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.

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.

Role of innate signalling pathways in the immunogenicity of alphaviral replicon-based vaccines.

BACKGROUND: Alphaviral replicon-based vectors induce potent immune responses both when given as viral particles (VREP) or as DNA (DREP). It has been suggested that the strong immune stimulatory effect induced by these types of vectors is mediated by induction of danger signals and activation of innate signalling pathways due to the replicase activity. To investigate the innate signalling pathways involved, mice deficient in either toll-like receptors or downstream innate signalling molecules were immunized with DREP or VREP. RESULTS: We show that the induction of a CD8+ T cell response did not require functional TLR3 or MyD88 signalling. However, IRF3, converging several innate signalling pathways and important for generation of pro-inflammatory cytokines and type I IFNs, was needed for obtaining a robust primary immune response. Interestingly, type I interferon (IFN), induced by most innate signalling pathways, had a suppressing effect on both the primary and memory T cell responses after DREP and VREP immunization. CONCLUSIONS: We show that alphaviral replicon-based vectors activate multiple innate signalling pathways, which both activate and restrict the induced immune response. These results further show that there is a delicate balance in the strength of innate signalling and induction of adaptive immune responses that should be taken into consideration when innate signalling molecules, such as type I IFNs, are used as vaccine adjuvant.

Toll-like receptor 3 promotes cross-priming to virus-infected cells.

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8alpha+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.

Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity.

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

Exosomes from antigen-pulsed dendritic cells induce stronger antigen-specific immune responses than microvesicles in vivo.

Extracellular vesicles (EV), including exosomes and microvesicles (MV), represent a rapidly expanding field of research with diagnostic and therapeutic applications. Although many aspects of EV function remain to be revealed and broad investigations are warranted, most published findings focus on only one vesicle category or a non-separated mix of EVs. In this paper, we investigated both MVs and exosomes from Ovalbumin (OVA)-pulsed dendritic cells for their immunostimulatory potential side-by-side in vivo. Only exosomes induced antigen-specific CD8+ T-cells, and were more efficient than MVs in eliciting antigen-specific IgG production. Further, mainly exosome-primed mouse splenocytes showed significant ex vivo interferon gamma production in response to antigen restimulation. Exosomes carried high levels of OVA, while OVA in MVs was barely detectable, which could explain the more potent antigen-specific response induced by exosomes. Moreover, exosomes induced increased germinal center B cell proportions, whereas MVs had no such effect. Immunisation with both vesicle types combined showed neither inhibitory nor synergistic effects. We conclude that DC-derived MVs and exosomes differ in their capacity to incorporate antigen and induce immune responses. The results are of importance for understanding the role of EVs in vivo, and for future design of vesicle-based immunotherapies and vaccines.

Exosomes from breast milk inhibit HIV-1 infection of dendritic cells and subsequent viral transfer to CD4+ T cells.

OBJECTIVE: To investigate whether exosomes derived from human breast milk or plasma confer protection against HIV-1 infection of monocyte-derived dendritic cells (MDDCs) and subsequent viral transfer to CD4 T cells. DESIGN: MDDCs were generated and milk and plasma-derived exosomes were isolated from healthy donors. To determine the capacity of exosomes to inhibit HIV-1 infection, MDDCs were preincubated with exosomes before exposure to HIV-1BaL. To investigate transfer of HIV-1 from MDDCs to CD4 T cells, MDDCs preincubated with exosomes and HIV-1BaL were cocultured with allogeneic CD4 T cells. To explore receptors used by MDDCs for binding of exosomes, blocking experiments were performed. METHODS: Productive HIV-1 infection was analysed in MDDCs and CD4 T cells by determining p24 expression by flow cytometry. Confocal microscopy and flow cytometry was used to investigate uptake of fluorescently labelled exosomes by MDDCs. RESULTS: Milk exosomes, but not plasma exosomes, bind MDDCs via DC-SIGN inhibiting HIV-1 infection of MDDCs and subsequent viral transfer to CD4 T cells. CONCLUSION: We propose that milk exosomes act as a novel protective factor against vertical transmission of HIV-1 by competing with HIV-1 for binding to DC-SIGN on MDDCs.

Cancer immunotherapy with exosomes requires B-cell activation.

Exosomes derived from dendritic cells (dexosomes) induce potent antitumor immune responses in mice. We have shown that the efficacy of dexosome-elicited antitumor immunity relies on the presence of both T- and B-cell dexosome-associated epitopes. Hence, the inclusion of B-cell epitopes in anticancer vaccines is crucial for the success of this immunotherapeutic intervention.

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.

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.

Comparative prime-boost vaccinations using Semliki Forest virus, adenovirus, and ALVAC vectors demonstrate differences in the generation of a protective central memory CTL response against the P815 tumor.

Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8(+) T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8(+) T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8(+) T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8(+) T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L(+) P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8(+) T cell response correlates with tumor protection.

RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates.

Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

MyD88 expression is required for efficient cross-presentation of viral antigens from infected cells.

While virus-infected dendritic cells (DCs) in certain instances have the capacity to activate naive T cells by direct priming, cross-priming by DCs via the uptake of antigens from infected cells has lately been recognized as another important pathway for the induction of antiviral immunity. During cross-priming, danger and stranger signals play important roles in modulating immune responses. Analogous to what has been shown for other microbial infections, virally infected cells may contain several pathogen-associated molecular patterns that are recognized by Toll-like receptors (TLRs). We analyzed whether the efficient presentation of antigens derived from infected cells requires the usage of MyD88, which is a common adaptor molecule used by all TLRs. For this study, we used murine DCs that were wild type or deficient in MyD88 expression and fibroblasts that were infected with an alphavirus replicon to answer this question. Our results show that when DCs are directly infected, they are able to activate antigen-specific CD8(+) T cells in a MyD88-independent manner. In contrast, a strict requirement of MyD88 for cross-priming was observed when virally infected cells were used as a source of antigen in vitro and in vivo. This indicates that the effects of innate immunity stimulation via the MyD88 pathway control the efficiency of cross-presentation, but not direct presentation or DC maturation, and have important implications in the development of cytotoxic T lymphocyte responses against alphaviral replicon infections.