IL-15 enhances CD8⁺ T-cell priming. (A left) In vitro CD40L-activated HLA-A*0201⁺ CD14⁺ DCs were loaded with MART-1(26-35) peptide (3μM) and cultured with autologous naive CD8⁺ T cells for 9 days with or without IL-15 (20 ng/mL). CD40L and IL-7 were added on day 0, and IL-2 was added on day 3. The expansion of Ag-specific CD8⁺ T cells was assessed by flow cytometry with the use of MART-1(26-35)–HLA-A*0201 tetramer. Data are representative of 8 independent experiments. (A right) Graph shows the absolute MART-1(26-35)–specific cells per milliliter of 8 independent experiments. Statistical analysis was performed using paired Student t test. (B) Allogeneic naive CD8⁺ T cells were primed by CD40L-activated dermal CD14⁺ DCs with or without IL-15 for 7 days and analyzed by flow cytometry for the intracellular expression of the effector molecules: granzyme A, granzyme B, and perforin. Gray histogram shows staining with an isotype control. Data are representative of 3 independent experiments. (C) Intracellular expression of the effector molecules, granzyme B and perforin, by allogeneic CD8⁺ T cells primed for 7 days by CD40L-activated skin LCs or dermal CD14⁺ DCs without or with increasing dose of IL-15 (1, 5, and 20 ng/mL, left panel). (Right panel) Expression level granzyme B and perforin cultured for 7 days with IL-15 (20 ng/mL) and no DCs. Data are representative of 3 independent experiments. (D) The cytotoxic activity of MART-1(26-35)–specific CD8⁺ T cells primed by in vitro peptide–loaded autologous CD14⁺ DCs for 2 consecutive stimulations with or without IL-15 as assessed in a standard ⁵¹Cr release assay against an HLA-A*0201⁺ melanoma cell line MEL526 expressing HLA-A*0201–MART-1 complexes at indicated E:T ratios. CD40L and IL-7 were added on day 0, and IL-2 was added on day 3 of the primary coculture. Data are expressed as mean of triplicate measurements for a single donor representative of 3 independent experiments with the use of different donors. (E) The cytotoxic activity of MART-1(26-35)–specific or gp100(209-217)–specific CD8⁺ T cells primed by in vitro peptide–loaded autologous CD14⁺ DCs for 2 consecutive stimulations with or without IL-15 as assessed in a standard ⁵¹Cr release assay against target cells expressing peptide HLA-A*0201 complexes at indicated E:T ratios. CD40L and IL-7 were added on day 0, and IL-2 was added on day 3 of the primary coculture. Graph shows percentage of maximal killing of 5 experiments with the use of different target cells and different donors. (F) Allogeneic CD8⁺ T cells were primed for 8 days with sorted in vitro CD40L-activated DC subsets. Plots show the level of CFSE dilution and IFN-γ expression after 6 hours of re-stimulation with fresh DCs in the presence of monensin and anti-CD28/49d. Data are representative of 3 independent experiments. (G) Histogram shows CD25, CD28, and CCR7 receptor expression level by the viable CFSE^loCD8⁺ T cells that were exposed to allogeneic CD40L-activated dermal CD14⁺ DCs with (black) or without (gray) soluble IL-15, at indicated DC/T-cell ratio and cytokine concentrations. Data are representative of 3 independent experiments.