Inability of plasmacytoid dendritic cells to directly lyse HIV-infected autologous CD4+ T cells despite induction of tumor necrosis factor-related apoptosis-inducing ligand

Abstract

The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-alpha) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4(+) T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-alpha/beta were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-alpha upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV(+) subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4(+) T cells from viremic HIV(+) subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-alpha, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV(+) CD4(+) T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4(+) T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-alpha in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4(+) T cells.

FIG. 1.

Increase in plasma levels of sTRAIL, but not IFN-α or IFN-β, in HIV-1-infected subjects after viral rebound during ART interruption. Soluble TRAIL (A), IFN-α (B), and IFN-β (C) levels were determined by ELISA in cryopreserved plasma samples from HIV-1-infected subjects at baseline (n = 21) and after 2, 4, and 6 weeks of ART interruption (n = 21). Horizontal bars represent the median values for each group, and brackets indicate statistical significance by Wilcoxon signed rank tests. ns, not significant; VL, viremia level (copies of HIV-1 RNA/ml). (D) Association between increased levels of TRAIL and T-cell activation was determined by the Spearman rank correlation. ETI, end of 6-week treatment interruption; BTI, beginning of 6-week treatment interruption.

FIG. 2.

Increase in sTRAIL but not IFN-α or IFN-β in HIV-1-infected subjects during chronic viremia or after ART-induced viral suppression. Soluble TRAIL (A), IFN-α (B), and ΙFΝ-β (C) levels were determined by ELISA in cryopreserved plasma samples from controls and HIV-1-infected subjects. Data from HIV-1⁺ subjects were segregated into viremic (n = 37) and aviremic (n = 32) groups. Horizontal bars represent the median values for each group, and brackets indicate statistical significance by Mann-Whitney U tests. ns, not significant.

FIG. 3.

Increased expression of mTRAIL but not IRF-7 or IFN-α by PDC from HIV-1-infected viremic subjects. mTRAIL (A) and intracellular IRF-7 (B) levels were determined by flow cytometry on freshly isolated PBMC-gated BDCA2/4⁺ PDC. Bar graphs represent composite data from 19 different donors. Representative diagrams for controls and HIV⁺ subjects are shown below the graphs. Medians and ranges for percentages of PDC expressing mTRAIL and fluorescence intensity (MFI) for IRF-7 are shown. (C) Determination of the intracellular expression of IFN-α and TNF-α was performed by intracellular flow cytometry on freshly isolated PBMC after resiquimod stimulation. (D) IFN-α production by PDC from 10 HIV⁺ subjects before and after ART interruption and from 16 controls in response to 18 h of HIV-AT2 stimulation was determined by ELISA. Brackets indicate statistical significance by Mann-Whitney U tests or Wilcoxon signed rank tests.

FIG. 4.

DR5 surface expression in cell lines and primary cells. (A) DR5 surface expression was determined on HIV-1-infected Sup-T1, uninfected Sup-T1, and K562 cells and the melanoma cell line WM793, used as the control. Gray histograms represent the isotype control, and open histograms represent DR5. (B) Representative staining for DR5 expression on freshly isolated CD8⁻ CD4⁺ gated T cells is shown for one control subject and one viremic subject. (C) Composite data showing medians and ranges for DR5 expression from 10 controls and 16 HIV-1 infected subjects. Brackets indicate statistical differences by Mann-Whitney U tests. ns, not significant. (D) Purified primary CD4⁺ T cells from a control donor were infected in vitro with HIV-1_NL4-3 for 4 days and stained for DR5 expression along with intracellular p24 Ag. One representative experiment of five is shown.

FIG. 5.

TLR engagement induces robust IFN-α production but does not modulate DR5 expression on CD4⁺ T cells. Freshly isolated PBMC were exposed to CpG-2216 (A) or HIV-1-infected or uninfected primary autologous CD4⁺ T cells at a 10:1 ratio (B) for 18 h, and DR5 expression was determined on gated CD3⁺ CD4⁺ T cells. (C and D) Cell free supernatants from panels A and B were tested for IFN-α by ELISA.

FIG. 6.

Purified PDC do not express perforin and cannot kill K562 target cells. (A) PDC were enriched to high purity from PBMC by negative selection with magnetic beads and stained with monoclonal antibodies to BDCA-2/4, lineage (LIN) cocktail mix, and CD56. The frequencies of PDC before and after purification and the frequency of contaminating CD56⁺ NK cells are shown. (B) Perforin expression in CD56⁺ CD3⁻ gated NK cells and BDCA-2⁺/4⁺ LIN⁻ gated PDC. (C) Purified PDC and NK cells were used as effector cells in a chromium release assay with DR5-expressing K562 targets in the presence or absence of 1,000 nM concanamycin A (CMA) at a 10:1 E:T ratio for 6 h (PDC) or 4 h (NK). Means ± standard errors of 4 different experiments are shown. DMSO, dimethyl sulfoxide.

FIG. 7.

PDC efficiently kill DR5-expressing Sup-T1 cells (infected or not with HIV) but lack lytic activity against HIV-infected primary autologous CD4⁺ T cells. (A) DR5 and p24 Ag expression in Sup-T1 cells. (B) Purified PDC and NK cells were incubated with ⁵¹Cr-labeled DR5-expressing Sup-T1 cells or HIV-infected Sup-T1 cells in the presence of 10 μg/ml control antibody or 10 μg/ml anti-TRAIL (B) or 1,000 nM CMA (C) for 6 h at a 10:1 E:T ratio. Results represent means ± standard errors for 4 different donors.

FIG. 8.

PDC accessory cell help is required to activate NK cells to lyse HIV-1-infected primary aCD4⁺ T cells. (A) Total PBMC, PDC-depleted PBMC, or purified PDC were incubated with HIV-1-infected or uninfected aCD4⁺ T cells for 18 h at a 10:1 E:T ratio, and supernatants were tested for IFN-α secretion by ELISA. (B) Staining with CD69 antibody is shown on CD3⁻ CD56⁻ gated NK cells. Gray histograms represent isotype-matched control antibody, and open histograms represent CD69.

FIG. 9.

PDC lack lytic activity against HIV-infected primary autologous CD4⁺ T cells. (A) Purified NK cells were incubated in the presence or absence of autologous PDC and the TLR9 agonist CpG-2216 for 18 h at a 10:1 NK cell/PDC ratio. Cells were then incubated with chromium-labeled primary HIV-1-infected aCD4⁺ T cells at a 10:1 E/T ratio for 6 h in a standard chromium release assay. (B) Resting or activated PDC or activated NK cells were incubated with chromium-labeled primary HIV-1-infected aCD4⁺ T cells at a 10:1 E/T ratio for 6 h. Means ± standard errors of 4 to 6 different experiments are shown.