Blocking DC-derived IL-15 inhibits priming of effector CD8⁺ T cells. (A) CD8⁺ T cells were labeled with CFSE and primed for 7 days with decreasing numbers of allogeneic CD40L-activated skin LCs. Neutralizing IL-15 mAb (clone MAB 647; R&D Systems) and anti–IL-15Rα (clone AF247; R&D Systems) or isotype-matched control mAbs were used as indicated. Dot plots showing the proportion of cells that diluted CFSE (CFSE^lo) were assessed by flow cytometry. Data are shown from 1 of 11 independent experiments. (B) Graph shows the proportions of CD8⁺ T cells that were primed by allogeneic CD40L-activated LCs at a DC/T-cell ratio of 1:1000 or lower, in the presence of neutralizing mAb to IL-15 or matched isotype control. Graph shows data of 11 independent experiments; P < .001. (C) Purified naive CD8⁺ T cells were primed for 7 days by allogeneic CD40L-activated skin LCs. Neutralizing anti–IL-15 mAb or a control mAb was added as indicated. The cultured cells were activated for 24 hours with fresh LCs. Monensin was added during the last 6 hours, and intracellular IFN-γ and IL-2 production were measured by flow cytometry. Data are representative of 3 independent experiments. (D) CFSE-labeled naive CD8⁺ T cells were primed for 7 days by allogeneic CD40L-activated skin LCs in the presence of neutralizing anti–IL-15 mAb or an isotype-matched control. Histograms show intracellular expression of the effector molecules granzyme A and granzyme B by the viable CD3⁺CD8⁺CFSE^lo T cells in response to o/n restimulation with fresh LCs as analyzed by flow cytometry. Data are representative of 3 independent experiments. (E) CD40L-activated skin LCs were used to prime allogeneic naive CFSE-labeled T cells. Neutralizing IL-15 or an isotype-matched control mAb was added to the coculture. After 7 days, the dilution of CFSE and intracellular Bcl-2 expression by the cultured CD8⁺ T cells were assessed by flow cytometry. Histogram shows the expression of Bcl-2 by the viable CD3⁺CD8⁺CFSE^lo T cells. Data are representative of 3 independent experiments. (F) CD8⁺ T cells were cultured with autologous peptide-loaded sorted in vitro HLA-A*0201⁺ LCs with a neutralizing anti–IL-15 mAb or an isotype-matched control. CD40L and IL-7 were added on day 0, and IL-2 was added on day 3. After 10 days, cells were restimulated for 24 hours with fresh DCs, and effector memory populations were analyzed by flow cytometry that was based on the costaining with CCR7 and CD45RA. Data are representative of 3 independent experiments. (G) In vitro HLA-A*0201⁺ DC subsets were used to prime autologous naive CD8⁺ T cells. CD40L and IL-7 were added on day 0, and IL-2 was added on day 3 of the primary coculture. Neutralizing mAb to IL-15 was added to the coculture of naive CD8⁺ T cells and in vitro LCs. Cells were analyzed after 2 consecutive stimulations by flow cytometry for the dilution of CFSE dye, frequency of MART-1 tetramer–binding cells, and intracellular expression of granzymes, perforin, and Bcl-2 proteins. Data are representative of ≥ 3 independent experiments. (H) CFSE-labeled naive CD8⁺ T cells were primed with allogeneic CD40L-activated dermal CD1a⁺ DCs at the indicated 1:40 DC/T-cell ratio and with anti–IL-15 or an isotype-matched control mAb. After 9 days, cells were harvested and restimulated for 6 hours with fresh autologous LCs in the presence of monensin and anti-CD28/CD49d to assess IFN-γ production and CD107a surface mobilization. (Bottom panel) Cells are gated on viable CD3⁺CD8⁺CFSE^lo T cells. Data are representative of 4 independent experiments.