Transfer of HIV-1 from infected DCs to CD4⁺ T cells. Sorted CD11c⁺ MDCs were exposed to HIV-1_BaL for 72 h, washed thoroughly, and incubated briefly with CMV or SEB before co-culture with sorted and CFSE-labeled autologous CD4⁺ T cells. (A–C) Co-cultures were analyzed for T cell proliferation (CFSE dilution) and frequency of HIV-1 infection (intracellular p24 staining) after 1.5, 3.5, and 7.5 d. The numbers in each gate indicate the percentages of p24⁺ cells out of all cells. (D) As a control, uninfected MDCs were co-cultured with T cells in the presence or absence of CMV or SEB to measure background staining for p24 and examine the rate of proliferation in the absence of HIV-1. The graphs show CD4⁺ T cells gated on the presence of CFSE labeling, excluding single CD11c⁺ cells to leave out MDCs from the analysis. One representative experiment of three is shown. The numbers in each gate indicate the percentages of p24⁺ cells out of all cells. (E) PDCs (open squares) and MDCs (filled squares) were exposed to different doses of HIV-1_BaL for 72 h before co-culture with T cells in the presence of SEB. The frequency of p24⁺ T cells was again measured after 3.5 d of co-culture. One representative experiment of four is shown.

Pathways for transfer of virus from DCs to T cells differ with virus exposure time. Sorted CD11c⁺ MDCs (A and B) and CD123⁺ PDCs (C and D) in the presence or absence of AZT were exposed to HIV-1_BaL (A and C) or HIV-1_IIIB (B and D) for 72 h with or without R-848 stimulation during the final 24 h. DCs were washed, pulsed with CMV or SEB, and co-cultured with autologous CD4⁺ CFSE-labeled T cells for 3.5 d. The frequency of p24⁺ T cells after co-culture was analyzed by flow cytometry. The graphs show the percentage of p24⁺ CD4⁺ T cells gated on the presence of CFSE labeling and on size, excluding single CD11c⁺/CD123⁺ DCs in 6–11 individual donors. The mean frequency of p24 expression is indicated in each column. Significant differences between the groups were assessed by Mann-Whitney's unpaired t test (**, P ≤ 0.005; *, P ≤ 0.05). (E) MDCs (black bars) and PDCs (white bars) were exposed to HIV-1_BaL for 6 or 72 h in the absence or presence of AZT. DCs were then washed and co-cultured with T cells in the presence of SEB for 3.5 d. The graphs show the frequency of p24⁺ T cells (mean ± SEM) after co-culture in three donors.

Responding T cells exhibit a higher viral replication. Sorted CD11c⁺ MDCs and CD123⁺ PDCs were exposed to HIV-1_BaL or HIV-1_IIIB for 72 h, washed thoroughly, and co-cultured in the presence of CMV antigen and Indinavir with autologous CFSE-labeled CD4⁺ T cells from a CMV-seropositive donor. Fractions of responding and nonresponding T cells (undivided cytokine-producing; dividing [including all divisions] cytokine-producing; dividing noncytokine-producing; and undivided noncytokine-producing) were sorted using flow cytometry. (A) T cells sorted after 3.5 d of co-culture with HIV-1_BaL–exposed MDCs were analyzed for their content of HIV-1 gag DNA copies per 10⁵ T cells using quantitative real time PCR. The graph shows the mean ± SEM of three donors. (B and C) T cells sorted after 10 h of co-culture with HIV-1–infected MDCs (B) or PDCs (C) were analyzed for their content of HIV-1 sssDNA (an early product of replication) using quantitative real time PCR. Delta count values demonstrate the numbers of cycles required for detection of HIV-1 sssDNA and were used as an estimate to examine replication activity in the various fractions. The graphs show the mean ± SEM of two donors.

Productive HIV-1_BaL and HIV-1_IIIB infection in MDCs and PDCs. (A) Sorted CD11c⁺ MDCs (red) and CD123⁺ PDCs (blue) were analyzed for their expression of CD4, CCR5, and CXCR4 after overnight culture. MDCs (B) and PDCs (C) were exposed to HIV-1_BaL or HIV-1_IIIB in the presence or absence of AZT for 72 h using medium as a control. The level of HIV-1 infection was determined by intracellular p24 staining and flow cytometry. Any contaminating monocytes were excluded from the analysis by gating on the cells that expressed CD11c (MDCs) or CD123 (PDCs), but not CD14. The numbers in each gate represent the percentages of p24⁺ cells out of all cells. DCs from 2 of 14 representative donors are shown. (D) MDCs (black bars) and PDCs (white bars) from 12 individual donors (mean ± SEM) were exposed to HIV-1_BaL or HIV-1_IIIB for 72 h and the supernatants were analyzed for IFN-α content by ELISA. Significant differences between the groups were assessed by Wilcoxon's paired t test (***, P ≤ 0.0005). (E) Histograms of the cell surface expression of the costimulatory molecule CD86 on MDCs and PDCs are shown for one representative donor at 72 h (gray region, medium; black line, HIV-1_BaL; green line, R-848 stimulation).

MDCs and PDCs transfer CCR5-using BaL and CXCR4-using IIIB with different efficiencies. Sorted CD11c⁺ MDCs (A and C) and CD123⁺ PDCs (B and D) were exposed to HIV-1_BaL or HIV-1_IIIB for 72 h, washed, and co-cultured with autologous CFSE-labeled CD4⁺ T cells with SEB for 3.5 d in the absence (A and B) or presence (C and D) of the protease inhibitor Indinavir. Cultures were analyzed for T cell proliferation (CFSE dilution) and frequency of HIV-1 infection (intracellular p24 staining). The graphs show CD4⁺ T cells gated on size and lack of CD11c or CD123 expression to exclude DCs from the analysis. The numbers in each gate indicate the percentages of p24⁺ cells out of all cells. One representative experiment of at least five is shown. (E) The graphs show the frequency of p24⁺ T cells (mean ± SEM) after co-culture with MDCs (black bars) or PDCs (white bars) exposed to the respective virus strains. The co-cultures were performed in the absence or presence of Indinavir, as indicated. Significant differences between the groups were assessed by Mann-Whitney's unpaired t test (*, P ≤ 0.05).

DCs mediate the spread of HIV-1 to antigen-specific CD4⁺ T cells. Sorted CD11c⁺ MDCs were exposed to HIV-1_BaL for 72 h, washed thoroughly, and co-cultured for 3.5 d in the presence of CMV antigen and Indinavir with autologous CFSE-labeled CD4⁺ T cells from a CMV-seropositive donor. The graphs show CD4⁺ T cells gated on the presence of CFSE labeling and on size, excluding CD11c⁺ MDCs. Each cell division was analyzed. IFN-γ TNF-α IL-2 represents cytokine production. (A) Cytokine-producing and noncytokine-producing T cells were analyzed for the frequency of HIV-1 infection by intracellular p24 staining (red, undivided cells; blue, first cell division; green, second cell division; orange, third cell division). Numbers represent the percentages of p24⁺ cells out of all cells in that cell division. (B) HIV-1–infected p24⁺ and p24⁻ T cells were analyzed for their capacity to produce cytokines (red, undivided cells; blue, first cell division; green, second cell division; orange, third cell division). Numbers represent the percentages of cytokine-producing cells out of all cells in that cell division. (C) The total responding (i.e., cytokine-producing and/or divided) T cells were compared with respect to their frequency of HIV-1 infection. The p24⁺- and p24⁻-responding T cells were further analyzed for intracellular cytokine content. The numbers in each gate indicate the percentages of those cells out of all cells. The figure shows 1 representative experiment of 10.

Conjugates of DCs and T cells are primary sites for viral production. Sorted CD11c⁺ MDCs (A) or CD123⁺ PDCs (B) were exposed to HIV-1_BaL or HIV-1_IIIB, respectively, for 72 h, washed thoroughly, and incubated briefly with CMV before co-culture with CFSE-labeled autologous CD4⁺ T cells for 3.5 d. The frequency of HIV-1 infection (p24) and cytokine production (IFN-γ TNF-α IL-2) were analyzed in the following four populations: single CD11c⁺/CD123⁺ DCs, single undivided or divided T cells, and conjugates of DCs and T cells. The numbers in each quadrant indicated the percentage of total cells in that quadrant. The figure shows 1 representative experiment of 10.