(A) Inclusion of a CD4⁺ binding moiety in exosomes (EXO^CD4-Tat) increased CD4⁺ T cell binding. Control Exosomes (Exo-C), EXO-Tat, or EXO^CD4-Tat exosomes were incubated with CD4⁺ T cells for 24 hours, and Western blot was used to compare intracellular Tat levels in cells treated with EXO-Tat or EXO^CD4-Tat exosomes. GAPDH was used as a cell lysate loading control. Western blot band intensity was measured using the Licor Odyssey software. (B) EXO^CD4-Tat exosomes specifically target CD4⁺ T cells. Exo-C, EXO-Tat, or EXO^CD4-Tat exosomes were incubated with PMBCs from healthy donors for 24 hours. The supernatants were removed by centrifugation. The cell pellets were prepared and probed with fluorescent conjugated antibodies, which recognize CD4 (green) or HA-tagged Tat (red). The top row shows CD4 staining (green). The middle row shows CD4 staining (green), Tat staining (red), and the merge of green and red. The bottom row shows CD4 staining (green), Tat staining (red), and the merge of green and red. The much stronger merged color orange indicates Tat protein containing exosomes binding to CD4⁺ T cells. Total magnification × 100. (C) A representative data set of 2 independent Western blot results is shown here, indicating EXO^CD4-Tat exosomes specifically target CD4⁺ cells. CD4^– PBMCs were prepared by depleting CD4⁺ cells from PBMCs using CD4 Dynabeads. CD4⁺ T cells were isolated from PBMCs using Dynabeads Untouched Human CD4⁺ T cells kit. CD4^– PBMCs and CD4⁺ T cells were mixed at the indicated ratio and incubated with or without the same amount of EXO^CD4-Tat exosomes (100 μg) for 24 hours. Cell-bound exosomes were measured by Western blot. (D) EXO^CD4-Tat exosomes reactivated latent HIV-1 from 3/3 patient samples, while control exosomes did not. The lowest limit of p24 quantification is 0.0075 pg/ml.