DCs present HIV-1 virion-derived antigens to HIV-specific (HS) CD4⁺ cells. (A) Protocol for generating HS CD4⁺ T-cell clones. Primary CD4⁺ T cells were primed with autologous monocyte-derived DCs pulsed with HIV_MN virions inactivated with aldrilthiol-2 (HIV_MN-AT-2). Cultures were restimulated with autologous DCs, pokeweed-stimulated (PW) blasts, or B-EBV cells pulsed with a pool of HIV-1 Gag-p24 peptides. Peptide-specific bulk cultures were then selected and HS clones derived by limiting dilution. (B) DCs exposed to HIV particles activate HS CD4⁺ cells. HLA-DRβ^*01⁺ imDCs were exposed to the indicated HIV-1 strains in the presence of AZT, and cocultivated with IV9 HS cells for about 8 hours. Activation of IV9 cells was assessed in an IFNγ ELISPOT assay. Results are depicted as the number of IFNγ⁺ cells for the indicated number of effectors. Left panel: the indicated viral strains (5.4 nM p24) were used. YU2b (R5 tropic) or Bru (X4 tropic) carry the gag1 epitope recognized by IV9 cells, whereas this sequence is naturally mutated in NL4-3 strain (X4 tropic) and HIV(VSV) pseudotypes. Right panel: HIV Env increases MHC-II HIV antigen presentation by DCs. DCs were exposed to wild-type HIV Bru or Env-deleted (BruΔenv) isogenic viruses (0.5 nM p24). (C) Dose-response analysis of HIV antigen presentation by DCs. DCs were exposed to increasing concentrations of HIV_MN-AT2 or, as a control, gag1 peptide, and tested as in panel B. For each panel, data are mean ± SD of triplicates and are representative of 3 independent experiments.