IntroductionDendritic cells (DCs) are antigen (Ag)-presenting cells (APCs) that function as biosensors of the cellular microenvironment by detecting the presence of signals that determine T-cell tolerance or immunity. 1,2 To accomplish this task, DCs acquire extracellular Ags by receptor-mediated endocytosis, macropinocytosis, or phagocytosis [3][4][5] ; by incorporation of microvesicles shed from the surface of neighboring cells, 6,7 and by their recently described interaction with nanovesicles (Յ 100 nm) termed "exosomes." [8][9][10][11][12] Exosomes are formed by reverse budding of the membrane of late endosomes [13][14][15] or multivesicular bodies (MVBs) and are released to the extracellular space by fusion of MVB with the plasma membrane. [13][14][15] Originally described in neoplastic cell lines, 16 exosomes also are produced by leukocytes and epithelial cells. [17][18][19][20][21][22] Although the function of exosomes still is poorly understood, exosomes are a source of Ag for APCs and participate in Ag presentation to T lymphocytes. 11,12 High concentrations of exosomes expressing major histocompatibility complex (MHC) and costimulatory molecules activate T-cell clones and T-cell lines weakly 10,13 and fail to stimulate naive T cells. 9,11 This impaired naive T-cell stimulatory ability of exosomes has been attributed to their low T-cell receptor-cross-linking capacity (inadequate for naive T-cell activation) and their small size and membrane composition. 10 However, in the presence of DCs, exosomes increase their ability to stimulate T cells. 10,11,23,24 The mechanism of interaction of extracellular exosomes with DCs is unknown. Although there is evidence that exosomes may transfer functional MHC-I/peptide complexes to DCs, 24 it is unclear whether exosomes cluster or fuse with DCs or if they are internalized and processed, as occurs with vesicles derived from apoptotic cells. [2][3][4][5] Herein we demonstrate that exosomes are internalized efficiently by DCs. Targeting of exosomes to DCs depends on ligands on the exosome and DC surface and is independent of complement factors. Once internalized by DCs, exosomes are sorted into recycling endosomes and then through late endosomes/lysosomes. By this mechanism, DCs process and present peptides derived from the internalized exosomes to T cells. In vivo, blood-borne exosomes are captured by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. In the steady state, uptake of circulating exosomes by splenic DCs does not induce DC maturation and does not prevent CD40-induced DC activation in vivo. Our results demonstrate that blood-borne allogeneic exosomes are efficiently targeted, internalized, and processed by splenic DCs in vivo, a phenomenon followed by presentation of exosome-derived allopeptides by CD8␣ ϩ DCs to CD4 ϩ T cells. Since allogeneic exosomes are a rich source of alloMHC and are targeted and processed in vivo by host DCs (without inducing their activation), intravenous administration of donor-derived exosomes may constitut...

The ability of dendritic cells (DC) to regulate Ag-specific immune responses via their influence on T regulatory cells (Treg) may be key to their potential as therapeutic tools or targets for the promotion/restoration of tolerance. In this report, we describe the ability of maturation-resistant, rapamycin (RAPA)-conditioned DC, which are markedly impaired in Foxp3− T cell allostimulatory capacity, to favor the stimulation of murine alloantigen-specific CD4+CD25+Foxp3+ Treg. This was distinct from control DC, especially following CD40 ligation, which potently expanded non-Treg. RAPA-DC-stimulated Treg were superior alloantigen-specific suppressors of T effector responses compared with those stimulated by control DC. Supporting the ability of RAPA to target effector T and B cells, but permit the proliferation and suppressive function of Treg, an infusion of recipient-derived alloantigen-pulsed RAPA-DC followed by a short postoperative course of low-dose RAPA promoted indefinite (>100 day) heart graft survival. This was associated with graft infiltration by CD4+Foxp3+ Treg and the absence of transplant vasculopathy. The adoptive transfer of CD4+ T cells from animals with long-surviving grafts conferred resistance to rejection. These novel findings demonstrate that, whereas maturation resistance does not impair the capacity of RAPA-DC to modulate Treg, it profoundly impairs their ability to expand T effector cells. A demonstration of this mechanism endorses their potential as tolerance-promoting cellular vaccines.

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Under steady-state conditions, internalization of self-antigens embodied in apoptotic cells by dendritic cells (DCs) resident in peripheral tissue followed by DC migration and presentation of self-peptides to T cells in secondary lymphoid organs are key steps for induction and maintenance of peripheral T-cell tolerance. We show here that, besides this traffic of apoptotic cells mediated by peripheral tissueresident DCs, splenic marginal zone DCs rapidly ingest circulating apoptotic leukocytes, process apoptotic cell-derived peptides into major histocompatibility complex class II (MHC-II) molecules, and acquire CD8␣ during their mobilization to T-cell areas of splenic follicles. Because apoptotic cells activate complement and some complement factors are opsonins for phagocytosis and play roles in the maintenance of peripheral tolerance, we investigated the role of complement receptors (CRs) in relation to phagocytosis of apoptotic cells by DCs. Apoptotic cell uptake by marginal zone DCs was mediated in part via CR3 (CD11b/CD18) and, to a lesser extent, CR4 (CD11c/CD18) and was reduced significantly in vivo in hypocomplementemic animals. Following phagocytosis of apoptotic cells, DCs exhibited decreased levels of mRNA and secretion of the proinflammatory cytokines interleukin 1␣ (IL-1␣), IL-1␤, IL-6, IL-12p70, and tumor necrosis factor ␣ (TNF-␣), without effect on the anti-inflammatory mediator transforming growth factor ␤1 (TGF-␤1). This selective inhibitory effect was at least partially mediated through C3bi-CD11b/CD18 interaction.

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...

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically-relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5–10×106/kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on day −2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n=6) and 113.5 days (p< 0.05) in DCreg-treated animals (n=6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95+ T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further pre-clinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

Tolerance induction against donor allo-antigens (allo-Ag