Dermal CD14⁺ DC-derived IL-10 and TGF-β1 prevent the generation of effector CTLs. (A) CFSE-labeled naive CD8⁺ T cells were stimulated with allogeneic CD40L-activated skin LCs (top panel) or dermal CD1a⁺ DCs (bottom panel) in the presence of IL-10 (10 ng/mL) and TGF-β1 (5 ng/mL). Graph shows the absolute CD8⁺ T-cell number or the proportion of CD8⁺ T cells that diluted CFSE dye (CFSE^lo) as measured after 7 days by flow cytometry. Boxes represent average results of 4 independent experiments; P < .001. (B) Flow cytometric analysis of granzyme B and perforin expression by the CD8⁺CFSE^lo T cells cultured for 7 days over allogeneic CD40L-activated skin LCs with (gray histogram) or without (black histogram) IL-10 and/or TGF-β1 as indicated. Results are representative of 3 independent experiments. (C) Flow cytometric analysis of CD8⁺ T-cell subsets (as indicated by the expression of CCR7 and CD45RA; top panel) and the activation marker CD25 (bottom panel) by CD8⁺ T cells cultured for 7 days over allogeneic CD40L-activated skin LCs conditioned with IL-10, TGF-β1, or a combination of the 2 cytokines. Data are representative of 3 independent experiments. (D) Flow cytometric analysis of surface receptors (CD25, CD28, and CD45RA) expression by CFSE^loCD8⁺ T cells cultured for 6 days with allogeneic CD40L-activated dermal CD14⁺ DCs with a neutralizing IL-10 mAb or an isotype-matched control after reactivation with fresh autologous DCs for 24 hours before the analysis. Data are representative of 3 independent experiments. (E) Similar experiment as in panel D. Dot plots show the expression of granzyme B (top panel) and perforin (bottom panel) by the cultured viable CD3⁺CD8⁺ T cells. Data are representative of 3 independent experiments. (F) Granzyme B (top panel) and perforin (bottom panel) expression as measured by the cultured viable CD3⁺CD8⁺ T cells in 3 independent experiments; P = .01 and P = .06, respectively. (G) CD8⁺ T cells were cultured with autologous peptide-loaded in vitro HLA-A*0201⁺ CD14⁺ DCs in the presence of neutralizing anti–IL-10 mAb or an isotype-matched control for 10 days. CD40L and IL-7 were added on day 0, and IL-2 was added on day 3. 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.