Figure 7. Pam3CSK4 increases HIV-1 capture and primarily transmits cell-bound HIV-1.

(A and B) Emigrant LCs were stimulated with Pam3CSK4 for 1 hour and inoculated with NL4.3 BaL. After 2 hours, cells were extensively washed. The cells were fixed, permeabilized, and stained for the LC-marker CD1a and HIV-1 p24. The cells were counterstained with isotype-specific Alexa antibodies (red, HIV-1 p24; green, CD1a). Single HIV-1 particles are indicated with arrows. Original magnification, ×630. A representative experiment out of 2 is depicted. (C and D) Epidermal single-cells suspensions were stimulated with Pam3CSK4. After 6 hours, cells were inoculated with single-cycle HIV-1. After 2 hours, the cell suspensions were washed, and subsequently cells were treated with trypsin at 37°C to remove cell-bound HIV-1 or a PBS control (C), or with HIV-1 neutralizing antibody b12 at 4°C to neutralize cell-bound, but not internalized, HIV-1 and an isotype control (D). Cells were washed and CCR5⁺ Jurkat T cells were added. At day 5, the cocultures were analyzed for GFP expression by flow cytometry. A representative experiment out of 3 is depicted. Errors bars represent the SD of duplicates. (E) Epidermal single-cells suspension was stimulated with TNF-α or Pam3CSK4 before being inoculated with different concentrations of HIV-1–eGFP. After 2 hours, the cell suspensions were washed, and CCR5⁺ Jurkat T cells were added. HIV-1 transmission was followed by flow cytometry. A representative experiment out of 2 donors is depicted. Error bars represent the mean ± SD of duplicates. TCID, tissue-culture infectious dose.