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Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC).Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-B-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IB, an rAd-encoding the dominant-negative form of IB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.As professional antigen-presenting cells (APC), dendritic cells (DC) exhibit the unique ability to stimulate both naive and memory T lymphocytes and play a critical role in central and peripheral T-cell tolerance (3,4,34,55,58). Their potential to determine the balance between immunity and tolerance makes DC targets for the therapeutic manipulation of immune responses against tumor cells or microorganisms or for the control of undesired immune reactions against allo-or autoantigens. In this respect, gene transfer approaches have been explored in an effort to potentiate the adjuvant (12, 29) or tolerogenic properties of DC (30,35,57). Recombinant adenovirus (rAd) has been demonstrated to be one of the most effective vehicles to deliver foreign DNA into DC (1,15,16,29,41,59,71). However, a fundamental problem with the use of replication-deficient rAd is that they generate the rapid development of natural killer (NK) cell and cytotoxic T-lymphocyte (CTL) responses that eliminate rAd-infected cells and induce neutralizing antibodies (Abs) that "limit" readministration of the same rAd serotype (65-67). The immunogenicity of rAd is a particular drawback when long-term transgene expression is required or when transduced DC are employed to generate antigen-specific tolerance for therapy of graft rejection or autoimmune diseases (26,30,35,(65)(66)(67). Although the mechanistic basis of rAd immunogenicity is unknown, evidence has accumulated...

IntroductionMyeloid dendritic cells (DCs) are crucial antigen-presenting cells (APCs) for primary T-cell responses. They arise from bone marrow (BM) precursors that colonize peripheral tissues through the blood or lymph. 1,2 Tissue-resident immature DCs are excellent at internalizing and processing antigen, but they exhibit low ability to stimulate naive T cells. Exposure to allergens, bacterial (lipopolysaccharide [LPS], CpG DNA motifs) or viral (dsRNA) components, proinflammatory cytokines (interleukin-1␤ , tumor necrosis factor-␣ [TNF-␣], interferon ␣ [IFN-␣], granulocytemacrophage colony-stimulating factor (GM-CSF), and cognate T-cell interactions are some of the stimuli that trigger DC differentiation. 1,2 The capacity of mature DCs to prime naive T lymphocytes and to promote their differentiation into different T-cell subsets is attributed to the up-regulation of surface major histocompatibility complex (MHC), costimulatory and adhesion molecules, and the ability to secrete IL-1, Although the capacity of DCs to produce an ample repertoire of cytokines is documented in humans and rodents, there is little information on how cytokine genes are expressed during DC ontogeny. 4 In this study, we analyzed a range of cytokine transcripts and their respective proteins in highly purified mouse BM-derived myeloid DCs (BM DCs) at different stages of cell differentiation. Immature BM DCs expressed higher levels of IL-1␣, IL-1␤, TNF-␣, transforming growth factor ␤1 (TGF-␤1), and macrophage migration inhibitory factor (MIF) transcripts/ protein. After spontaneous differentiation in culture, the DCs up-regulated the levels of IL-6 and IL-15 mRNA and transcribed mRNA for IL-12p35, IL-12p40, and IL-18 de novo. Similar findings were found at the protein level by flow cytometry or enzyme-linked immunoabsorbent assay (ELISA).We also investigated the changes in the cytokine repertoire of BM DCs terminally differentiated with LPS or after CD40 cross-linking. Both stimuli increased the levels of IL-6, IL12p40, IL-15, and TNF-␣ transcripts/intracellular protein. However, only LPS markedly up-regulated the transcription of IL-1␣, IL-1␤, IL-12p35, and MIF genes. Although LPS or CD40 ligation augmented the T-cell allostimulatory capacity of DC, only LPS shifted the balance of naive T helper (Th) from a mixed Th1/Th2 population to Th1 cells, a result that agrees with the fact that only LPS was able to up-regulate the transcription of IL-12p35 in BM DCs. These results also demonstrate that, depending on the stimuli that induce the terminal differentiation of DCs, their T-cell stimulatory activity (signal 2) can be dissociated from their Th cell-driving ability (signal 3). 28 It appears that one of the key factors that regulates the Th cell-driving ability of myeloid DCs is the ability of the Materials and methods Experimental animalsTen-to 12-week-old C57BL/10 (B10; H2K b , IA b , IE Ϫ ) and C3H/He (C3H; H2K k , IA k , IE k ) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the pathogen-free Central...

There is evidence that donor-derived dendritic cells (DC), particularly those at a precursor/immature stage, may play a role in the immune privilege of liver allografts. Underlying mechanisms are poorly understood. We have examined the influence of in vitro generated mouse liver-derived DC progenitors (DCp) on proliferative, cytotoxic, and Th1/Th2 cytokine responses induced in allogeneic T cells. Liver DCp, propagated in GM-CSF from C57B10 mice (H2b), induced only minimal proliferation, and weak cytotoxic responses in allogeneic (C3H; H2k) T cells compared with mature bone marrow (BM)-derived DC. Flow-cytometric analysis of intracellular cytokine staining revealed that mature BM DC, but not liver DCp, elicited CD4+ T cell production of IFN-γ. Intracellular expression of IL-10 was very low in both BM DC- and liver DCp-stimulated CD4+ T cells. Only stimulation by liver DCp was associated with IL-10 secretion in primary MLR. Notably, these liver DCp cocultured with allogeneic T cells stained strongly for IL-10. Following local (s.c.) injection in allogeneic recipients, both BM DC and liver DCp homed to T cell areas of draining lymph nodes and spleen, where they were readily detected by immunohistochemistry up to 2 wk postinjection. Liver DCp induced clusters of IL-10- and IL-4-secreting mononuclear cells, whereas Th2 cytokine-secreting cells were not detected in mice injected with mature BM DC. By contrast, comparatively high numbers of IFN-γ+ cells were induced by BM DC. Modulation of Th2 cytokine production by donor-derived DCp may contribute to the comparative immune privilege of hepatic allografts.