PRO 2000 inhibits MDDC-mediated transfer of HIV_BaL to CD4⁺ T cells and infection in MDDC-CD4⁺ T-cell cocultures. (A) For transfer experiments, MDDC were mixed with HIV_BaL preincubated with PRO 2000 (100 μg/ml) or medium for 1 h at 37°C. After 2 h of incubation, MDDC were washed and added to PHA-activated CD4⁺ T cells. As a control, MDDC were cultured without T cells after exposure to the virus. Alternatively, T cells were infected directly with cell-free HIV_BaL in the presence or absence of PRO 2000, washed, and cultured with or without the compound. Supernatants were collected at the beginning of the culture (day 0) and on days 5, 7, 9, and 14. Results of a representative experiment (mean ± standard deviation [SD] of the results of duplicate wells) are shown. (B) Summary of the results of three to five transfer experiments (mean ± SD) done as described for panel A is shown. Percent inhibition of CD4⁺ T-cell infection by PRO 2000 in transfer experiments and significant differences relative to the level of infection in the absence of the compound (control; 0% inhibition) are shown. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (C) For coculture experiments, MDDC were mixed with virus in the presence or absence of PRO 2000 (100 μg/ml) and immediately added to PHA-activated CD4⁺ T cells. After 2 h of incubation, MDDC and T cells were washed and plated with or without PRO 2000. Supernatants were collected as described for panel A. Results representative of four experiments (mean ± SD of the results of duplicate wells) are shown.

HIV_BaL capture by MDDC is inhibited in the presence of PRO 2000. HIV_BaL binding at 4°C (A) and binding/internalization by MDDC at 37°C (B) in the presence of PRO 2000 (100 μg/ml; means ± standard deviations of the results of triplicate wells) are shown. The minimum levels of p24 detected in binding and binding/internalization experiments were 1.1 ng/ml and 0.82 ng/ml, respectively. Mean background values were subtracted. Results representative of two binding and three binding/internalization experiments are shown.

MDDC cytokine profile after short- and long-term exposure to PRO 2000. (A) MDDC were exposed to PRO 2000 (100 μg/ml) for 1 h, washed, and replated in the absence (PRO 2000 1h_Med 48 h) or presence of LPS (PRO 2000 1h_LPS 48 h) at 100 ng/ml for 48 h. Controls included MDDC cultured in the presence of LPS or medium alone. Cell-free supernatants were collected at the end of the culture. (B) The experiments were performed as described for panel A except that MDDC were exposed to PRO 2000 for 48 h prior to culture in the absence (PRO 2000 48h_Med 48 h) or presence of LPS (PRO 2000 48h_LPS 48 h). In the experiments whose results are shown in panel C, LPS was added simultaneously with PRO 2000 for 48 h. The indicated cytokines were measured with a Beadlyte human multicytokine detection system. Each supernatant was tested in duplicate. Of note is that the differences in baseline cytokine production in experiments described for panels A, B, and C derive from the fact that there are differences in the experimental setups, including washouts versus no washout and total duration of MDDC culture prior to the measurements. A summary of the results (mean pg/ml ± standard deviation) of three to four experiments is shown. *, P < 0.05; ***, P < 0.001.

PRO 2000 inhibits MDDC-mediated RD HIV_JRFL transfer to HeLa CD4⁺ CCR5⁺ cells and infection in MDDC-HeLa CD4⁺ CCR5⁺ cell cocultures. (A and B) In transfer experiments, MDDC or mMDDC were mixed with RD HIV_JRFL preincubated with PRO 2000 or medium for 1 h at 37°C. After 2 h of incubation, MDDC were washed and plated with HeLa CD4⁺ CCR5⁺ cells. The results of a representative experiment (mean ± standard deviation [SD] of the results of duplicate wells) (A) and the summary of three to eight experiments (mean ± SD) (B) are shown. Percent inhibition of HeLa CD4⁺ CCR5⁺ cell infection by PRO 2000 in transfer experiments and significant differences are shown relative to infection in the absence of the compound (0% inhibition). (C) In coculture experiments, MDDC were mixed with RD HIV_JRFL preincubated with PRO 2000, immediately added to target cells, and then incubated in the presence or absence of the compound for 2 h. Then, medium containing the virus and the compound was aspirated and cells were cocultured in fresh medium with or without PRO 2000 for 72 h. HeLa CD4⁺ CCR5⁺ cells were infected with cell-free RD HIV_JRFL in the presence or absence of PRO 2000, washed, and incubated with or without the compound. Percent inhibition of infection by PRO 2000 and significant differences are shown relative to level of infection in the absence of the compound (0% inhibition). A summary of the results of three to eight experiments (mean ± SD) is shown. R.L.U., relative light units; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

PRO 2000 does not cause cytotoxicity in MDDC and CD4⁺ T-cell cultures. MDDC or PHA-activated CD4⁺ T cells were cultured in the presence of PRO 2000 for 4 days at 37°C. Cytotoxicity was determined by MTS assay (Promega). Data are means ± standard deviations of the results of triplicate wells and represent two experiments each with MDDC and T cells.

PRO 2000 inhibits R5 envelope fusion. Fusion assay with R5 envelope-expressing cells was performed in the presence of PRO 2000 or T20. Data are means ± standard deviations of the results of triplicate wells. Mean background value was subtracted (results of one of two to three experiments are shown). R.L.U, relative light units.