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.