HIV-2 is not propagated in mDCs even after 96 h. (A) Directly isolated CD11c⁺ mDCs were exposed to HIV-1 or HIV-2 isolates for 0, 24, 48, 72, or 96 h and assessed for infection by intracellular p24 expression. Plots for a single donor are shown. (B) p24 expression over time is shown for mDCs exposed to HIV-1 or HIV-2 isolates. Data are for one of three donors. (C) qPCR was used to detect HIV-1 or HIV-2 gag expression in mDCs exposed to virus for 24, 48, 72, or 96 h. One representative experiment of three is shown.

HIV-2 exposure does not induce full maturation of mDCs or pDCs. CD11c⁺ mDCs and CD123⁺ pDCs were exposed to HIV-1 or HIV-2 isolates for 72 h, with or without TLR7/8 stimulation for the final 24 h. Cell surface expression of the costimulatory molecule CD86 on mDCs (A) and pDCs (B) was determined by flow cytometry. Histograms from one representative donor show data for the medium control (filled area), virus alone (blue line), or virus plus TLR7/8 (red line). (C) pDCs from five or six individual donors were exposed to HIV-1 or HIV-2 isolates for 72 h in the presence (closed bars) or absence (open bars) of TLR7/8 stimulation for the final 24 h. Supernatants were analyzed for IFN-α content by ELISA, and data are shown as means ± standard deviations.

HIV-2-specific CD4⁺ T cells contain more viral DNA than do other memory CD4⁺ T cells in vivo. HIV-2-specific (black), CD45RO⁺ CD57⁻ (gray), and CD45RO⁺ CD57⁺ (open) memory CD4⁺ T cells were sorted by flow cytometry from PBMC from seven HIV-2-positive donors. Numbers of HIV-2 viral DNA copies were determined by qPCR and are shown as numbers of gag copies/10⁵ cells. Also shown on the right y axis are the percentages of infected cells, assuming one viral DNA copy per cell.

DCs are not productively infected by HIV-2. Sorted CD11c⁺ mDCs (A) and CD123⁺ pDCs (B) were exposed to HIV-1 BaL or IIIB or HIV-2 CBL-20, RH2-3, RH2-14, or RH2-13 for 72 h. Contaminating monocytes were excluded by gating cells that expressed CD11c or CD123 in the absence of CD14. The level of infection was determined by intracellular p24 staining. The number in each gate represents the proportion of total cells that were p24 positive. This experiment was repeated for a number of individual donors, and the mean percentage and SEM of p24-positive cells detected after 72 h of virus exposure are shown for CD11c⁺ mDCs (n = 10 donors) (C) and CD123⁺ pDCs (n = 6 donors) (D). DNAs were also extracted from virus-exposed DCs at 72 h postexposure and analyzed for the amount of full-length HIV-1 or HIV-2 gag DNA per 10⁵ cells, using qPCR. Graphs show means and SEM for six donors of mDCs (E) and pDCs (F) analyzed by qPCR. mDCs were infected at an MOI of 0.05, and pDCs were infected at an MOI of 0.25 for all viruses. Significant differences between infections of mDCs or pDCs by BaL versus the other viruses were assessed by the Mann-Whitney test (*, P < 0.0001; #, P < 0.005; ^, P < 0.01; +, P < 0.05; NS, not significant). (G) The positive PCR signal for pDCs exposed to CBL-20 was inhibited by the addition of an anti-CD4 MAb. Means and SEM of PCR signals from three separate experiments using three concentrations of anti-CD4 antibody are shown. (H) CD8-depleted CD4⁺ T cells from five healthy donors were exposed to HIV-1 BaL or HIV-2 CBL-20, RH2-3, RH2-14, or RH2-13 at an MOI of 0.05 for 72 h, and the level of infection was determined by intracellular p24 staining. Means and SEM are shown. (I) Median fluorescence intensities of anti-p24 staining for CBL-20 (gray line)- and BaL (black line)-infected or uninfected (dashed line) CD4⁺ T cells.

Stimulation of DCs does not induce productive infection by HIV-2. Sorted CD11c⁺ mDCs and CD123⁺ pDCs were exposed to HIV-1 or HIV-2 isolates for 72 h, with or without stimulation with a TLR7/8 agonist. HIV-1 or HIV-2 gag DNA was quantified by qPCR for mDCs (A) or pDCs (B) exposed to virus for a total of 72 h, with the addition of TLR7/8 stimulation in the final 24 h, or in mDCs (C) or pDCs (D) exposed to TLR7/8 for 12 h prior to 72 h of virus exposure. Bar graphs show mean numbers of gag copies/10⁵ mDCs for three donors ± standard deviations. O/N, TLR7/8 stimulation in the 12 h prior to infection.

HIV-2-exposed mDCs do not transfer virus to autologous CD4⁺ T cells. Sorted CD11c⁺ mDCs from CMV-seropositive donors were exposed to HIV-1 or HIV-2 isolates for 72 h, washed thoroughly, and incubated with autologous CFSE-labeled CD4⁺ T cells in the presence of CMV lysate for 3.5 days. (A) CD4⁺ T cells were divided into four populations based on cytokine production and proliferation history. Cytokine-positive cells produced IFN-γ, IL-2, or TNF-α. (B) Each of the four subpopulations of autologous CD4⁺ T cells was analyzed for productive infection by intracellular p24 staining of CBL-20, RH2-3, and BaL cocultures. Numbers represent the percentages of p24⁺ T cells among all cells in the populations. Plots are shown for one representative experiment of two. Percentages of p24⁺ cells from each T-cell subpopulation are shown for cocultures where DCs were exposed for 72 h (C) or 6 h (D) and are shown as means ± standard deviations for two donors.