DC-SIGN trafficking and MHC-II-restricted HIV-1 antigen presentation. (A) Surface levels of DC-SIGN wild type (WT) and dileucine mutant (LL/AA) in B-cell lines. B-BRE cells (left panel) and B-420 cells (right panel) were transduced with a lentiviral vector coding for WT and LL/AA DC-SIGN, yielding B-BRE-DCS WT and B-420-DCS WT cells and B-BRE-DCS LL/AA and B-420-DCS LL/AA cells, respectively. In the latter, the dileucine sorting motif present in the cytoplasmic tail of DC-SIGN has been mutated. Cells were stained with anti-DC-SIGN Abs and analyzed by flow cytometry. An isotypic mAb was used as a negative control (dotted line). (B) Activity of B-BRE derivatives as stimulators of HS clone IV9. B-BRE, B-BRE-DCS WT, and B-BRE-DCS LL/AA cells were exposed to HIV_MN-AT2, HIV_Bru (4 nM p24), or gag1 peptide (22 nM). Cells were then cocultivated with IV9 cells for 8 hours. Activity of IV9 cells was tested in an IFNγ ELISPOT assay. Data are mean ± SD of triplicates and are representative of 3 independent experiments. (C) Activity of B-420 derivatives as stimulators of HS clone L11. B-420, B-420-DCS WT, and B-420-DCS LL/AA cells were exposed to increasing concentrations HIV_MN-AT2. Cells were then cocultivated with L11 cells for 6 hours. Activity of L11 cells was tested by measuring TNFα production by flow cytometry, as described in Figure 4. Results are presented as the percentage of TNFα-positive cells within CD4⁺ cells. Data are mean ± SD of duplicates and are representative of 3 independent experiments.