DC-SIGN promotes MHC-II-restricted HIV antigen presentation. (A) Reactivity of HS clone IV9. B-BRE and B-BRE-DCS cells were used as stimulators in an IFNγ ELISPOT assay. The effectors were autologous IV9 HS cells that recognize the gag1 epitope. Stimulating cells were exposed to the indicated concentrations of HIV_MN-AT2. (B) Reactivity of HS clone L11. B-420 and B-420-DCS cells were used as stimulator cells in an IFNγ ELISPOT assay. The effectors were autologous L11 HS cells that recognize the gag2 epitope. B cells were exposed to HIV_MN-AT2, HIV_Bru (both at 10 nM p24), and HIV_Yu2b (4 nM p24), or to the cognate gag2 peptide (100 nM). (A-B) Data are mean ± SD of triplicates and are representative of 3 independent experiments. (C) Analysis of the role of DC-SIGN by flow cytometry. B-420 and B-420-DCS cells were used as stimulators in an intracellular cytokine assay measuring the activity of autologous L11 HS cells. Stimulating cells were exposed to HIV_MN-AT2 or to the cognate gag2 peptide at the indicated concentrations. After 6 hours of coculture, cells were stained for surface CD4 and intracellular TNFα, and analyzed by flow cytometry. Numbers in the right corner indicate the percentage of TNFα-positive cells among L11 cells, which are CD4⁺. B-420 and B-420-DCS cells are CD4^-. Isotypic mAbs (Ig) were used as a negative control. Data are representative of 3 independent experiments.