data from seven samples are shown. Data in a are representative of four experiments for each condition tested. Data in b-d are mean values with SEs of six experiments for each condition tested. **, P < 0.01 determined by a two-tailed Student t test