Exosomes secreted from monocyte-derived dendritic cells support in vitro naive CD4+ T cell survival through NF-(kappa)B activation

We investigated the effect of exosomes secreted from human monocyte-derived dendritic cells (Mo-DCs), which are generated from PBMCs in response to treatment with GM-CSF and IL-4, on naive CD4+ T cell survival in vitro. Exosomes isolated from culture supernatants of Mo-DCs (>90% purity) were purified with anti-HLA-DP, -DQ, -DR-coated paramagnetic beads. Purified exosomes prolonged the survival of naive CD4+ T cells (>98% purity) in vitro. Treatment with neutralizing mAb against HLA-DR significantly decreased the supportive effect of purified exosomes on CD4+ T cell survival. Exosomes increased nuclear translocation of NF-(kappa)B in naive CD4+ T cells, and NF-(kappa)B activation was significantly suppressed by anti-HLA-DR mAb or NF-(kappa)B inhibitor pyrrolidine dithiocarbamate (PDTC). In addition, PDTC inhibited the effect of exosomes on naive CD4+ T cell survival. Thus, exosomes secreted by Mo-DCs appear to support naive CD4+ T cell survival via NF-(kappa)B activation induced by interaction of HLA-DR and TCRs.     

MHC class II+ exosomes in plasma suppress inflammation in an antigen-specific and Fas ligand/Fas-dependent manner

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells – the influence of phosphoglycans

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells--the influence of phosphoglycans.

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

Exosome-based immunotherapy

Exosomes are small membrane vesicles originating from late endosomes and secreted by hematopoietic and epithelial cells in culture. Exosome proteic and lipid composition is unique and might shed some light into exosome biogenesis and function. Exosomes secreted from professional antigen-presenting cells (i.e., B lymphocytes and dendritic cells) are enriched in MHC class I and II complexes, costimulatory molecules, and hsp70–90 chaperones, and have therefore been more extensively studied for their immunomodulatory capacities in vitro and in vivo. This review will present the main biological features pertaining to tumor or DC-derived exosomes, will emphasize their immunostimulatory function, and will discuss their implementation in cancer immunotherapy.Abbreviations APC antigen-presenting cell - ASI active specific immunotherapy - CTL cytotoxic T lymphocyte - DC dendritic cell - FDC follicular dendritic cell - MD-DC monocyte-derived dendritic cell - GMP good manufacturing procedure - HLA human leukocyte antigen - HSP heat shock protein - MHC major histocompatibility complex - MVB multivesicular body - ExAs ascitis-derived exosomes - DEX DC-derived exosome - TEX tumor cell–derived exosome This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Autocrine IFN-γ promotes naive CD8 T cell differentiation and synergizes with IFN-α to stimulate strong function

Autocrine IFN-γ signaling is important for CD4 differentiation to Th1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show in this paper that naive murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at ～8 h and declining by 24 h. The autocrine IFN-γ signals for upregulation of expression of T-bet and granzyme B and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, whereas IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or type I IFN.     

IL-10 producing CD8+ T cells in human infection with Leishmania guyanensis

Cytokines are increasingly recognized as important components of the cellular immune responses to intracellular pathogens. In this study, we analyzed the production of TGF-β, IL-10 and IFN-γ by PBMC of unexposed naïve subjects and LCL patients after stimulation with live Leishmania guyanensis (L.g.). We demonstrated that IFN-γ is produced in controls and LCL patients, IL-10 only in LCL patients and TGF-β only in naïve subjects. Furthermore, in naive subjects, neutralization of TGF-β induced IL-10 production. IL-10 produced in naïve subjects when TGF-β is neutralized or in LCL patients did not modify the IFN-γ production but inhibit reactive nitrogen species production. Analysis of the phenotype of IL-10 producing cells in naive subjects when TGF-β is neutralized clearly showed that they are memory CD45RA⁻ CD8⁺ T cells. In LCL patients, IL-10 producing cells are both CD45RA⁻ CD4 and CD8⁺ T cells. The role of these IL-10 producing CD8⁺ T cells in the development of the diseases should be carefully evaluated.     

外体（exosome）在肿瘤免疫治疗中的应用

外体（exosome）是由细胞分泌至细胞外的膜性小囊泡。研究发现,来源于树突状细胞和来源于肿瘤细胞的外体富集MHCⅠ/Ⅱ类分子、协同刺激分子、热激蛋白70和90等多种活性分子和肿瘤抗原,在体内外免疫调节中起关键作用,以外体为基础的肿瘤免疫治疗已成为热点。我们简要介绍外体的特征、分离制备和临床应用研究。     

Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells

The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell‐to‐cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT‐PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.     

Isolation and proteomic analysis of exosomes secreted by human cancer cells in vitro

Exosomes are 20-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro and in vivo. Since exosomes contain both RNA (mRNA and microRNA) and proteins, which can be transferred to another cell, and be functional in that new environment, these vesicles may be involved in the communication between cells. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo suggest their participation in pathological situations. Our purpose here is to describe methods for the production, purification, and proteomic characterization of exosomes derived from human cancer cells in vitro. Based on exosomes' unique lipidic composition, we have developed the new approach to increase production of exosomes by cells in vitro. Secondly, we have developed quality control by laser correlation spectroscopy for exosomal assays based on the amount of MHC class I and CD63 molecules on their surface. At last, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used after 2D electrophoresis for the proteomic analysis of exosomes derived from cancer cell lines. This study describes the protein composition of brain tumor cell-derived exosomes in more detail.     

Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

Exosomes: Isolation,characterization, and biomedical applications

Exosomes are nano-sized bioactive vesicles of 30–150 nm in diameter. They are secreted by exocytosis of nearly all type of cells in to the extracellular fluid. Thereby, they can be found in many biological fluids. Exosomes regulate intracellular communication between cells via delivery of their cargo which include lipids, proteins, and nucleic acid. Many desirable features of exosomes made them promising candidates in several therapeutic applications. In this review, we discuss the use of exosomes as diagnostic tools and their possible biomedical applications. Additionally, current techniques used for isolation, purification, and characterization of exosomes from both biological fluids and in vitro cell cultures were discussed.     

Dendritic Cells from Spleen, Mesenteric Lymph Node and Peyer's Patch Can Induce the Production of Both IL-4 and IFN-γ from Primary Cultures of Naive CD4+ T Cells in a Dose-Dependent Manner

Dendritic cells (DCs) as antigen presenting cells can stimulate naive CD4⁺ T cells and initiate the primary immune response which controls Th1/Th2 development. It has been suggested that DCs derived from different tissues have distinct properties. We investigated whether DCs from mesenteric lymph nodes (MLN), Peyer's patches (PP) and spleen (SPL) could induce different responses of naive CD4⁺ T cells to varying doses of antigen by using a co-culture system of DCs and T cells. DCs from each tissue induced IL-4 secretion from naive CD4⁺T cells in the presence of low dose antigenic peptide, and induced IFN-γ production at high doses of antigen. When purified CD11c⁺/B220^? DCs were used, MLN-derived DCs induced a higher amount of IFN-γ secretion from naive CD4⁺ T cells, compared with SPL-derived DCs. We could not detect large differences in the expressions of costimulatory molecules on the surface of these two populations of DCs. On the other hand, we found that large amounts of IL-12 were secreted from MLN DCs in an antigen dose-dependent fashion. In conclusion, DCs from SPL, MLN and PP can induce the production of both IL-4 and IFN-γ from naive CD4⁺ T cells, depending on antigen dose. MLN-derived CD11c⁺/B220^? DCs induce higher IFN-γ production from naive CD4⁺ T cells than SPL-derived DCs, through efficient IL-12 secretion.     

Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production

Both Th1 and Th17 cells have been implicated in the pathogenesis of inflammatory bowel disease and experimental colitis. However, the complex relationship between Th1 and Th17 cells and their relative contributions to the pathogenesis of inflammatory bowel disease have not been completely analyzed. Although it has been recently shown that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in the pathogenesis of colitis are still largely unknown. In this study, we report that Th17 cells from CBir1 TCR transgenic mice, which are specific for an immunodominant microbiota Ag, are more potent than Th1 cells in the induction of colitis, as Th17 cells induced severe colitis, whereas Th1 cells induced mild colitis when transferred into TCRβxδ(-/-) mice. High levels of IL-12 and IL-23 and substantial numbers of IFN-γ(+) Th1 cells emerged in the colons of Th17 cell recipients. Administration of anti-IL-17 mAb abrogated Th17 cell-induced colitis development, blocked colonic IL-12 and IL-23 production, and inhibited IFN-γ(+) Th1 cell induction. IL-17 promoted dendritic cell production of IL-12 and IL-23. Furthermore, conditioned media from colonic tissues of colitic Th17 cell recipients induced IFN-γ production by Th17 cells, which was inhibited by blockade of IL-12 and IL-23. Collectively, these data indicate that Th17 cells convert to Th1 cells through IL-17 induction of mucosal innate IL-12 and IL-23 production.     

Radio-attenuated leishmanial parasites as immunoprophylactic agent against experimental murine visceral leishmaniasis

The present study intends to evaluate the role of radio-attenuated leishmania parasites as immunoprophylactic agents for experimental murine visceral leishmaniasis. BALB/c mice were immunized with gamma (γ)-irradiated Leishmania donovani. A second immunization was given after 15 days of first immunization. After two immunizations, mice were infected with virulent L. donovani promastigotes. Protection against Kala-azar (KA) was estimated from spleen and liver parasitic burden along with the measurement of nitrite and superoxide anion generation by isolation of splenocytes and also by T-lymphocyte helper 1(Th1) and T-lymphocyte helper 2(Th2) cytokines release from the experimental groups. It was observed that BALB/c mice having prior immunization with radio-attenuated parasites showed protection against L. donovani infection through higher expression of Th1 cytokines and suppression of Th2 cytokines along with the generation of protective free radicals. The group of mice without prior priming with radio-attenuated parasites surrendered to the disease. Thus it can be concluded that radio-attenuated L. donovani may be used for.     

Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers

Exosomes are tiny vesicles (30–150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years. Here, we describe the strategies for isolation of exosomes from human blood serum and urine, characterization of their RNA cargo by sequencing, and present the initial data on exosome labelling and uptake tracing in a cell culture model. The value of exosomes for clinical applications is discussed with an emphasis on their potential for diagnosing and treating neurodegenerative diseases and brain cancer.     

Lack of signaling by IL-4 or by IL-4/IL-13 has more attenuating effects on Leishmania amazonensis dorsal skin--than on footpad-infected mice

Lesion development in tegumentary leishmaniasis is markedly influenced by the inoculation site and the type and number of injected infective forms. This and the yet unclear contribution of Th2 cytokines as susceptibility factors to Leishmania amazonensis infection prompted us to investigate the roles of IL-4, IL-13 and IL-10 on C57BL/6 and BALB/c mice infected in the footpad (paw) or rump with low-dose L. amazonensis purified-metacyclics. Wild-type (WT) mice of either strain developed, in the rump, a single large ulcerated lesion whereas paw lesions never ulcerated and were much smaller in C57BL/6 than in BALB/c mice. However, rump-inoculated IL-4-deficient (IL-4(-/-)) C57BL/6 mice did not develop any visible lesions although parasites remained in the dermis and lymph nodes, even after systemic IL-10-receptor blocking. By comparison, all IL-4(-/-) BALB/c mice developed rump ulcers. Strikingly, only 30% of rump-infected IL-4Rα(-/-) BALB/c mice developed lesions. IL-4(-/-) mice had higher IFN-γ and lower IL-10 and IL-13 levels than WT mice. Paw-infected IL-4Rα(-/-) BALB/c mice developed minimal paw lesions. While other factors contributing to L. amazonensis susceptibility cannot be discounted, our results indicate that absent signalling by IL-4 or by IL-4/IL-13 have more intense attenuating effects on rump than on paw lesions but do not eradicate parasitism.