HIV-1 transmission to CD4⁺ T cells is receptor and fusion dependent, and results in productive infection. (A) HIV-1–infected MDMs were cocultured with autologous CD4⁺ T cells for 1 to 12 hours, followed by harvesting of the T cells with cold 5 mM EDTA/PBS. Cells were subsequently stained for CD3 and intracellular CA-p24 and analyzed by FC to determine the percentage of HIV-1–positive T cells. Data shown represent means of 3 independent experiments, and error bars represent SEM. (B) HIV-1–infected MDMs were cocultured with CD4⁺ T cells that had been preincubated with several inhibitors (1 hour, 37°C), followed by harvesting of the T cells after 10 hours, and staining for FC as in panel A. Alternatively, T cells were not added to MDMs directly, but were separated by a transwell (0.3-μm pore size; Costar, Cambridge, MA) to prevent cell-cell contact. Data represent the mean of triplicates in a single experiment and error bars represent + 1 SD. *P < .02; **P < .03; ***P < .001; ****P < .001, ANOVA. (C) Detection of HIV-1 reverse transcription using qPCR. HIV-1–infected MDMs were cocultured with autologous CD4⁺ T cells for 0, 6, 12, and 24 hours, followed by gentle aspiration of the T cells with warm RPMI, lysis, and DNA isolation and purification. qPCR using HIV-1 pol primers was performed to measure de novo viral DNA synthesis. Data were normalized to human β-globin. Data represent the mean of triplicates in a single experiment and error bars represent + 1 SD and *P < .02, ANOVA. (D) Replication of HIV-1 after transmission. CD4⁺ T cells were collected from 12-hour cocultures with HIV-1–infected MDMs, depleted of all MDMs with CD14 beads (Miltenyi-Biotec), and cultured for an additional week (10⁵/well/250 μL). Viral replication was detected by p24 released into the supernatant by ELISA. Data represent the mean of quadruplicates in a single experiment and error bars represent + 1 SD.