Hi149

DCs were harvested on day 5 to 6 of culture, and their immature phenotype CD14⁻CD1a⁺CD209⁺ (M5E2, Biolegend; HI149 and DCN46, both BD Bioscience) was confirmed by flow cytometry analysis. Autologous CD14⁻ PBLs were thawed and added to DCs at a 5:1 ratio to achieve the DC:PBL coculture, which was then stimulated with the immunomodulators at the indicated concentrations (SI Appendix, Table S1).

After fixation, cells were permeabilized in PBS supplemented with 0.5% saponin (Sigma) and 1.0% BSA (Sigma) for 10 min. To assess LC infection and transmission to U87 cells, cells were stained with PE‐conjugated p24 (KC57, Beckman Coulter) in combination with APC‐conjugated CD1a (HI149, BD Biosciences). For some experiments, NL4.3eGFP‐Bal was used and HIV‐1‐1eGFP expression was measured. HIV‐1 acquisition was defined as % p24⁺ CD1a‐positive cells (infection of LCs) or % p24⁺ CD1a‐negative cells (transmission of LCs to U87.CD4.CCR5). For surface expression of maturation markers, cells were stained in Tris‐buffered saline supplemented with 1.0% BSA with PE‐conjugated CD80 (L307.4, BD Pharmingen), FITC‐conjugated CD86 (2331, BD Pharmingen), PE‐conjugated CCR7 (3D12, BD Pharmingen), PE‐conjugated langerin (DCGM4, Beckman Coulter) or CD1a (HI149, BD Biosciences) in different combinations. Cells were quantified by flow cytometry on FACS Canto II (BD Biosciences), and results were analyzed using FlowJo software. Surface marker expression is defined by mean fluorescent intensity (MFI).