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The discovery of regulated trafficking of extracellular vesicles (EVs) has added a new dimension to our understanding of local and distant communication among cells and tissues. Notwithstanding the expanded landscape of EV subtypes, the majority of research in the field centers on small and large EVs that are commonly termed exosomes, microvesicles and apoptotic cell-derived vesicles. In the context of pregnancy, EV-based communication has a special role in the crosstalk among the placenta, maternal and fetal compartments, with most studies focusing on trophoblastic EVs and their effect on other placental cell types, endothelial cells, and distant tissues. Many unanswered questions in the field of EV biology center on the mechanisms of vesicle biogenesis, loading of cargo molecules, EV release and trafficking, the interaction of EVs with target cells and the endocytic pathways underlying their uptake, and the intracellular processing of EVs inside target cells. These questions are directly relevant to EV-based placental-maternal-fetal communication and have unique implications in the context of interaction between two organisms. Despite rapid progress in the field, the number of speculative, unsubstantiated assumptions about placental EVs is concerning. Here we attempt to delineate existing knowledge in the field, focusing primarily on placental small EVs (exosomes). We define central questions that require investigative attention in order to advance the field.

Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs.We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs.We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts.When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.

SUMMARY Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors., Graphical Abstract, In Brief The neurokinin 1 receptor (NK1R) induces Ca2+ flux in excitable cells. Here, Morelli et al. show that NK1R signaling in T cells promotes optimal Ca2+ flux triggered by TCR stimulation, which is necessary to sustain T cell survival and the efficient Th1- and Th17-based immunity that is relevant for immunotherapies based on pro-inflammatory neuropeptides.

Increasing evidence indicates the existence of a complex cross-regulation between the most important biosensors of the human body: The immune and nervous systems. Cytokines control body temperature and trigger autoimmune disorders in the central nervous system, whereas neuropeptides released in peripheral tissues and lymphoid organs modulate inflammatory (innate) and adaptive immune responses. Surprisingly, the effects of nerve fibers and the antidromic release of its pro-inflammatory neuropeptides on the leukocytes of the immune system that mediate graft rejection are practically unknown. In the transplantation field, such area of research remains practically unexplored. A recent study by Riol-Blanco et al has revealed new details on how nociceptive nerves regulate the pro-inflammatory function of leukocytes in peripheral tissues. Although the mechanism(s) by which neuroinflammation affects the immune response against the allograft remains unknown, recent data suggest that this new area of research is worth exploring for potential development of novel complementary therapies for prevention/treatment of graft rejection.

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 × 10(6) /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 (p0.05) 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 preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the alloresponse and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.

The influence of GM + IL-4 and Flt3 ligand (FL) on phenotype and function of BM-derived DC from Lewis rats was investigated. GM + IL-4-induced DC, despite expression of CD80/CD86, were less stimulatory than FL-induced DC that expressed low CD80/CD86 and were efficient stimulators of allogeneic T cells. GM + IL-4 DC were CD11b + OX62 lo , whereas FL DC were CD11b lo OX62 + . Following activation, GM + IL-4 DC produced IL-10 and IL-6, but no IL-12p70, and were resistant to further maturation. FL DC produced IL-12p70, IFN-α/β, IL-10 and IL-6 and underwent maturation. Repeated stimulation of T cells with GM + IL-4 DC inhibited proliferation, cytokine production and induced early T cell apoptosis. FL DC-activated T cells produced large amounts of IFN-γ/IL-10 and exhibited late T cell apoptosis/necrosis. In vivo, GM + IL-4 DC induced alloAg-specific hyporesponsiveness following T cell restimulation. These results demonstrate that GM + IL-4 DC display intrinsic regulatory properties, inducing passive-cell-death in T cells with potential for inactivation/regulation of alloreactive T cells in transplantation.

Dendritic cells (DC) are professional antigen (Ag) presenting cells (APC) that trigger the anti-donor T-cell response that causes allograft rejection. During the past decade several laboratories have employed in vitro generated DC with tolerogenic potential for prolongation of allograft survival. This minireview describes the development of a second-generation of DC-based strategies for transplantation tolerance based on the delivery in situ of donor allogeneic (allo)-Ag to quiescent DC of graft recipients by means of donor-derived apoptotic cells or exosomes. Donor leukocytes in early apoptosis are rich in allo-Ag, are internalized efficiently by recipient DC in vivo and deliver immunosuppressive signals to DC. Administration (i.v.) of donor apoptotic leukocytes prolongs bone marrow engraftment and cardiac allografts survival in mice by exerting a profound down-regulatory effect on the anti-donor T-cell response. Exosomes are nanovesicles (

CCR and CC chemokine expression in relation to Flt3 ligand- induced renal dendritic cell mobilization. Background. We investigated the expression and function of CC chemokine receptors (CCR) on highly-purified kidney and blood dendritic cells isolated from mice in which dendritic cells were mobilized with fms-like tyrosine 3 kinase ligand (Flt3L). Methods. CCR and CC chemokine expression were de- termined by RNase protection assay or flow cytometry, and dendritic cell migratory responses assayed using Transwell chambers. Chemokine production in renal tissue was detected by immunofluorescence staining. Trafficking of fluorochrome- labeled dendritic cells was monitored in vivo. Results. Freshly-isolated renal dendritic cells expressed mRNA for CCR1, 2, 5, and 7 and CCR1 and 5 protein. They did not migrate to inducible chemokines—CCL3 (macrophage inflammatory protein (MIP)-1a), CCL5 (regulated upon acti- vation, normal T cell expressed and secreted (RANTES)), or CCL20 (MIP-3a). Following lipopolysaccharide (LPS) stimula- tion, the dendritic cells down-regulated CCR1, 2, and 5 expres- sion, up-regulated or sustained signals for CCR7, and migrated to the constitutively expressed ligands CCL19 (MIP-3b) and CCL21 (secondary lymphoid tissue chemokine). Normal kid- neys expressed weak message for CCL2, 3, and 4, with stronger signals for CCL5 and 19. Intrarenal CCL5 production was en- hanced by Flt3L administration, in association with marked increases in interstitial CD45 + mononuclear cells. Mobilized blood dendritic cells migrated to CCR2 and CCR5 ligands and trafficked to renal intertubular sites following adoptive (intra- venous) transfer. Their migration to the CCR5 ligand MIP-1b (CCL4) and homing to kidneys of Flt3L-treated recipients were inhibited by CCR5 antagonism. Conclusion. These data implicate specific CCR and their lig- ands in regulation of the dendritic cell constituency of the kid- ney. CCR5 antagonism inhibits their directed migration and intrarenal accumulation.

Ethylcarbodiimide (ECDI) couples soluble antigens (Ag) to lymphoid cells bestowing tolerizing potential. We examined whether ECDI-treated allogeneic dendritic cells (DC) could promote Ag-specific T cell unresponsiveness and prolong graft survival. Exposure of murine myeloid DC to ECDI did not affect surface immunophenotype but reduced their ability to cluster with T cells, enhanced their apoptotic death, and markedly reduced their allostimulatory activity. Anti-donor proliferative and cytotoxic T cell responses of mice primed with ECDI-treated DC were markedly inhibited. Secretion of both Th1 (IFNγ) and Th2 cytokines (IL-5, IL-10) was suppressed. Cardiac allograft survival in mice preconditioned with a single injection of ECDI-DC was prolonged significantly. These results indicate that ECDI-treated DC promote T cell unresponsiveness to donor alloAgs and prolong transplant survival. The effects are not associated with sparing of Th2 responses, but may reflect inhibitory effects of apoptotic donor DC on host immune reactivity.

In addition to their potential to modulate the maturation, interleukin-12 producing capacity and Th-cell-polarizing ability of dendritic cells, there is new direct evidence that prostaglandins (PGs) regulate chemokine receptor expression, cell migration and apoptosis of these important professional antigen-presenting cells. We discuss these new insights in relation to how PGs might influence innate and adaptive immune responses.

A role for dendritic cells (DC) as critical regulators of immune reactivity has become increasingly recognized. There is evidence in rodent models that donor-derived DC, particularly in the immature state, can prolong organ allograft survival and even induce donor-specific tolerance. To allow the potential tolerogenic properties of these cells to be evaluated more fully with a view to clinical testing, it is necessary to identify DC subsets in nonhuman primates. We have identified the putative rhesus monkey equivalents of circulating human DC subset precursors as lineage(-), HLA-DR(+), CD123(lo),CD11c(hi)(pDC1) and lineage(-), HLA-DR(+), CD123(hi),CD11c(lo)(pDC2). Testing of these DC populations both in vitro and in vivo, as well as in transplant models in combination with conventional or experimental immunosuppressive reagents, will aid the development of novel strategies for the promotion of allo-antigen specific tolerance in transplantation.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play crucial roles as initiators and modulators of adaptive immune responses. Although DC-based vaccines have been utilized successfully to generate cytolytic T-cell activity against tumor antigens (Ags), evidence has accumulated that DCs also have potent capabilities to tolerize T cells in an Ag-specific manner. DCs cultured in the laboratory can suppress auto- or alloimmunity. Current and prospective strategies to promote this inherent tolerogenic potential of DCs might prove to be important for the therapy of transplant rejection and autoimmune diseases.

Background Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FceRI cross-linking by antigen-IgE complexes (FceRI-activated mast cells [FceRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FceRI-MC–mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. Objective We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FceRI-MC–mediated allergic disorders. Methods Bone marrow–derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R −/− mice were used to investigate the effects of NK1R signaling on FceRI-MCs. BMMCs generated from Tac1 −/− mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R −/− , and c-Kit W-sh/W-sh mice reconstituted with WT or NK1R −/− BMMCs were used to evaluate NK1R signaling on FceRI-MC–mediated passive local and systemic anaphylaxis and on airway inflammation. Results FceRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. Conclusions FceRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FceRI-MC–mediated disorders.

Exosomes are nanovesicles produced in the late endosome and secreted by various cell types, including dendritic cells (DC) and other antigen presenting cells. Studies of tumor-derived exosomes have demonstrated that they are capable of suppressing host anti- tumor immune response and placental-derived exosomes play an important role in establishing maternal immune tolerance toward the fetus. We have demonstrated previously that exosomes derived from DC transfected with adenoviral vectors containing anti- inflammatory molecules such as IL-10, IL-4 and FasL are capable of suppressing antigen-specific immune responses in multiple disease models, such as delayed type hypersensitivity and collagen-induced arthritis. In this study, we examine the ability of DC and DC-derived exosomes to halt the progression of diabetes in the NOD model of autoimmune diabetes. Exosomes were isolated by differential centrifugation from the enriched culture medium of bone marrow derived DC from NOD/LTJ mice transduced with either an adenoviral vector encoding IL-4 or a control empty vector. NOD/LTJ mice were treated via tail vein injection with either the transduced DC or the exosome preparation. The DC transduced with IL-4 were able to prevent the onset of diabetes with a single treatment to either 5- or 10-week old NOD mice, similar to our previous results. Interesting, treatment of NOD/LTJ with DC-derived exosomes also prevented the onset of hyperglycemia. These results suggest that exosomes from immunosuppressive DC are able to block progression of diabetes in NOD mice and thus could be used clinically to treat type I diabetes.

We have previously demonstrated the ability of dendritic cells (DC) genetically modified by adenoviral infection to express interleukin (IL)-10, IL-4 and Fas ligand (L) to: i) reduce inflammation in a model of delayed type hypersensitivity (DTH), ii) suppress the onset on collagen-induced arthritis (CIA) and iii) reduce the severity of established arthritis in mice. We now have demonstrated that exosomes derived from genetically modified, bone marrow- derived DC can reduce the severity and the incidence of established CIA and inhibit inflammation in a model of DTH in mice. The ability of the DC and DC-derived exosomes to suppress the DTH response was MHC Class II, B7.1/B7.2 and Fas/FasL dependent. In addition to exosomes from DC, we tested whether serum exosomes elicit a similar immuno-regulatory effect in a murine model. Our results have demonstrated that exosomes isolated from serum of mice immunized against the model antigen KLH, but not from OVA-immunized mice, were able to suppress KLH-specific DTH inflammatory responses when injected into the footpad. The anti- inflammatory effect of the MHC class II+ serum-derived exosomes was dependent upon the presence of FasL on the exosomes surface and Fas in the recipient mouse. These results demonstrate that administration of both exosomes derived from genetically modifiedDC and from serum are able to regulate the outcome of the T cell response in vivo in an antigen-specific manner through MHC and Fas/FasL-dependent mechanisms. These results may explain the efficacy of certain types of exosomes for treatment of established CIA and suppression of the DTH inflammatory response. Thus DC- and serum-derived exosomes could be used as therapeutic tools for the treatment of T cell-mediated autoimmune disorders and inflammatory diseases.