Human immunodeficiency virus type 1 Vpr impairs dendritic cell maturation and T-cell activation: implications for viral immune escape

Abstract

Antigen presentation and T-cell activation are dynamic processes involving signaling molecules present in both APCs and T cells. Effective APC function and T-cell activation can be compromised by viral immune evasion strategies, including those of human immunodeficiency virus type 1 (HIV-1). In this study, we determined the effects of HIV-1 Vpr on one of the initial target of the virus, dendritic cells (DC), by investigating DC maturation, cytokine profiling, and CD8-specific T-cell stimulation function followed by a second signal. Vpr impaired the expression of CD80, CD83, and CD86 at the transcriptional level without altering normal cellular transcription. Cytokine profiling indicated that the presence of Vpr inhibited production of interleukin 12 (IL-12) and upregulated IL-10, whereas IL-6 and IL-1beta were unaltered. Furthermore, DC infected with HIV-1 vpr+ significantly reduced the activation of antigen-specific memory and recall cytotoxic-T-lymphocyte responses. Taken together, these results indicate that HIV-1 Vpr may in part be responsible for HIV-1 immune evasion by inhibiting the maturation of costimulatory molecules and cytokines essential for immune activation.

Figures

FIG. 2.

Immunophenotyping of DC infected with HIV-1 vpr+ and HIV-1 vpr−. Immature DC were infected with HIV-1 vpr+ or HIV-1 vpr− as described in Materials and Methods, stimulated with either LPS or cells expressing CD40L, and subsequently analyzed for phenotype by direct flow cytometry. Infected DC were stimulated with irradiated CD40L-expressing J558 cells and assessed for CD80, CD83, and CD86 cell surface molecules using directly conjugated specific antibodies and isotype controls followed by intracellular p24 staining with IgG control, as described earlier. Viable DC were gated on forward and side scatter dot blots and analyzed for surface expression of DC markers in p24-negative uninfected (left upper quadrant) or p24-positive infected (right upper quadrant) populations. The bottom row represents corresponding IgG controls. The overlay column represents the histograms for CD40L-stimulated DC infected with HIV-1 vpr− (black histogram) and CD40L-stimulated DC infected with HIV-1 vpr+ (dark gray histogram). NT, no treatment. The data are representative of four similar experiments.

FIG. 3.

Induction of apoptosis by CD40L- or LPS-stimulated DC infected with HIV-1 vpr− or vpr+. DC were infected and stimulated as described in Materials and Methods. Induction of apoptosis following infection and stimulation was detected by staining the DC with PI and annexin V-FITC antibodies. (Top left) Gating of DC on forward and side scatter dot blots. (Top right) UV-irradiated DC as a control. (Bottom) Apoptotic DC populations with no treatment (NT), CD40L-stimulated uninfected, and HIV-1 vpr−- and vpr+-infected and stimulated DC. The number in the upper right quadrant indicates the percent necrotic cells (PI and annexin V positive). The lower right quadrant in each blot indicates the apoptotic population (annexin V-positive cells). (B) Expression of total and cleaved caspase 3 in DC by immunoblot analysis. The results are representative of three similar experiments.

FIG. 4.

Downregulation of DC costimulatory molecule mRNA expression by real-time RT-PCR. DC were cultured and infected as previously described, followed by 4 hours of incubation with LPS. Real-time RT-PCR was carried out on an ABI 7000 using primers and probes specific for CD80 (A), CD83 (B), CD86 (C), or the internal control RPLPO (D). The figure is representative of data attained from experiments performed in triplicate with three separate donors. The CT used to calculate the relative ratio was the cycle number (x axis) at which probe-specific fluorescence crossed the threshold line (dark horizontal line) as set by ABI PRISM 7000 Sequence Detection System software. Colored lines are defined by adjacent labels.

FIG. 5.

Effect of HIV-1 Vpr on particle uptake by immature DC. Immature DC were infected with HIV-1 vpr+ or HIV-1 vpr− as described in Materials and Methods and cultured for 3 days. Following 24-h CD40L (top row) or LPS (bottom row) stimulation, the cells were incubated with FITC-dextran for 50 min at 37°C or 4°C, and antigen uptake was assessed by flow cytometry. The filled light-gray histograms represent antigen uptake at 37°C; the white histograms indicate antigen uptake at 4°C. The value inside each histogram is the percent endocytosis at 37°C. Antigen uptake by unstimulated and uninfected DC at 37°C was considered to be 100%, calculated based on the corresponding MFI. NT, no treatment. The data are representative of four similar experiments, each performed in triplicate.

FIG. 6.

Production of proinflammatory cytokines from infected DC, followed by CD40L-induced maturation. DC treated with HIV-1 vpr− or HIV-1 vpr+ were stimulated with CD40L (top two rows) or LPS (bottom two rows) for 24 h. DC culture supernatants were collected and assayed for IL-12p70, IL-1β, IL-6, and IL-10 using a cytokine bead array kit in a Luminex 100. NT, no treatment. The results are representative of six independent experiments, each performed in triplicate. *, P < 0.02 compared to DC infected with HIV-1 vpr− and stimulated with CD40L or LPS.

FIG. 7.

Antigen presentation by virus-infected DC measured by IFN-γ ELISPOT. (A to D) HLA-A2-specific uninfected DC and DC infected with either HIV-1 vpr+ or HIV-1 vpr− were loaded with different A2-specific cytotoxic-T-cell peptides (tyrosinase, gp100, and p24gag) and subsequently coincubated with a CD8+ peptide-specific T-cell line in an IFN-γ ELISPOT plate. Antigen-specific immune response was measured by the ability of the T cells to produce IFN-γ as determined by ELISPOT assay. The results are expressed as IFN-γ SFCs per 106 cells. The data represent one out of two independent experiments performed in triplicate. (E and F) DC derived from an HLA-A*0201 donor with influenza virus (Flu) and chronic EBV infections were cultured and infected with vpr− or vpr+ viruses and stimulated with CD40L. The cells were then pulsed with EBV or influenza virus for 2 hours before responder cells were added. Autologous CD14 T cells derived from the same EBV- and influenza virus-infected donor were added at ratios of 1:10 and 1:5 (DC:T cell) for 18 h. The antigen-specific T-cell response was measured by IFN-γ ELISPOT assay using specific antibodies. NT, no treatment. The data are representative of three similar experiments, each performed in triplicate. *, P value < 0.02 compared to HIV-1 vpr−-infected cells.

FIG. 1.

Virus preparation, infection, and expression of viral proteins in infected DC. (A) VSV-G Env-pseudotyped HIV-1 vpr+ and HIV-1 vpr− were prepared as described in Materials and Methods and further characterized for the presence of Vpr by immunoblot analysis using p24- and Vpr-specific antibodies. (B) PBMCs were isolated from healthy donors. CD14+ monocytes were purified by positive selection using anti-CD14 monoclonal antibody-coated magnetic microbeads. The purity of the cells isolated for myeloid-derived monocytes was tested by flow cytometry using CD14- and CD1a-specific antibodies. Expression of CD14 and CD1a on day 0 (top) and day 6 (bottom) of culture in the presence of IL-4 and GM-CSF. The dark-gray histogram represents the corresponding IgG control. The white histogram represents expression of CD14- and CD1a-positive cells. This experiment was repeated several times. (C) PBMC-derived CD14+ monocytes were isolated and cultured with GM-CSF and IL-4 to generate DC. On day 4, the cells were infected with HIV-1 vpr+ and HIV-1 vpr− at an MOI of 2.0 and incubated further in DC culture medium in the presence (+) and absence (−) of AZT (1 μM) with an IgG control. Three days postinfection, the cells were analyzed by flow cytometry to identify the number of infected cells using p24-FITC antibody and FACS analysis. DC were cultured in the presence (white histogram) and absence (light-gray histogram) of AZT (1 μM) with an IgG control (dark gray histogram). (D) Infected DC were lysed and immunoblotted to detect the presence of the viral proteins Gag (p24) and Vpr; α-tubulin was used as an internal loading control. (E) Quantitation of virus particles released into the culture medium by infected DC in the presence and absence of AZT was done by p24 ELISA. Each experiment was repeated at least six times, and similar results were obtained.