Human immunodeficiency virus type 1 activates plasmacytoid dendritic cells and concomitantly induces the bystander maturation of myeloid dendritic cells

Abstract

In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-alpha/beta) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11c+ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Blood-purified pDCs and CD11c+ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-alpha and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11c+ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate naïve CD4+ T cells, albeit less efficiently than CD11c+ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses.

FIG. 1.

HIV-1 activates pDCs but not CD11c⁺ DCs. pDCs and CD11c⁺ DCs were obtained by magnetic bead depletion and fluorescence-activated cell sorting. (A) pDCs were cultured with Mv, 300 ng of HIV-1_MN/ml, or 20 ng of IL-3/ml. All HIV-1 amounts are based on p24 equivalents per milliliter. After 16 h, pictures of cultures were taken by using an inverted microscope. (B) pDCs were cultured with 20 ng of IL-3/ml, 300 ng of HIV-1 (right panel, MN, left panel, JR-CSF)/ml, AT-2 HIV-1 (right panel, MN, left panel, JR-CSF), Mv, or 10 μM R-848 while CD11c⁺ DCs were cultured alone, with HIV-1, AT-2 HIV-1, Mv, or R-848. After 16 h, expression of CCR7, HLA-DR, CD80, CD83, and CD86 was measured. CCR7 expression was not assessable on CD11c⁺ DCs because the FL2 channel was saturated by PE-conjugated anti-CD11c MAb used to sort these cells. DC survival can be estimated by the percentage of gated cells on the forward scatter/side scatter dot plot. Numbers on histogram plots represent means of fluorescence intensity. (C) pDCs were cultured with Mv, 300 ng of HIV-1_MN/ml, 500 ng of HIV-1_NL4-3/ml, or HIV-1_Δenv. After 16 h, expression of CD83 was measured. (D) Presence of IFN-α and TNF-α in culture supernatants as measured by ELISA. Each figure is representative of at least three different experiments demonstrating similar trends.

FIG. 1.

HIV-1 activates pDCs but not CD11c⁺ DCs. pDCs and CD11c⁺ DCs were obtained by magnetic bead depletion and fluorescence-activated cell sorting. (A) pDCs were cultured with Mv, 300 ng of HIV-1_MN/ml, or 20 ng of IL-3/ml. All HIV-1 amounts are based on p24 equivalents per milliliter. After 16 h, pictures of cultures were taken by using an inverted microscope. (B) pDCs were cultured with 20 ng of IL-3/ml, 300 ng of HIV-1 (right panel, MN, left panel, JR-CSF)/ml, AT-2 HIV-1 (right panel, MN, left panel, JR-CSF), Mv, or 10 μM R-848 while CD11c⁺ DCs were cultured alone, with HIV-1, AT-2 HIV-1, Mv, or R-848. After 16 h, expression of CCR7, HLA-DR, CD80, CD83, and CD86 was measured. CCR7 expression was not assessable on CD11c⁺ DCs because the FL2 channel was saturated by PE-conjugated anti-CD11c MAb used to sort these cells. DC survival can be estimated by the percentage of gated cells on the forward scatter/side scatter dot plot. Numbers on histogram plots represent means of fluorescence intensity. (C) pDCs were cultured with Mv, 300 ng of HIV-1_MN/ml, 500 ng of HIV-1_NL4-3/ml, or HIV-1_Δenv. After 16 h, expression of CD83 was measured. (D) Presence of IFN-α and TNF-α in culture supernatants as measured by ELISA. Each figure is representative of at least three different experiments demonstrating similar trends.

FIG. 2.

pDCs activated by HIV-1 migrates toward CCL19. pDCs were cultured with 20 ng of IL-3/ml, 300 ng of HIV-1 (MN or JR-CSF)/ml, 300 ng of AT-2 HIV-1 (MN or JR-CSF)/ml, or 10 μM R-848. After 16 h, chemotaxis toward 25 ng of CCL19/ml was measured by using a transwell assay. Shown are representative results from four different experiments demonstrating similar trends.

FIG. 3.

pDCs activated by HIV-1 activate CD11c⁺ DCs. (A) CD11c⁺ DCs and pDCs were cultured alone or together in the presence of Mv or 300 ng of HIV-1_MN/ml. After 16 h, surface expression of CD83, CD80, and CD86 was measured using CD11c⁺ as a marker to discriminate DC subsets. (B) CD11c⁺ DCs were cultured alone, with 300 ng of HIV-1_MN/ml, 10 μM R-848, or a 1:2 dilution of supernatants of pDCs or CD11c⁺ DCs cultured for 16 h with Mv or 300 ng of HIV-1_MN/ml. After overnight culture, expression of CD83 by CD11c⁺ DCs was measured. (C) CD11c⁺ DCs were cultured in the absence or presence of neutralizing antibodies against IFN-α, IFN-β, TNF-α, and IFN-α/-β receptor and with supernatants of pDCs previously cultured for 16 h with 300 ng of HIV-1_MN/ml. After overnight culture, expression of CD83 by CD11c⁺ DCs was measured. (D) Immature monocyte-derived DCs were cultured alone, with 10 μM R-848, or a 1:3 dilution of supernatants of pDCs or CD11c⁺ DCs cultured for 16 h with Mv, 300 ng of HIV-1 (right panel, MN; left panel, JR-CSF)/ml or 300 ng of AT-2 HIV-1 (right panel, MN; left panel, JR-CSF)/ml. After overnight culture, expression of CD83 by monocyte-derived DCs was measured. (E) Immature monocyte-derived DCs were cultured alone, with R-848, 300 ng of HIV-1_MN/ml, or a 1:3 dilution of supernatants of pDCs or CD11c⁺ DCs cultured during 16 h with Mv or 300 ng of HIV-1_MN/ml. After 40 h, expression of CD83 by monocyte-derived DCs was measured. Each image is representative of at least three different experiments.

FIG. 3.

pDCs activated by HIV-1 activate CD11c⁺ DCs. (A) CD11c⁺ DCs and pDCs were cultured alone or together in the presence of Mv or 300 ng of HIV-1_MN/ml. After 16 h, surface expression of CD83, CD80, and CD86 was measured using CD11c⁺ as a marker to discriminate DC subsets. (B) CD11c⁺ DCs were cultured alone, with 300 ng of HIV-1_MN/ml, 10 μM R-848, or a 1:2 dilution of supernatants of pDCs or CD11c⁺ DCs cultured for 16 h with Mv or 300 ng of HIV-1_MN/ml. After overnight culture, expression of CD83 by CD11c⁺ DCs was measured. (C) CD11c⁺ DCs were cultured in the absence or presence of neutralizing antibodies against IFN-α, IFN-β, TNF-α, and IFN-α/-β receptor and with supernatants of pDCs previously cultured for 16 h with 300 ng of HIV-1_MN/ml. After overnight culture, expression of CD83 by CD11c⁺ DCs was measured. (D) Immature monocyte-derived DCs were cultured alone, with 10 μM R-848, or a 1:3 dilution of supernatants of pDCs or CD11c⁺ DCs cultured for 16 h with Mv, 300 ng of HIV-1 (right panel, MN; left panel, JR-CSF)/ml or 300 ng of AT-2 HIV-1 (right panel, MN; left panel, JR-CSF)/ml. After overnight culture, expression of CD83 by monocyte-derived DCs was measured. (E) Immature monocyte-derived DCs were cultured alone, with R-848, 300 ng of HIV-1_MN/ml, or a 1:3 dilution of supernatants of pDCs or CD11c⁺ DCs cultured during 16 h with Mv or 300 ng of HIV-1_MN/ml. After 40 h, expression of CD83 by monocyte-derived DCs was measured. Each image is representative of at least three different experiments.

FIG. 3.

pDCs activated by HIV-1 activate CD11c⁺ DCs. (A) CD11c⁺ DCs and pDCs were cultured alone or together in the presence of Mv or 300 ng of HIV-1_MN/ml. After 16 h, surface expression of CD83, CD80, and CD86 was measured using CD11c⁺ as a marker to discriminate DC subsets. (B) CD11c⁺ DCs were cultured alone, with 300 ng of HIV-1_MN/ml, 10 μM R-848, or a 1:2 dilution of supernatants of pDCs or CD11c⁺ DCs cultured for 16 h with Mv or 300 ng of HIV-1_MN/ml. After overnight culture, expression of CD83 by CD11c⁺ DCs was measured. (C) CD11c⁺ DCs were cultured in the absence or presence of neutralizing antibodies against IFN-α, IFN-β, TNF-α, and IFN-α/-β receptor and with supernatants of pDCs previously cultured for 16 h with 300 ng of HIV-1_MN/ml. After overnight culture, expression of CD83 by CD11c⁺ DCs was measured. (D) Immature monocyte-derived DCs were cultured alone, with 10 μM R-848, or a 1:3 dilution of supernatants of pDCs or CD11c⁺ DCs cultured for 16 h with Mv, 300 ng of HIV-1 (right panel, MN; left panel, JR-CSF)/ml or 300 ng of AT-2 HIV-1 (right panel, MN; left panel, JR-CSF)/ml. After overnight culture, expression of CD83 by monocyte-derived DCs was measured. (E) Immature monocyte-derived DCs were cultured alone, with R-848, 300 ng of HIV-1_MN/ml, or a 1:3 dilution of supernatants of pDCs or CD11c⁺ DCs cultured during 16 h with Mv or 300 ng of HIV-1_MN/ml. After 40 h, expression of CD83 by monocyte-derived DCs was measured. Each image is representative of at least three different experiments.

FIG. 4.

pDCs activated by HIV-1 remain poor activators of naïve CD4⁺ T cells. (A) pDCs were cultured with Mv and IL-3, with 300 ng of HIV-1_MN/ml, or with 10 μM R-848, while CD11c⁺ DCs were cultured with Mv, HIV-1_MN, or with R-848. After 16 h DCs were washed and cocultured with allogeneic naïve CD45RA⁺ CD4⁺ T cells at different T-cell/DC ratios. After 4 days, [³H]thymidine was added for 12 h. Cells were then harvested to assess proliferation. (B) pDCs and CD11c⁺ DCs were cultured with naïve CD4⁺ T cells at a ratio of 1:30. After 7 days the T cells were restimulated with PMA and ionomycin in presence of brefeldin A and were analyzed by intracellular flow cytometry for production of IFN-γ or IL-4. The percentage of T cells producing cytokines is depicted in the FACS profiles. Each image is representative of at least three different experiments.