Figure 4. TNF-α and Pam3CSK4 enhance HIV-1 transmission ex vivo.

(A) Epidermal single-cell suspensions were stimulated with TNF-α and Pam3CSK4, and after 2 hours, the cells were inoculated with HIV-1–eGFP. After 2 hours the cells were washed and CCR5⁺ Jurkat T cells were added for 7 days and analyzed for GFP expression by flow cytometry. A representative experiment out of 2 donors is depicted. (B) Epidermal sheets were stimulated with TNF-α, Pam3CSK4, LTA, LPS, or flagellin for 6 hours, and ex vivo transmission was determined as in Figures 1 and 3. A part of the migrated cells was extensively washed before addition of the CCR5⁺ Jurkat T cells (right panel). HIV-1 transmission is depicted as percentage of CCR5⁺ Jurkat T cells positive for GFP expression. Error bars represent the mean ± SD of duplicates. A representative experiment out of 2 donors is depicted. (C) Epidermal sheets were stimulated with TNF-α and Pam3CSK4 before incubation with HIV-1–eGFP. After 3 days, the migrated cells were washed and LCs were isolated by CD1a-selection using magnetic beads. The CD1a⁺ and CD1a^– fraction were added to CCR5⁺ Jurkat T cells. The cocultures were monitored by flow cytometry at different days. Error bars represent the mean ± SD of duplicates. A representative experiment out of 2 is shown.