TLR7/8-signaling enhances DCIR-mediated primary CD8⁺ T-cell response by mDCs. (A) Blood-derived mDCs from an HLA-A201⁺ donor were targeted with 17nM of anti–DCIR-MART-1 or a control IgG4-MART-1 fusion proteins, activated with CD40L (100 ng/mL), CL075 (1 μg/mL), poly I:C (5 μg/mL), or LPS (50 ng/mL) and cocultured with autologous naive CD8⁺ T cells for 10 days. The expansion of MART-1–specific CD8⁺ T cells was measured with a specific HLA-A201-MART-1(26-35) tetramer. Data are of 2 independent experiments with 2 different donors. (B) Blood-derived mDCs from an HLA-A201⁺ donor were targeted with 30nM of anti–DCIR-MART-1 fusion protein or anti–DCIR-p24, activated with either CD40L or TLR7/8 agonists, and cocultured with autologous naive CD8⁺ T cells for 10 days. (Top panel) The proportions of HLA-A201-MART-1(26-35) peptide tetramer-positive CD8⁺ T cells expanded by purified blood mDCs cultured with anti–DCIR-MART-1 fusion protein and activated with either CD40L or TLR7/8 agonist. (Bottom panel) The proportions of HLA-A201-HIV gag p24(151-159) peptide tetramer-positive CD8⁺ T cells expanded by purified blood mDCs targeted with anti–DCIR-p24 fusion protein and activated with either CD40L or TLR7/8 agonist. Data are of 2 independent experiments with 2 different donors. (C) The expression of intracellular effector molecules Granzyme B and perforin was assessed by flow cytometry on CD8⁺ T cells primed by IFN-α DCs targeted with 10nM of anti–DCIR-MART-1 or IgG4-MART-1 fusion proteins and activated with CD40L, CL075, or a combination of CD40L and CL075. The expression on the antigen specific MART-1(26-35)-positive cells was analyzed by co staining with the corresponding HLA-A201-tetramer. Data are representative of 2 independent experiments. (D) The frequency of MART-1–specific CD8⁺ T cells, as measured with a specific HLA-A201-MART-1(26-35) tetramer, after expansion with anti–DCIR-MART-1–targeted DCs that were activated with CD40L, TLR7/8 ligand, or a combination of CD40L and TLR7/8 ligand. IgG4-MART-1 fusion protein or no antigen conditions served as controls. Each dot represents a single experiment. (E top panel) IFN-α DCs were targeted with 17nM of anti–DCIR-MART-1 or a control IgG4-MART-1 fusion proteins, activated with CD40L (100 ng/mL), CL075 (1 μg/mL), poly I:C (10 μg/mL), or LPS (50 ng/mL) and cocultured with autologous naive CD8⁺ T cells. Ten days later, cells were restimulated with fresh DCs that were loaded with 15mer overlapping peptides derived from the MART-1 protein. Plots show the level of intracytoplasmic IFN-γ by CD8⁺ T cells after 5-hour stimulation in the presence of monensin. Data are representative of 3 independent experiments. (Bottom panel) Anti–DCIR-p24 or a control IgG4-p24 fusion proteins were used as a model antigen. (F) IFN-α DCs were targeted with 113nM anti–DCIR-MART-1 fusion protein activated with either CD40L (100 ng/mL) or CL075 (1 μg/mL) and cocultured with autologous naive CD8⁺ T cells. Ten days later, cells were restimulated with fresh DCs that were loaded with 15mer overlapping peptides derived from the MART-1 protein. The levels of IL-4, IL-5, IL-13, IFN-γ, TNF-α, and IL-12p40 were measured by Luminex in the culture supernatant after 24 hours. The graph represents mean ± SD; n = 3. (G) IFN-α DCs were targeted with 10nM anti–DCIR-MART-1 (▴) or a control IgG4-MART-1 () fusion proteins activated with either CD40L (100 ng/mL) or CL075 (1 μg/mL), or a combination of CD40L and CL075 and cocultured with autologous naive CD8⁺ T cells. Coculture in the absence of an antigen served as an additional control (□). Ten days later, cells were restimulated with fresh IFN-α DCs that were loaded with MART-1 fusion protein and analyzed by flow cytometry for their intracellular cytokine production. Graphs show the frequency of IFN-γ (left panel) and IFN-γ⁺TNF-α⁺ (right panel) producing CD8⁺ T cells primed by DCIR-targeted, or control IFN-α DCs after 5-hour restimulation in the presence of monensin and 0.25 μg/mL of anti-CD28/CD49d mAb (n = 3).