Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.     

Curcumin reverses breast tumor exosomes mediated immune suppression of NK cell tumor cytotoxicity

An important characteristic of tumors is that they at some point in their development overcome the surveillance of the immune system. Tumors secrete exosomes, multivesicular bodies containing a distinct set of proteins that can fuse with cells of the circulating immune system. Purified exosomes from TS/A breast cancer cells, but not non-exosomal fractions, inhibit (at concentrations of nanograms per ml protein) IL-2-induced natural killer (NK) cell cytotoxicity. The dietary polyphenol, curcumin (diferuloylmethane), partially reverses tumor exosome-mediated inhibition of natural killer cell activation, which is mediated through the impairment of the ubiquitin-proteasome system. Exposure of mouse breast tumor cells to curcumin causes a dose-dependent increase in ubiquitinated exosomal proteins compared to those in untreated TS/A breast tumor cells. Furthermore, exosomes isolated from tumor cells pretreated with curcumin have a much attenuated inhibition of IL-2 stimulated NK cell activation. Jak3-mediated activation of Stat5 is required for tumor cytotoxicity of IL-2 stimulated NK cells. TS/A tumor exosomes strongly inhibit activation of Stat5, whereas the tumor exosomes isolated from curcumin-pretreated tumor cells have a lowered potency for inhibition of IL-2 stimulated NK cell cytotoxicity. These data suggest that partial reversal of tumor exosome-mediated inhibition of NK cell tumor cytotoxicity may account for the anti-cancer properties of curcumin.     

Effect of mesenchymal stem cell-derived exosomes on the induction of mouse tolerogenic dendritic cells

Dendritic cells (DCs) orchestrate innate inflammatory responses and adaptive immunity through T-cell activation via direct cell–cell interactions and/or cytokine production. Tolerogenic DCs (tolDCs) help maintain immunological tolerance through the induction of T-cell unresponsiveness or apoptosis, and generation of regulatory T cells. Mesenchymal stromal cells (MSCs) are adult multipotent cells located within the stroma of bone marrow (BM), but they can be isolated from virtually all organs. Extracellular vesicles and exosomes are released from inflammatory cells and act as messengers enabling communication between cells. To investigate the effects of MSC-derived exosomes on the induction of mouse tolDCs, murine adipose-derived MSCs were isolated from C57BL/6 mice and exosomes isolated by ExoQuick-TC kits. BM-derived DCs (BMDCs) were prepared and cocultured with MSCs-derived exosomes (100 μg/ml) for 72 hr. Mature BMDCs were derived by adding lipopolysaccharide (LPS; 0.1μg/ml) at Day 8 for 24 hr. The study groups were divided into (a) immature DC (iDC, Ctrl), (b) iDC + exosome (Exo), (c) iDC + LPS (LPS), and (d) iDC + exosome + LPS (EXO + LPS). Expression of CD11c, CD83, CD86, CD40, and MHCII on DCs was analyzed at Day 9. DC proliferation was assessed by coculture with carboxyfluorescein succinimidyl ester-labeled BALB/C-derived splenocytes p. Interleukin-6 (IL-6), IL-10, and transforming growth factor-β (TGF-β) release were measured by enzyme-linked immunosorbent assay. MSC-derived exosomes decrease DC surface marker expression in cells treated with LPS, compared with control cells ( ≤ .05). MSC-derived exosomes decrease IL-6 release but augment IL-10 and TGF-β release (p ≤ .05). Lymphocyte proliferation was decreased (p ≤ .05) in the presence of DCs treated with MSC-derived exosomes. CMSC-derived exosomes suppress the maturation of BMDCs, suggesting that they may be important modulators of DC-induced immune responses.     

Bone marrow precursors of nonobese diabetic mice develop into defective macrophage-like dendritic cells in vitro

The NOD mouse spontaneously develops autoimmune diabetes. Dendritic cells (DC) play a crucial role in the autoimmune response. Previous studies have reported a defective DC generation in vitro from the NOD mouse bone marrow (BM), but a deviated development of myeloid precursors into non-DC in response to GM-CSF was not considered. In this study, we demonstrate several abnormalities during myeloid differentiation of NOD BM precursors using GM-CSF in vitro. 1) We found reduced proliferation and increased cell death in NOD cultures, which explain the previously reported low yield of DC progeny in NOD. Cell yield in NOR cultures was normal. 2) In a detailed analysis GM-CSF-stimulated cultures, we observed in both NOD and NOR mice an increased frequency of macrophages, identified as CD11c(+)/MHCII(-) cells with typical macrophage morphology, phenotype, and acid phosphatase activity. This points to a preferential maturation of BM precursors into macrophages in mice with the NOD background. 3) The few CD11c(+)/MHCII(high) cells that we obtained from NOD and NOR cultures, which resembled prototypic mature DC, appeared to be defective in stimulating allogeneic T cells. These DC had also strong acid phosphatase activity and elevated expression of monocyte/macrophage markers. In conclusion, in this study we describe a deviated development of myeloid BM precursors of NOD and NOR mice into macrophages and macrophage-like DC in vitro. Potentially, these anomalies contribute to the dysfunctional regulation of tolerance in NOD mice yet are insufficient to induce autoimmune diabetes because they occurred partly in NOR mice.     

Endothelial acid ceramidase in exosome-mediated release of NLRP3 inflammasome products during hyperglycemia: Evidence from endothelium-specific deletion of Asah1 gene

Exosomes have been demonstrated to be one of the mechanisms mediating the release of intracellular signaling molecules to conduct cell-to-cell communication. However, it remains unknown whether and how exosomes mediate the release of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome products such as interleukin-1 beta (IL-1β) from endothelial cells. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of multivesicular bodies (MVBs) to control the exosome-mediated release of NLRP3 inflammasome products during hyperglycemia. Using a streptozotocin (STZ)-induced diabetes mouse model, we found that endothelium-specific AC gene knockout mice (Asah1^fl/fl/EC^cre) significantly enhanced the formation and activation of NLRP3 inflammasomes in coronary arterial ECs (CECs). These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein compared to wild-type (WT/WT) littermates. We also observed the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was augmented in STZ-treated Asah1^fl/fl/EC^cre mice compared to WT/WT mice, which was accompanied by an increased IL-1β release of exosomes. In the primary cultures of CECs, we demonstrated that AC deficiency markedly enhanced the formation and activation of NLRP3 inflammasomes, but significantly down-regulated tight junction proteins when these cells were exposed to high levels of glucose. The CECs from Asah1^fl/fl/EC^cre mice had decreased MVB-lysosome interaction and increased IL-1β–containing exosome release in response to high glucose stimulation. Together, these results suggest that AC importantly controls exosome-mediated release of NLRP3 inflammasome products in CECs, which is enhanced by AC deficiency leading to aggravated arterial inflammatory response during hyperglycemia.     

Estrogen receptor alpha, but not beta, is required for optimal dendritic cell differentiation and [corrected] CD40-induced cytokine production

Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17beta-estradiol (E(2)) has been shown to promote the differentiation of DCs from bone marrow (BM) precursors in vitro. However, the estrogen receptor (ER) involved in this effect has not yet been characterized. Using recently generated ERalpha- or ERbeta-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERalpha, but not ERbeta, is required for normal DC development from BM precursors cultured with GM-CSF. We show that reduced numbers of DCs were generated in the absence of ERalpha activation and provide evidence for a cell-autonomous function of ERalpha signaling in DC differentiation. ERalpha-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERalpha-deficient DCs failed to up-regulate MHC class II and CD86 molecules, which could account for their reduced capacity to prime naive CD4(+) T lymphocytes. Although they retained the ability to express CD40 and to produce proinflammatory cytokines (e.g., IL-12, IL-6) upon TLR engagement, ERalpha-deficient DCs were defective in their ability to secrete such cytokines in response to CD40-CD40L interactions. Taken together, these results provide the first genetic evidence that ERalpha is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.     

Exosomes derived from dendritic cells

Dendritic cells (DC) are potent antigen presenting cells and the only ones capable of inducing primary cytotoxic immune responses both in vivo and vitro. DCs secrete a 60-80 nm membrane vesicle population of endocytic origin, called exosomes. The protein composition of exosomes was analyzed using a systematic proteomic approach. Besides MHC and costimulatory molecules, exosomes bear several adhesion proteins, probably involved in their specific targeting. Exosomes also accumulate several cytosolic factors, most likely involved in exoxome's biogenesis in late endosomes. Like DCs, exosomes induce potent anti tumor immune responses in vivo. Indeed, a single injection of DC-derived exosomes sensitized with tumor peptides induced the eradication of established mouse tumors. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome treated mice, and depletion of CD8+ T cells in vivo inhibited the anti tumor effect of exosomes. These results strongly support the implementation of human DC-derived exosomes for cancer immunotherapy.     

Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells

Th2-type inflammation spontaneously shown in Bcl6-knockout (KO) mice is mainly caused by bone marrow (BM)-derived nonlymphoid cells. However, the function of dendritic cells (DCs) in Bcl6-KO mice has not been reported. We show in this article that the numbers of CD4(+) conventional DCs (cDCs) and CD8α(+) cDCs, but not of plasmacytoid DCs, were markedly reduced in the spleen of Bcl6-KO mice. Generation of cDCs from DC progenitors in BM cells was perturbed in the spleen of irradiated wild-type (WT) mice transferred with Bcl6-KO BM cells, indicating an intrinsic effect of Bcl6 in cDC precursors. Although cDC precursors were developed in a Bcl6-KO BM culture with Fms-like tyrosine kinase 3 ligand, the cDC precursors were more apoptotic than WT ones. Also p53, one of the molecular targets of Bcl6, was overexpressed in the precursors. The addition of a p53 inhibitor to Bcl6-KO BM culture protected apoptosis, suggesting that Bcl6 is required by cDC precursors for survival by controlling p53 expression. Furthermore, large numbers of T1/ST2(+) Th2 cells were naturally developed in the spleen of Bcl6-KO mice. Th2 skewing was accelerated in the culture of WT CD4 T cells stimulated with Ags and LPS-activated Bcl6-KO BM-derived DCs, which produced more IL-6 and less IL-12 than did WT DCs; the addition of anti-IL-6 Abs to the culture partially abrogated the Th2 skewing. These results suggest that Bcl6 is required in cDC precursors for survival and in activated DCs for modulating the cytokine profile.     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

Generation of antigen-presenting cells from tumor-infiltrated CD11b myeloid cells with DNA demethylating agent 5-aza-2′-deoxycytidine

Tumor-recruited CD11b myeloid cells, including myeloid-derived suppressor cells, play a significant role in tumor progression, as these cells are involved in tumor-induced immune suppression and tumor neovasculogenesis. On the other hand, the tumor-infiltrated CD11b myeloid cells could potentially be a source of immunostimulatory antigen-presenting cells (APCs), since most of these cells represent common precursors of both dendritic cells and macrophages. Here, we investigated the possibility of generating mature APCs from tumor-infiltrated CD11b myeloid cells. We demonstrate that in vitro exposure of freshly excised mouse tumors to DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (decitabine, AZA) results in selective elimination of tumor cells, but, surprisingly it also enriches CD45⁺ tumor-infiltrated cells. The majority of “post-AZA” surviving CD45⁺ tumor-infiltrated cells were represented by CD11b myeloid cells. A culture of isolated tumor-infiltrated CD11b cells in the presence of AZA and GM-CSF promoted their differentiation into mature F4/80/CD11c/MHC class II-positive APCs. These tumor-derived myeloid APCs produced substantially reduced amounts of immunosuppressive (IL-13, IL-10, PGE₂), pro-angiogenic (VEGF, MMP-9) and pro-inflammatory (IL-1beta, IL-6, MIP-2) mediators than their precursors, freshly isolated tumor-infiltrated CD11b cells. Vaccinating naïve mice with ex vivo generated tumor-derived APCs resulted in the protection of 70% mice from tumor outgrowth. Importantly, no loading of tumor-derived APC with exogenous antigen was needed to stimulate T cell response and induce the anti-tumor effect. Collectively, our results for the first time demonstrate that tumor-infiltrated CD11b myeloid cells can be enriched and differentiated in the presence of DNA demethylating agent 5-aza-2′-deoxycytidine into mature tumor-derived APCs, which could be used for cancer immunotherapy.     

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

Cytokines in the generation and maturation of dendritic cells: recent advances

Dendritic cells (DCs) are extremely efficient antigen presenting cells (APCs) that are potent stimulators of both T and B cell-mediated immune responses. Although DCs are normally present in very small numbers in the peripheral blood (PB), recent advances have made it possible to generate relatively large numbers of cells in culture. DCs can be differentiated in vitro from various cellular sources, including bone marrow (BM), cord blood (CB) and PB mononuclear cells (PBMCs). Although a wide variety of conditions have been reported to be able to support DC generation, the majority of research and clinical protocols to date differentiate DCs from precursors using granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with either tumor necrosis factor-(TNF-)alpha or interleukin (IL)-4. However, a diverse array of cytokines has been shown to be able to induce DC differentiation under a variety of conditions. According to recent reports, cytokines such as IL-2, IL-6, IL-7, IL-13, IL-15 and hepatocyte growth factor (HGF), in combination or even, in some cases, alone, can contribute to the generation of DCs from either monocytes or CD34+ cells. Although the majority of cytokine combinations include GM-CSF, some do not. For example, Flt3 ligand (FL), in conjuction with IL-6 (in the absence of GM-CSF), has been reported to be able to induce DC differentiation from BM cells in a murine system. Other agents can play a dual role in DC activity. CD40 ligand (CD40L), as a single agent, has been shown to be able to generate DCs from PB monocytes, while numerous other reports have also demonstrated its role as a potent maturation factor. In contrast, for other cytokines such as IL-16 or IL-17, although there is no data for a role in DC generation, they have been reported to be involved in promoting DC maturation in vitro as defined by upregulation of costimulatory molecules, major histocompatibility complex (MHC) antigens and antigen presenting/T lymphocyte stimulatory capacity. Furthermore, cytokines such as stem cell factor (SCF) and FL have been shown to dramatically enhance in vivo DC recovery. The wide variety of cytokines and conditions that have been shown to be able to influence DC differentiation and activity to amply demonstrate the extreme heterogeneity found in the DC population, something that is reflected in the diverse phenotypes, functions and ontogeny displayed by DCs. This diversity may account for the large number of roles that have been attributed to DCs in the development and function of the immune system and, in turn, emphasizes the potential as well as the challenges of modifying specific aspects of the immune response system by manipulating specific DC subpopulations.     

Downregulation of endogenous STAT3 augments tumoricidal activity of interleukin 15 activated dendritic cell against lymphoma and leukemia via TRAIL

Effector functions in tumor resistance by dendritic cells (DCs) are less well characterized. In this study, we describe that the murine DCs upon stimulation with recombinant IL-15 in vitro or in vivo, expresses TNF superfamily member TRAIL which mediates cytotoxicity and growth inhibition against a murine lymphoma called Dalton lymphoma (DL) via apoptosis. Presence of tumor lysate or intact tumor cells significantly reduces the DC mediated tumoricidal effect, possibly via masking and down-regulating TRAIL in DCs. The antitumor effect of DC derived TRAIL was further augmented by deactivation of STAT3 in tumor cells by cucurbitacin I, which makes it more susceptible to DC derived TRAIL Treatment of tumor cells with cucurbitacin I upregulates TRAIL receptor expression in addition to activation of caspases. Compared to naïve DCs, DCs from tumor bearing mice are significantly impaired in TRAIL expression and consequent antitumor functions against DL which was partially restored by activation with IL-15 or LPS. Priming with recombinant IL-15 prolongs the survival of tumor bearing mice treated with cucurbitacin I. Naïve peripheral blood DCs derived from chronic myeloid leukemia (CML) patients have significant impairment in expression of TRAIL and consequent tumoricidal properties against TRAIL sensitive lymphoma cell lines and primary tumor cells compared to normal control.     

Acidic polysaccharides isolated from Phellinus linteus induce phenotypic and functional maturation of murine dendritic cells

Acidic polysaccharides (PL) isolated from Phellinus linteus are known to stimulate the proliferation of T lymphocytes and humoral immune functions to act as a polyclonal activator of B cells, and to inhibit tumor growth and metastasis. However, little is known about their immunomodulating effects or the effects of its mechanisms on murine bone marrow (BM)-derived dendritic cells (DC). In this study, it profoundly increased CD80, CD86, MHC I, and MHC II expression in murine, GM-CSF and IL-4 stimulated, BM-derived myeloid DC. The ability of unstimulated DC to uptake dextran was higher than that of PL- or LPS-stimulated DC. We analyzed the concentration of IL-12 secreted by DC using flow cytometry and ELISA. Untreated DC secreted a low concentration of IL-12, while PL- or LPS-stimulated DC secreted higher levels of IL-12 than untreated DC. There were no remarkable differences in the concentrations of IL-12 produced by PL- or LPS-stimulated DC. However, polymyxin B (PB; an LPS inhibitor) effectively inhibited the surface molecules and IL-12 production induced by LPS, but had no effect on the PL in DC. PL-treated DC were much more potent antigen-presenting cells in allogeneic immune response than untreated DC. PL treatment not only formed morphologically mature DC but also induced predominant migration to lymphoid tissues. Moreover, the inhibitors of protein tyrosine kinase (PTK) or protein kinase C (PKC) significantly blocked the expression of surface molecules and IL-12 production in PL-stimulated DC. Treatment of DC with PL directly induced PKC activity and phosphorylated PTK. Furthermore, CD11b and/or CD18 partially mediated PL-induced DC maturation.     

Exosomes released from Mycoplasma infected tumor cells activate inhibitory B cells

Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.     

Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.     

Tumor-derived exosomes confer antigen-specific immunosuppression in a murine delayed-type hypersensitivity model

Exosomes are endosome-derived small membrane vesicles that are secreted by most cell types including tumor cells. Tumor-derived exosomes usually contain tumor antigens and have been used as a source of tumor antigens to stimulate anti-tumor immune responses. However, many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms, most of which are antigen-independent. In the present study we used a mouse model of delayed-type hypersensitivity (DTH) and demonstrated that local administration of tumor-derived exosomes carrying the model antigen chicken ovalbumin (OVA) resulted in the suppression of DTH response in an antigen-specific manner. Analysis of exosome trafficking demonstrated that following local injection, tumor-derived exosomes were internalized by CD11c+ cells and transported to the draining LN. Exosome-mediated DTH suppression is associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN. The tumor-derived exosomes examined were also found to inhibit DC maturation. Taken together, our results suggest a role for tumor-derived exosomes in inducing tumor antigen-specific immunosuppression, possibly by modulating the function of APCs.     

Developmental Regulation by Cytokines of Bone Marrow-Derived Dendritic Cells and Epidermal Langerhans Cells

Dendritic cells (DC) are specialized antigen-presenting cells involved in T cell-mediated immune responses. Differentiation and functional maturation of the DC are now known to be regulated by various cytokines, including TGF-β1. The experiments of this study examined the effect of other cytokines, such as IL-4, IL-10 and IL-6, on the differentiation and maturation of bone marrow (BM)-derived DC (BM-DC) and epidermal Langerhans cells (LC). When IL-6 or IL-10 was added to cultures of BM cells in the presence of GM-CSF, both cytokines, as in the case of TGF-β1, suppressed the maturation of DC in terms of the expression of adhesion and costimulatory molecules and T cell-stimulating activity. In contrast, IL-4 was not suppressive but rather supportive for the differentiation of DC. However, these suppressive cytokines hardly counteracted the maturation-inducing activity of TNF-α when added to cultures of immature DC. In addition, they appeared to block the overmaturation of DC, which is characterized by a loss of MHC class II molecules. Regarding LC maturation in epidermal cell cultures, IL-6 and IL-10 were inhibitory for the expression of CD86 and CD80 in a dose-dependent fashion. Unlike BM-DC, LC maturation was slightly enhanced by TGF-β1. The protein antigen-presentation by LC to Th1 clone was not affected by IL-6, but slightly reduced by IL-10. These results suggest that each cytokine contributes to regulate the differentiation and maturation of DC at a different developmental stage.