Overactivation of plasmacytoid dendritic cells inhibits antiviral T-cell responses: a model for HIV immunopathogenesis

Abstract

A delicate balance between immunostimulatory and immunosuppressive signals mediated by dendritic cells (DCs) and other antigen-presenting cells (APCs) regulates the strength and efficacy of antiviral T-cell responses. HIV is a potent activator of plasmacytoid DCs (pDCs), and chronic pDC activation by HIV promotes the pathogenesis of AIDS. Cholesterol is pivotal in maintaining HIV envelope integrity and allowing HIV-cell interaction. By depleting envelope-associated cholesterol to different degrees, we generated virions with reduced ability to activate pDCs. We found that APC activation was dissociated from the induction of type I IFN-α/β and indoleamine-2,3-dioxygenase (IDO)-mediated immunosuppression in vitro. Extensive cholesterol withdrawal, resulting in partial protein and RNA loss from the virions, rendered HIV a more powerful recall immunogen for stimulating memory CD8 T-cell responses in HIV-exposed, uninfected individuals. These enhanced responses were dependent on the inability of cholesterol-depleted HIV to induce IFN-α/β.

Figure 1

Envelope cholesterol withdrawal impairs HIV-induced IFN-α production and IDO activity. IFN-α (A) and IFN-β (B) were quantified by ELISA in the supernatants of PBLs from healthy donors (n = 19) cultured with HIV or HIV treated with different concentrations of βCD (20, 80, or 120mM) or medium alone (CTRL). (C) The concentrations of kynurenine (KYN) and tryptophan (TRP) were measured by HPLC (n = 25); the ratio between KYN and TRP is shown; horizontal dotted line indicates the sensitivity threshold of the assay. (D) IFN-α in supernatants from PBLs cultured in the presence of HIV, βCD80-HIV, or βCD120-HIV normalized according to viral RNA concentrations (n = 3). In all graphs, horizontal bars represent median values; in panels A-C, boxes indicate the interquartile range and vertical lines extend to 90th and 10th percentiles. In panel D, vertical lines indicate interquartile range. *P values that remained significant after Hochberg correction for multiple comparisons.

Figure 2

Uptake of HIV and βCD-HIV by APCs. Flow cytometric histograms (left) and whisker plots (right) showing staining for DL488 in pDCs (A), monocytes (B), and mDCs (c) from PBLs (n = 10) cultured in presence of DL488-labeled HIV or HIV treated with different concentrations of βCD (20, 80, or 120mM) and unlabeled HIV or medium alone (CTRL). In all histograms, vertical dotted lines indicate thresholds of positive staining based on unlabeled HIV control; 1 example of 10 experiments is shown. In all whisker plots, horizontal bars represent median values; vertical lines indicate interquartile range. *P values that remained significant after Hochberg correction for multiple comparisons.

Figure 3

Expression of T-cell activation markers after PBMC exposure to HIV and βCD-treated HIV. (A) Flow cytometric contour plots showing detection of T cells (CD3⁺; left panel) and distinction of T-cell subsets based on CD4 and CCR5 expression (right panel). (B) Flow cytometric histograms of CD38 (top panels) and CD69 (bottom panels) expression on the gated T-cell subpopulations in the different culture conditions (vertical dotted lines indicate thresholds of positive staining based on isotype controls; 1 example of 15 experiments is shown). (C) CD38 (top panel) and CD69 (bottom panel) expression measured as frequency of expressing T cells in the different culture conditions for each T-cell subset (n = 15). Horizontal bars represent median values; vertical lines extend to 75th and 25th percentiles. P values show comparisons with control. *P values that remained significant after Hochberg correction for multiple comparisons.

Figure 4

Induction of HIV-specific IFN-γ-producing memory CD8 T-cell responses by cholesterol-depleted HIV. A) Frequency of IFN-γ–producing CD8 T cells in HESN (n = 10) and HIV⁺ (n = 10) patients after exposure of PBLs to βCD-treated and untreated HIV in the presence of costimulating anti-CD28 Abs. (B) Frequency of IFN-γ–producing CD8 T cells in HESN (n = 5) and HIV⁺ (n = 5) patients after exposure of PBLs to HIV in the presence or absence of blocking Abs against αIFNAR2. (C) Frequency of IFN-γ–producing CD8 T cells in HESN (n = 3) and HIV⁺ (n = 3) patients after exposure of PBLs to βCD120-HIV in the presence or absence of rIFN-α. In all plots, gray dots represent HESN patients and white dots represent HIV⁺ patients (each individual is indicated by a dot). Horizontal bars represent the median values for HESN patients (gray bars in panels B and C) and HIV⁺ patients (white bars in panels B and C). *P values that remained significant after Hochberg correction for multiple comparisons.

Figure 5

Double-threshold model for pDC stimulation and its effect on the course of viral infections. (A) During acute infections or the acute phase of most chronic infections, pDCs are activated beyond the threshold at which they exert antiviral activity, but pDC stimulation is rapidly reduced to maintain APC activity in the absence of proapoptotic and antiproliferative mechanisms. This allows the development and preservation of efficient T-cell responses that clear or control the infection. (B) If pDC activation is not controlled at the end of the acute phase (as during HIV infection), antiproliferative and proapoptotic mechanisms are kept active and undermine the maintenance of memory T-cell responses, favoring viral persistence.