Murine and human LPS-stimulated RAPA-DCs promote increased IFN-γ production associated with increased IL-4 in alloreactive CD4⁺ T cells. Murine CTR- and RAPA-DCs (B6; H-2^b), treated as indicated, were used to stimulate normal BALB/c CD4⁺ T cells (H-2^d) for 5 days. (A) After T-cell restimulation with PMA and ionomycin in the presence of Golgi plug, intracellular IL-4 and IFN-γ were determined by flow cytometry. Compared with LPS-stimulated CTR-DCs, which greatly increased the incidence of IFN-γ⁺CD4⁺ cells, LPS-stimulated RAPA-DCs promoted less IFN-γ expression and increased the incidence of IL-4⁺ CD4⁺ T cells. Data are representative of 4 independent experiments. Numbers are percentage CD4⁺ cells. (B) Cytokines secreted during BALB/c CD4⁺ T-cell stimulation were assessed by Luminex on day 5 culture supernatants. The mean ± SEM from 2 independent experiments are shown. *P < .05. (C) In vivo comparison of the T-cell stimulatory and early skewing capacity of allopeptide-presenting CTR- and RAPA-DCs indicated that LPS-exposed RAPA-DCs (C) had reduced stimulatory capacity and (D) did not increase IFN-γ⁺ in allospecific CD4⁺ T cells. (D) Assessment of adoptively transferred CD4⁺CD90.1⁺Vβ6⁺ 1H3.1 T cells, specific for the IEα-derived allopeptide IEα_52-68 in I-A^b, 5 days after intravenous injection of the indicated DCs presenting BALB/c allopeptides, revealed that RAPA-DCs, even when LPS-treated, induced less expansion than CTR-DCs (gated: CD3⁺CD4⁺). (D) Further examination of 1H1.3 cells (gated: CD90.1⁺Vβ6⁺) revealed that LPS-stimulated RAPA-DCs did not increase the incidence of IL-4⁺ cells over that induced by CTR-DCs. (E-F) Hu monocyte-derived CTR- and RAPA-DCs generated from CD14⁺ monocytes were stimulated for 24 hours with LPS (0.1 μg/mL). These DCs were incubated with allogeneic PBMCs (1:10 ratio) for 6 days, followed by 4 hours of restimulation with PMA and ionomycin in the presence of Golgi plug. (E-F) Two representative profiles showing IFN-γ and IL-4 production by alloreactive CD4⁺ T cells from 2 different individual responders to the same set of LPS-exposed CTR- and RAPA-DCs. (G-H) Overall IFN-γ (P < .001) and IL-4 (P = .03) production by alloreactive CD4⁺ T cells incubated with LPS-stimulated Hu CTR-DCs or RAPA-DCs. Data are from 7 independent experiments.