Although they display poor CD4⁺ T-cell allostimulatory capacity, mouse and human RAPA-DCs produce increased IL-12p70 when exposed to LPS. BM-derived mDCs were generated from B6 mice with GM-CSF and IL-4, in the presence (RAPA-DCs) or absence of RAPA (CTR-DCs) and then isolated and purified on day 7 of culture. These DCs were incubated for 18 hours in media alone (Unstim) or with 0.1 μg/mL LPS. (A) RAPA-DCs, compared with similarly stimulated CTR-DCs, showed inferior capacity to induce proliferation of alloreactive CD4⁺ BALB/c T cells, even after exposure to LPS. The data are mean ± SD from sample replicates of 1 experiment representative of more than 4 performed. (B) Intracellular staining for IL-12p40 revealed that DC generation in RAPA promoted an increase in IL-12p40 expression after exposure to LPS. Numbers on dot plots indicate percentage of CD11c⁺ cells. (C) Comparison of the percentages (mean ± SD) of IL-12p40⁺CD11c⁺ cells from 2 experiments representative of more than 6 performed. Enzyme-linked immunosorbent assay (D-E) or Luminex (F) analysis of culture supernatants revealed significantly increased secretion of IL-12p70 by LPS-stimulated RAPA-DCs, whereas the release of IL-23 (E) and IL-10 (F) was reduced. (A-F) *P < .05. The data shown are representative of more than 3 experiments performed. (G-H) Hu DCs were generated from CD14⁺ monocytes in the presence (Hu RAPA-DCs) or absence (Hu CTR-DCs) of RAPA. (G) Like their murine counterparts, unstimulated Hu RAPA-DCs and those stimulated for 18 hours with LPS showed a reduced capacity to stimulate allogeneic T cells in MLR. [³H]-thymidine (TdR) incorporation for each condition was normalized to that obtained with unstimulated Hu CTR-DCs for each experiment. Data represent mean percentage ± SD across multiple experiments (Unstim, n = 10; LPS, n = 4). *P < .05. (H) Hu RAPA-DCs also displayed significantly increased IL-12p70 secretion after exposure to LPS (0.1 μg/mL) Data are mean ± SD from multiple experiments (Unstim, n = 9; LPS, n = 5). *P < .05.