American Society of Hematology

Figure 5.

LCs do not secrete sufficient amounts of IL-12p70 to activate NK cells. (A) IL-12p40, IL-12p70, IL-15, and IL-18 secreted during maturation by 2 × 105 LCs, DDC-IDCs, and moDCs/mL were measured by ELISA. The culture supernatants required 10-fold concentration for detection of IL-15, but IL-12 and IL-18 could be measured directly. The values represent the averaged means ± SEMs from separate ELISAs for IL-12p40 (n = 3), IL-12p70 (n = 5), IL-18 (n = 3), and IL-15 (n = 5), in which duplicates were tested for each data point in each assay. In addition, surface expression of IL-15R-α was evaluated on mature moDCs, LCs, and IDC-DDCs. One representative of 3 independent experiments is shown. (B) Proliferation of purified peripheral blood NK cells was assessed by 3H-thymidine incorporation (Y-axis; note log scale) after culture with monocyte-derived dendritic cells (moDC) or CD34+ HPC-derived Langerhans cells (LC) or dermal-interstitial dendritic cells (DDC-IDC) for 6 days. Data points for the indicated NK-cell-dendritic cell ratio (X-axis; NK/DC from 3:1 to 1000:1) represent the average of duplicate microwell cultures and their standard deviation. NK-cell/DC cultures were supplemented with 500 pg/mL rIL-12 (DC + IL-12 ○) or not (DC only •). The data are representative of 5 independent experiments. (C) LCs, DDC-IDCs, and moDCs were cocultured with peripheral blood NK cells for 7 days at an NK/DC ratio of 10:1. These primary cultures were supplemented with 500 pg/mL rIL-12 (DC + IL-12 ○) or not (DC only •). NK cytotoxicity of the cultures was determined by 51Cr release assay against the MHC class I-negative target LCL721.221. Radiolabeled LCL721.221 targets were exposed for 4 to 6 hours to the NK-cell effectors, which were the responder lymphocytes from the original or primary DC/NK cocultures. The effectors were added as total lymphocytes, without adjustment for specific numbers of NK-cell blasts, to ascertain differences between the different DCs in stimulating NK cells in the primary cultures. The effector-target ratio is indicated along the X-axis, and specific lysis is plotted against the Y-axis. The data are representative of 5 independent experiments.

LCs do not secrete sufficient amounts of IL-12p70 to activate NK cells. (A) IL-12p40, IL-12p70, IL-15, and IL-18 secreted during maturation by 2 × 10⁵ LCs, DDC-IDCs, and moDCs/mL were measured by ELISA. The culture supernatants required 10-fold concentration for detection of IL-15, but IL-12 and IL-18 could be measured directly. The values represent the averaged means ± SEMs from separate ELISAs for IL-12p40 (n = 3), IL-12p70 (n = 5), IL-18 (n = 3), and IL-15 (n = 5), in which duplicates were tested for each data point in each assay. In addition, surface expression of IL-15R-α was evaluated on mature moDCs, LCs, and IDC-DDCs. One representative of 3 independent experiments is shown. (B) Proliferation of purified peripheral blood NK cells was assessed by ³H-thymidine incorporation (Y-axis; note log scale) after culture with monocyte-derived dendritic cells (moDC) or CD34⁺ HPC-derived Langerhans cells (LC) or dermal-interstitial dendritic cells (DDC-IDC) for 6 days. Data points for the indicated NK-cell-dendritic cell ratio (X-axis; NK/DC from 3:1 to 1000:1) represent the average of duplicate microwell cultures and their standard deviation. NK-cell/DC cultures were supplemented with 500 pg/mL rIL-12 (DC + IL-12 ○) or not (DC only •). The data are representative of 5 independent experiments. (C) LCs, DDC-IDCs, and moDCs were cocultured with peripheral blood NK cells for 7 days at an NK/DC ratio of 10:1. These primary cultures were supplemented with 500 pg/mL rIL-12 (DC + IL-12 ○) or not (DC only •). NK cytotoxicity of the cultures was determined by ⁵¹Cr release assay against the MHC class I-negative target LCL721.221. Radiolabeled LCL721.221 targets were exposed for 4 to 6 hours to the NK-cell effectors, which were the responder lymphocytes from the original or primary DC/NK cocultures. The effectors were added as total lymphocytes, without adjustment for specific numbers of NK-cell blasts, to ascertain differences between the different DCs in stimulating NK cells in the primary cultures. The effector-target ratio is indicated along the X-axis, and specific lysis is plotted against the Y-axis. The data are representative of 5 independent experiments.

This Feature Is Available To Subscribers Only