Blocking ICAM-1 on DCs and LFA-1 on primary CD4⁺ T cells inhibits DC-mediated HIV-1 transmission. (A) ICAM-1 is important for DC-mediated HIV-1 transmission to Hut/CCR5 cells. DCs were pretreated with anti-ICAM-1 before incubation with HIV-Luc/JRFL (MOI of 0.1). HIV-1-pulsed DCs were washed and cocultured with Hut/CCR5 cells. HIV-1 infection was detected at 3 dpi by measuring the luciferase activity in cell lysates. Asterisks indicate significant differences compared with medium controls (P < 0.01). (B) DCs and GHOST/R5 cells were separately preincubated with anti-ICAM-1. DCs were incubated with HIV-Luc/JRFL, washed, and cocultured with GHOST/R5 cells for 3 days before the luciferase activity in cell lysates was measured. (C) Inhibition of DC-mediated HIV-1 transmission of HIV-Luc/JRFL to primary CD4⁺ T cells with MAbs against ICAM-1 and LFA-1. Medium was used as a negative control. Asterisks indicate significant differences compared with medium controls (P < 0.05). All data show the means ± standard deviations of triplicate samples. One representative experiment out of four is shown. cps, counts per second.

ICAM-1 knockdown in target GHOST/R5 cells does not reduce DC-mediated HIV-1 transmission. (A) Knockdown of ICAM-1 in GHOST/R5 cells. GHOST/R5 cells were transfected with ICAM-1-specific siRNA or nonspecific (NS) siRNA. ICAM-1 expression on cell surfaces was measured by flow cytometry at 3 days posttransfection. Isotype-matched IgGs were used as negative controls of immunostaining. (B) HIV-Luc/JRFL infection of ICAM-1-silenced GHOST/R5 cells and control cells. (C) DC-mediated transmission of HIV-Luc/JRFL to ICAM-1-silenced GHOST/R5 cells relative to control cells. Luciferase activity of cell lysates was measured at 3 dpi. The data represent the means ± standard deviations. One representative experiment out of three is shown. cps, counts per second.

Blocking ICAM-2 and ICAM-3 on DCs and primary CD4⁺ T cells does not inhibit DC-mediated HIV-1 transmission. (A) DCs or primary CD4⁺ T cells were preincubated separately with neutralizing MAbs (10 μg/ml) against ICAM-2 and ICAM-3 before incubation with HIV-Luc/JRFL (MOI of 0.1). Cells were washed and cocultured, and HIV-1 transmission was detected at 3 dpi by measuring the luciferase activity in cell lysates. The data show the means ± standard deviations. One representative experiment out of three is shown. cps, counts per second. (B) Specific blocking effects of MAbs to ICAM-2 and ICAM-3. Activated primary CD4⁺ T cells were separately treated with medium or neutralizing MAbs (10 μg/ml) against ICAM-2 and ICAM-3 at room temperature for 30 min and then stained, respectively, with fluorescein isothiocyanate-conjugated anti-ICAM-2 or anti-ICAM-3 (2 μg/ml) at 4°C for 30 min. Isotype-matched IgGs were used as negative controls for staining.

Surface expression of ICAMs and their ligands on DCs and various types of HIV-1 target cells. iDCs, mDCs, Hut/CCR5 cells, PHA-activated primary CD4⁺ T cells, and GHOST/R5 cells were examined for cell surface expression of ICAM-1, -2, and -3 (A) and ligands of ICAMs (B). The asterisk in panel A indicates significantly increased expression of ICAM-1 on mDCs relative to expression on iDCs (P < 0.01, based on results from at least three independent experiments). Cells were stained with specific MAbs or isotype-matched IgG controls and analyzed by flow cytometry. Similar results with primary DCs and CD4⁺ T cells were obtained using cells derived from at least three different donors. One representative experiment out of three is shown.

ICAM-1 knockdown in DCs impairs DC-mediated HIV-1 transmission to primary CD4⁺ T cells. iDCs from donor 1 (A) and iDC and mDCs from donor 2 (B) were separately nucleofected with ICAM-1-specific siRNA or nonspecific (NS) siRNA. ICAM-1 expression on cell surfaces was measured by flow cytometry at 3 days postnucleofection. Isotype-matched IgGs were used as negative controls for immunostaining. (C and D) ICAM-1 silencing in DCs significantly decreases DC-mediated HIV-1 transmission with Hut/CCR5 cells (C) or primary CD4⁺ T cells (D). DC-alone samples were used as negative controls. Asterisks indicate significant differences compared to nonspecific siRNA controls (P < 0.05). The data represent the means ± standard deviations. One representative experiment out of three is shown. cps, counts per second.

Ectopic expression of ICAM-3 in GHOST/R5 target cells does not enhance their susceptibility to DC-mediated HIV-1 transmission. (A) Surface expression levels of ICAM-3, CD4, and CCR5 on ICAM-3-negative and -positive GHOST/R5 cells were measured by flow cytometry. Isotype-matched IgGs were used as negative controls for immunostaining. (B) Comparable HIV-Luc/JRFL infection of ICAM-3-negative and -positive GHOST/R5 cells. (C) iDC- and mDC-mediated transmission of HIV-Luc/JRFL to ICAM-3-negative and -positive GHOST/R5 cells. Luciferase activity of cell lysates was measured at 3 dpi. cps, counts per second. (D) DC-mediated transmission of replication-competent HIV-1 to ICAM-3-negative and -positive GHOST/R5 cells. DCs were pulsed with HIV-1_NLAD8 and then cocultured separately with ICAM-3-negative and -positive GHOST/R5 cells. Suspension DCs were removed after 10 h in the cocultivation by aspiration and washing, and adherent GHOST/R5 cells were cultured for 3 days. (E) Comparable infection of ICAM-3-negative and -positive GHOST/R5 cells with HIV-1_NLAD8. In both panels D and E, HIV-1 p24 levels in supernatants were measured at 3 dpi. The data represent the means ± standard deviations. One representative experiment out of three is shown. (F) HIV-1 synapse formation between DCs and ICAM-3-negative and-positive GHOST/R5 cells. DCs were incubated with GFP-Vpr-labeled HIV-1_NLAD8 (green), washed, and cultured separately with ICAM-3-negative and -positive GHOST/R5 cells for 1 h. White asterisks indicate GHOST/R5 cells and GHOST/R5/ICAM-3 cells at the virological synapses. (G) Quantitative image analysis of the virological synapses formed between DCs and ICAM-3-negative or -positive GHOST/R5 cells.

A proposed model for ICAM-ligand interactions in DC-mediated HIV-1 transmission. Our results suggest that ICAM-1-LFA-1 interaction is critical for DC-mediated HIV-1 transmission to CD4⁺ T cells. However, multifactorial interactions between ICAMs and their ligands expressed on DCs and CD4⁺ T cells may play an important role in the formation of immunological synapses and the induction of immune responses. TCR, T-cell receptor; MHC-II, major histocompatibility complex class II molecules. For simplicity, additional DC-T-cell interactions are not depicted.