In vitro derived dendritic cells trans-infect CD4 T cells primarily with surface-bound HIV-1 virions

Abstract

In the prevailing model of HIV-1 trans-infection, dendritic cells (DCs) capture and internalize intact virions and transfer these virions to interacting T cells at the virological synapse. Here, we show that HIV-1 virions transmitted in trans from in vitro derived DCs to T cells principally originate from the surface of DCs. Selective neutralization of surface-bound virions abrogated trans-infection by monocyte-derived DCs and CD34-derived Langerhans cells. Under conditions mimicking antigen recognition by the interacting T cells, most transferred virions still derived from the cell surface, although a few were transferred from an internal compartment. Our findings suggest that attachment inhibitors could neutralize trans-infection of T cells by DCs in vivo.

Figure 1. Mature MDDCs Transmit HIV-1 to T Cells Primarily via the trans Pathway

(A–C) NL4–3 or 81A virions were bound to MDDCs. After washing, the cells were added to activated autologous T cells immediately or after 1 to 5 d of culture at 37 °C to allow HIV-1 transmission to T cells for 24 h. The number of transmission events was measured by monitoring the appearance of infected T cells detected by p24^Gag intracellular immunostaining. (A) Transmission of 81A virions from immature MDDCs to T cells over time. FACS plots represent the population of T cells (CD3⁺CD1a^–) analyzed for intracellular Gag and CD4 expression. (B) Effects of maturation on HIV-1 transmission from MDDCs to T cells. Values are percentages of all transmission events over 5 d. (C) Relative contribution of trans pathway or de novo pathway in transmission of virus from immature or mature MDDCs to T cells. Data are averaged from MDDCs derived from nine donors. Transmission events from days 1 to 5 were added to determine the number of transmission events occurring via the de novo pathway. (D and E) NL4–3 or 81A virions containing BlaM-Vpr were bound to MDDCs at 4 °C. After washing, the cells were added to autologous T cells immediately (T₀) or after incubation for 10 to 120 min at 37 °C. HIV-1 transmission was measured with a virion-based fusion assay after gating on CD3⁺CD4⁺ cells. (D) HIV-1 transmission from MDDCs to T cells at T₀. FACS plots show CD3⁺CD4⁺CD1a^– cells analyzed for virion fusion. To control for specificity, MDDCs were incubated with T cells and entry inhibitors (500 nM TAK-779 or 500 nM AMD3100). (E) Effect of time on NL4–3 and 81A transmission from MDDCs to T cells. The curve is representative of four experiments.

Figure 2. HIV-1 Virions Transmitted In Trans from MDDCs to T Cells Are Sensitive to sCD4 and Pronase

(A) Immature and mature MDDCs were incubated at 4 °C with NL4–3 virions containing BlaM-Vpr, washed, and incubated for 30 min at 37 °C to allow virion internalization or held at 4 °C. MDDCs were treated (or not) with sCD4 at 4 °C to inactivate cell-surface virions. After extensive washes, loaded MDDCs were added to autologous PBLs to allow HIV-1 trans-infection of T cells. Fusion was measured with the virion-based fusion assay after gating on the CD3⁺CD4⁺ cells. The histogram presents a representative experiment independently performed five times with similar results. (B) sCD4 treatment of MDDCs loaded with HIV-1 virions does not inhibit transfer of virions loaded after treatment. Two sets of HIV-1 virions containing BlaM-Vpr were successively bound to immature MDDCs and neutralized with sCD4. After incubation with autologous CD4 T cells, fusion to CD3⁺CD4⁺ cells was assessed. (C) MDDCs were sequentially incubated with two reporter viruses (1 μg of p24^Gag each). GFP-HIV was bound and internalized, but CFP-HIV was bound only. Loaded MDDCs were incubated with autologous PBLs for 2 d. FACS plots show T cells analyzed for GFP and CFP expression when MDDCs were untreated or treated with sCD4 at 4 °C between or after loading of the reporter viruses. (D) GFP-HIV and CFP-HIV were either successively bound to MDDCs or GFP-HIV was, in addition, internalized by MDDCs during a 30-min incubation at 37 °C. MDDCs were then treated with increasing concentrations of pronase. Curves represent the number of T cells expressing CFP or GFP expressed as a percentage of the untreated samples. This graph presents a representative experiment with mature MDDCs that was independently performed three times with both immature and mature MDDCs.

Figure 3. HIV-1 Transmitted from SEB-Stimulated MDDCs to T Cells Remains Mostly Sensitive to sCD4

NL4–3 virions containing BlaM-Vpr were bound to SEB-pulsed MDDCs. The MDDCs were then incubated or not at 37 °C for the indicated time, treated or not with sCD4 at 4 °C, and incubated with purified autologous CD4 T cells. (A–C) Viral transfer was measured by analyzing HIV-1 fusion to CD4⁺CD3⁺ T cells after 2 h of culture (A) and by measuring productive infection of the T cells by intracellular Gag immunostaining 3 d later (B and C). (B) Histogram depicts one representative experiment performed three times with cells from three independent donors. (C) HIV-1 transfer to resting T cells from DCs pulsed with SEB and treated with sCD4 over time. This experiment was performed in triplicate; similar results obtained with MDDCs from two additional donors.