Macrophages transmit HIV-1 to CD4⁺ T cells across a virological synapse-like structure. Human MDMs were differentiated on glass coverslips for 7 days prior to infection with 6 ng CA-p24 of the HIV-1_BaL isolate for a further 7 days. MDMs were subsequently cocultured with 5 × 10⁵ PHA/IL-2–activated autologous CD4⁺ T cells that were prestained for CD4. Unattached T cells were carefully removed by aspiration at various times after coculture and incubated on poly-l-lysine–treated coverslips for 1 hour followed by fixation in 4% PFA. MDMs were gently washed with warm RPMI, and were fixed together with remaining attached T cells. Samples were subsequently permeabilized and stained for Gag and Env, and analyzed by LSCM. (A) HIV-1–infected MDMs in the absence of T cells. We used clone C49 to stain Gag-p17, which recognizes p17 cleaved from p55, representing mature virions. The right-hand panel is a magnified image of the boxed region from the merged image. A white line is drawn to indicate the MDM cell membrane in panels A and B. (B,C) MDM–T-cell cocultures stained for CD4, Gag, and Env. (D) Detached CD4⁺ T cells stained for HIV-1 Gag and CD4. White scale bars in panels A-D represent 10 μm. (E) EM analysis of the contact zone between HIV-1–infected MDMs and CD4⁺ T cells. Cells were cocultured for 5 hours in the presence of trace amounts of PHA (0.06 μg/mL) to stabilize clustered cells for sample preparation. M represents MDMs; T, CD4⁺ T cell; *, virus-containing vacuolar compartment; and V, HIV-1 particles; arrows point to closely apposed plasma membranes of MDMs and T cells. Note that many of the cell and viral membranes appear dense and strongly contrasted by the presence of ruthenium red label. Black scale bars represent 1 μm.