Dendritic cell precursors are permissive to dengue virus and human immunodeficiency virus infection

Abstract

CD14(+) interstitial cells reside beneath the epidermis of skin and mucosal tissue and may therefore play an important role in viral infections and the shaping of an antiviral immune response. However, in contrast to dendritic cells (DC) or blood monocytes, these antigen-presenting cells (APC) have not been well studied. We have previously described long-lived CD14(+) cells generated from CD34(+) hematopoietic progenitors, which may represent model cells for interstitial CD14(+) APC. Here, we show that these cells carry DC-SIGN and differentiate into immature DC in the presence of granulocyte-macrophage colony-stimulating factor. We have compared the CD14(+) cells and the DC derived from these cells with respect to dengue virus and human immunodeficiency virus type 1 (HIV-1) infection. Both cell types are permissive to dengue virus infection, but the CD14(+) cells secrete the anti-inflammatory cytokine interleukin 10 and no tumor necrosis factor alpha. Regarding HIV, the CD14(+) cells are permissive to HIV-1, release higher p24 levels than the derived DC, and more efficiently activate HIV Pol-specific CD8(+) memory T cells. The CD14(+) DC precursors infected with either virus retain their DC differentiation potential. The results suggest that interstitial CD14(+) APC may contribute to HIV-1 and dengue virus infection and the shaping of an antiviral immune response.

FIG. 1.

Generation of CD14⁺ cells from CD34⁺ hematopoietic progenitors. (A) Schematic overview of the culture procedures to obtain CD14⁺ preDC and DC. CD34⁺ progenitors isolated from umbilical cord blood were cultured in complete medium containing SCF, GM-CSF, and TNF-α (4, 7). After 5 days, the cells were harvested, washed, and cultured for 6 days in M-CSF. CD14⁺ cells were purified and allowed to differentiate to DC in the presence of GM-CSF-IL-4-TGF-β (GIT). Alternatively, DC were obtained by growing the day 5 cell population in GM-CSF-IL-4. Shown is the expression profile of CD1a versus CD14 of the cell population obtained after culture in M-CSF and before cell purification. (B) The antigenic phenotype of CD14⁺ positively selected cells was determined by flow cytometry using specific antibodies. The data are representative of more than five cord blood samples. All markers (except for intracellular factor XIIIa and CD68) were detected at the cell surface. White histograms represent isotype controls, and specific labeling is shown in gray.

FIG. 2.

CD14⁺ cells differentiate into DC. (A) CD14⁺ DC precursors were grown for 3 days in either GM-CSF, GM-CSF-IL-4, or GM-CSF-IL-4-TGF-β, and the expression of CD1a versus CD14 was assessed by flow cytometry. The percentage of CD1a⁺ CD14⁻ cells is shown. The data are representative of five donors. (B) CD14⁺ DC precursors were cultured for 3 or 5 days in GM-CSF-IL-4-TGF-β, and the percentage of CD1a⁺ CD14⁻ cell was assessed each day. Shown are the results for two different donors. (C) CD1a⁺ CD14⁻ cells, obtained either by culturing CD14⁺ DC precursors in GM-CSF-IL-4-TGF-β (preDC>DC) or directly from CD34⁺ progenitors in GM-CSF-IL-4 (CD34>DC), were stimulated with LPS, and the expression of CD80, CD83, and HLA-DR was monitored by flow cytometry before and after LPS stimulation. (D) The different APC were cultured in graded doses with allogeneic cord blood-purified CD4⁺ T cells. T-cell proliferation was measured by [³H]methyl-thymidine incorporation and expressed as mean cpm ± standard deviation for triplicate wells. PreDC were untreated (preDC), stimulated with LPS (preDC+LPS), differentiated into DC without (preDC>DC) or with (preDC>DC+LPS) subsequent LPS stimulation, and compared to control DC derived from CD34⁺ progenitors and stimulated with LPS (CD34>DC + LPS).

FIG. 3.

CD14⁺ DC precursors are infected by DV type 2. (A) CD14⁺ DC precursors (preDC) and CD14⁺ precursor-derived DC (preDC>DC) were incubated with UV-irradiated or live DV at different MOI, as indicated. At 2 days postinfection, the number of virions in the cell supernatant was measured by plaque assay. The data are representative of three different donors. (asterisk, undetected). (B) CD14⁺ DC precursors (preDC) and CD14⁺ precursor-derived DC (preDC>DC) or CD34⁺ progenitor-derived DC (CD34>DC) were infected with UV-irradiated or live DV at MOI 5. Two days after infection, production of IL-10 (black bars) and TNF-α (grey bars) was measured in the supernatants. The data are representative of three different donors. (asterisk, undetected). (C) CD14⁺ DC precursors (preDC) and CD14⁺ precursor-derived DC (preDC>DC) were infected with UV-irradiated or live DV at different MOI, as indicated. Three days later, the cell surface expression of CD83 was assessed by flow cytometry. The data are representative of three different donors. (D) Two hours after infection of preDC with DV at an MOI of 1, the cells were cultured in GM-CSF-IL-4-TGF-β. Differentiation into CD1a⁺ CD14⁻ immature DC was tested 1 and 2 days afterwards by monitoring CD1a and CD14 expression. The data are representative of three different donors.

FIG. 4.

CD14⁺ DC precursors are infected by HIV_BAL and present an HIV-encoded epitope to specific CD8⁺ T cells. (A) CD14⁺ DC precursors (preDC) and CD14⁺ precursor-derived DC (preDC>DC) or CD34⁺ progenitor-derived DC (CD34>DC), as well as DC obtained from circulating monocytes (MDDC), were cultured with macrophage-tropic HIV_BAL. As controls, preDC were mock treated (preDC Mock) or infected with T-cell-tropic HIV_LAI (preDC + HIV-LAI). After 5 days, supernatant HIV-Gag levels were measured using a p24-specific ELISA. The points represent the p24 level in each experiment, and the horizontal bars represent the mean of the data. (B) HLA-A2⁺ CD14⁺ DC precursors (preDC) and CD34⁺ progenitor-derived DC (CD34>DC) were either mock treated (white bars) or infected with HIV_BAL (gray bars). After 7 days, the cells were washed, and 2 × 10⁴ cells/well were cultured with a Pol_476-484-specific HLA-A2-restricted CD8⁺ T-cell line (T cell:APC ratio of 1:1). The number of IFN-γ-secreting T cells was measured by enzyme-linked immunospot assay and is expressed as the mean ± standard deviation of triplicate tests, representative of three different experiments. (C) Cell surface phenotypic analysis of mock-treated (preDC mock) or HIV-infected CD14⁺ DC precursors (preDC + HIV). Three days after infection or mock treatment, the cells were cultured for 3 days in GM-CSF-IL-4-TGF-β, and their differentiation into immature DC was assessed by monitoring the expression of different markers. White histograms represent isotype controls, and specific labeling is shown in gray. The data are representative of two different donors.