Ulf Gehrmann

Antigen-loaded exosomes alone induce Th1-type memory through a B cell–dependent mechanism

Exosomes are nanovesicles harboring proteins important for antigen presentation. We compared the potency of differently loaded exosomes, directly loaded with OVA(323-339) peptide (Pep-Exo) or exosomes from OVA-pulsed DCs (OVA-Exo), for their ability to induce specific T-cell proliferation in vitro and in vivo. Both Pep-Exo and OVA-Exo elicited specific transgenic T-cell proliferation in vitro, with the Pep-Exo being more efficient. In contrast, only OVA-Exo induced specific T-cell responses in vivo highlighting the importance of indirect loading strategies in clinical applications. Coadministration of whole OVA overcame the unresponsiveness with Pep-Exo but still elicited a lower response compared with OVA-Exo. In parallel, we found that OVA-Exo not only augmented the specific T-cell response but also gave a Th1-type shift and an antibody response even in the absence of whole OVA. We detected IgG2a and interferon-gamma production from splenocytes showing the capability of exosomes to provide antigen for B-cell activation. Furthermore, we found that B cells are needed for exosomal T-cell stimulation because Bruton tyrosine kinase-deficient mice showed abrogated B- and T-cell responses after OVA-Exo immunization. These findings reveal that exosomes are potent immune regulators and are relevant for the design of vaccine adjuvants and therapeutic intervention strategies to modulate immune responses.

Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration

BACKGROUND Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity. OBJECTIVE We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis. METHODS The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system. RESULTS Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-β1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-β1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma. CONCLUSION Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment.

Dendritic cell-derived exosomes need to activate both T and B cells to induce antitumor immunity.

Exosomes are secreted membrane nanovesicles of endosomal origin and are considered potential cancer vaccine vectors. Phase I clinical trials have been successfully conducted with tumor peptide–loaded exosomes derived from dendritic cells (dexosomes), and a phase II clinical trial is ongoing. However, much is still unknown regarding the in vivo role of dexosomes and whether their immunogenicity can be enhanced. We previously reported that dexosomes induce CD4+ T cell responses in a B cell–dependent manner, suggesting that immunization with dexosomes carrying only T cell peptides induce suboptimal immune responses. In this study, we show that CD8+ T cell responses were induced in vivo when mice were immunized with protein-loaded, but not peptide-loaded, dexosomes. We also show that the cytotoxic T cell response was totally dependent on CD4+ T cells and, interestingly, also on B cells. Mice deficient in complement activation and Ag shuttling by B cells have lower responses to protein-loaded dexosomes, showing involvement of these B cell–mediated mechanisms. Finally, protein-loaded dexosomes were superior in protecting against tumor growth. In conclusion, proper activation of CD4+ T and B cells needs to be considered when designing cancer vaccines to ensure full potential of the treatment.

Effects of subtoxic concentrations of TiO2 and ZnO nanoparticles on human lymphocytes, dendritic cells and exosome production

Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investigate if commercially available TiO(2) and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO(2) or ZnO nanoparticles at concentrations from 1 to 100μg/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO(2) nanoparticles. Non-toxic exposure, 10μg/mL, to TiO(2) and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO(2) nanoparticles induced a down regulation of FcγRIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on FcγR-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cultures. TiO(2) or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO(2) and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions.

Nanovesicles from Malassezia sympodialis and Host Exosomes Induce Cytokine Responses – Novel Mechanisms for Host-Microbe Interactions in Atopic Eczema

Background Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. Objective To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. Methods Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. Results We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-α responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-α responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-α but not IL-4 in undepleted PBMC. Conclusions Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE.

Synergistic Induction of Adaptive Antitumor Immunity by Codelivery of Antigen with α-Galactosylceramide on Exosomes

Exosomes and the invariant NKT (iNKT) immune cell ligand α-galactosylceramide (αGC) may offer novel tools for cancer immunotherapy. In this study, we investigated whether exosomes loaded with αGC can activate iNKT cells and potentiate a cancer-specific adaptive immune response. αGC loaded exosomes readily activated iNKT cells both in vitro and in vivo. Exosomes loaded with αGC plus the model antigen ovalbumin (OVA) induced potent NK and γδ T-cell innate immune responses, and they also synergistically amplified T- and B-cell responses that were OVA specific. In contrast to soluble αGC, which anergizes iNKT cells, we found that αGC/OVA-loaded exosomes did not induce iNKT cell anergy but were more potent than soluble αGC + OVA in inducing adaptive immune responses. In an OVA-expressing mouse model of melanoma, treatment of tumor-bearing mice with αGC/OVA-loaded exosomes decreased tumor growth, increased antigen-specific CD8(+) T-cell tumor infiltration, and increased median survival, relative to control mice immunized with soluble αGC + OVA alone. Notably, an additional injection of αGC/OVA-loaded exosomes further augmented the treatment effects. Our findings show that exosomes loaded with protein antigen and αGC will activate adaptive immunity in the absence of triggering iNKT-cell anergy, supporting their application in the design of a broad variety of cancer immunotherapy trials.

Harnessing the exosome-induced immune response for cancer immunotherapy.

In recent years exosomes have emerged as potent stimulators of immune responses and as agents for cancer therapy. Exosomes can carry a broad variety of immunostimulatory molecules depending on the cell of origin and in vitro culture conditions. Dendritic cell-derived exosomes (dexosomes) have been shown to carry NK cell activating ligands and can be loaded with antigen to activate invariant NKT cells and to induce antigen-specific T and B cell responses. Dexosomes have been investigated as therapeutic agents against cancer in two phase I clinical trials, with a phase II clinical trial currently ongoing. Dexosomes were well tolerated but therapeutic success and immune activation were limited. Several reports suggest that multiple factors need to be considered in order to improve exosomal immunogenicity for cancer immunotherapy. These include antigen-loading strategies, exosome composition and exosomal trafficking in vivo. Hence, a better understanding of how to engineer and deliver exosomes to specific cells is crucial to generate strong immune responses and to improve the immunotherapeutic potential of exosomes.

Zymosan suppresses leukotriene C4 synthase activity in differentiating monocytes: antagonism by aspirin and protein kinase inhibitors

Cysteinyl leukotrienes (cysLTs) are potent proinflammatory mediators with particular relevance for asthma. However, control of cysLT biosynthesis in the time period after onset of acute inflammation has not been extensively studied. As a model for later phases of inflammation, we investigated regulation of leukotriene (LT) C4 synthase (LTC4S) in differentiating monocytes, exposed for several days to fungal zymosan. Incubations with LTA4 revealed 20‐fold increased LTC4S activity during differentiation of monocytic Mono Mac 6 (MM6) cells, which was reduced by 80% in the presence of zymosan (25 µg/ml, 96 h). Zymosan (48 h) similarly attenuated LTC4S activity of primary human monocyte‐derived macrophages and dendritic cells. Several findings indicate phosphoregulation of LTC4S: increased activity during MM6 cell differentiation correlated with reduced phosphorylation of 70‐kDa ribosomal protein S6 kinase (p70S6K), which could phosphorylate purified LTC4S; the p70S6K inhibitor rapamycin (20 nM) doubled LTC4S activity of undifferentiated MM6 cells, and protein kinase A and C inhibitors (H‐89, CGP‐53353, and staurosporine) reversed the zymosan‐induced suppression of LTC4S activity. Finally, zymosan (48 h) up‐regulated PGE2 biosynthesis, and aspirin (10 µM) or prostaglandin E2 (PGE2) receptor antagonists counteracted the zymosan effect. Our results suggest a late PGE2‐mediated phosphoregulation of LTC4S during microbial exposure, which may contribute to resolution of inflammation, with implications for aspirin hypersensitivity.—Esser, J., Gehrmann, U., Salvado, M. D., Wetterholm, A., Haeggstrom,J. Z., Samuelsson, B., Gabrielsson, S., Scheynius, A., Râdmark, O. Zymosan suppresses leukotriene C4 synthase activity in differentiating monocytes: antagonism by aspirin and protein kinase inhibitors. FASEBJ. 25, 1417–1427 (2011). www.fasebj.org

Extracellular nanovesicles released from the commensal yeast Malassezia sympodialis are enriched in allergens and interact with cells in human skin

Malassezia sympodialis is a dominant commensal fungi in the human skin mycobiome but is also associated with common skin disorders including atopic eczema (AE). M. sympodialis releases extracellular vesicles, designated MalaEx, which are carriers of small RNAs and allergens, and they can induce inflammatory cytokine responses. Here we explored how MalaEx are involved in host-microbe interactions by comparing protein content of MalaEx with that of the parental yeast cells, and by investigating interactions of MalaEx with cells in the skin. Cryo-electron tomography revealed a heterogeneous population of MalaEx. iTRAQ based quantitative proteomics identified in total 2439 proteins in all replicates of which 110 were enriched in MalaEx compared to the yeast cells. Among the MalaEx enriched proteins were two of the M. sympodialis allergens, Mala s 1 and s 7. Functional experiments indicated an active binding and internalization of MalaEx into human keratinocytes and monocytes, and MalaEx were found in close proximity of the nuclei using super-resolution fluorescence 3D-SIM imaging. Our results provides new insights into host-microbe interactions, supporting that MalaEx may have a role in the sensitization and maintenance of inflammation in AE by containing enriched amounts of allergens and with their ability to interact with skin cells.

Potentiating antitumor immunity with αGC-loaded exosomes.

Anticancer immunotherapy is a promising treatment modality since it bears the potential of being highly specific, but effective clinical applications are still under development. We have recently described an exosome-based strategy for co-delivery of α-galactosylceramide and a tumor-associated antigen that synergistically potentiates tumor-specific adaptive immune responses while preventing the anergy of invariant natural killer T (iNKT) cells. We propose that the next generation of exosome-based immunotherapies should involve iNKT-cell ligands to induce a broad, amplified and sustainable antitumor immune response.