Cross-priming of Mart-1 and HIV gag p24 protein by anti-DCIR fusion mAb. (A) Skin-derived LCs from an HLA-A201⁺ donor were purified and cultured for 10 days with autologous purified T cells in the presence of 30nM anti–DCIR.doc-coh.MART-1 or IgG4.doc-coh.MART-1 conjugate mAbs. DCs were activated with CD40L. MART-1–specific CD8⁺ T-cell expansion was measured with a specific HLA-A201-MART-1(26-35) tetramer. (B) Anti–DCIR-MART-1 or IgG4-MART-1 (25nM) fusion proteins were used to target monocyte-derived IFN-α DCs. DCs were activated with CD40L and cultured with naive autologous CD8⁺ T cells. After 10 days, cells were restimulated for 24 hours with fresh DCs loaded with peptides derived from MART-1 protein or with unloaded DCs as a control. Plot shows the percentage of primed CD8⁺ T cells coexpressing IFN-γ and CD107a in response to a specific MART-1 peptide cluster. (C) CD34⁺-derived LCs were targeted with DCIR-MART-1 or control IgG4-MART-1 fusion proteins and cultured with naive CD8⁺ T cells for 9 days. Graph represents the percentage of cells coexpressing Granzyme B and perforin as analyzed at the end of the culture by flow cytometry. Values in the graph are the average of triplicates ± SD. Data are representative of 2 independent experiments. (D) Anti–DCIR-p24 or control IgG4-p24 (25nM) fusion proteins were used to target CD34⁺-derived LCs. DCs were activated with CD40L and cultured with naive autologous CD8⁺ T cells. After 2 consecutive stimulations, the proliferated cells were sorted and restimulated for 24 hours with fresh LCs and HIV gag p24 protein to evaluate IFN-γ secretion by Luminex. Cells with no protein served as a control. Values are average of duplicates. Data are representative of 2 independent experiments.