Exosomes - the future of vaccination?

Exosomes are small membrane vesicles that are secreted by cells as means of intercellular communication. They are typically between 50 and 100 nm in diameter and originate from the endosomal compartment of cells. Exosomes have been considered a potential novel cell- free therapeutic agent since exosomes are capable of antigen presentation. Indeed, exosomes from dendritic cells can activate the innate and adaptive immune systems, can establish protective immunity in various models of infectious diseases and can eradicate established tumors in mice. However, using exosomes in the clinic has proven difficult and need optimization to induce a sufficient immune response. Research now focuses on a) using exosomes as biomarkers and diagnostic tools for neoplastic diseases and b) understanding the exosomal immune response and finding ways to increase the immunogenicity of exosomes. This thesis aimed at 1) clarifying mechanisms important for the exosomal immune response, 2) identifying new ways to increase immunogenicity of exosomes, and 3) studying the relevance of exosomes in a human inflammatory disease, atopic eczema (AE). We report that exosomes from murine bone marrow-derived dendritic cells (DCs) can induce CD4+ T cell responses in a B cell-dependent manner. By comparing exosomes loaded with the whole ovalbumin (OVA) protein to exosomes loaded with the dominant CD4+ T cell epitope we found that only whole OVA-loaded exosomes could induce memory T and B cell responses in vivo. Interestingly, T cell activation was absent in Bruton kinase knockout (btk-/-) mice lacking a functional B cell compartment. Further, we found that bone marrow DC-derived exosomes express CD1d and can activate natural killer T cells (NKT cells) in vitro and in vivo. Activation of NKT cells subsequently amplified innate NK cell and γδ T cell responses as well as OVA-specific CD4+ T cell, CD8+ T cell and B cell responses. Our data suggest that exosome-induced antibody production is linked to subsequent activation of T follicular helper cells, germinal center B cells and plasma cells. In a third study, using exosomes from human monocyte-derived DCs, macrophages and plasma, we discovered a novel inflammatory property of exosomes. We found that exosomes contain enzymes of the leukotriene pathway and that they could produce high amounts of leukotriene B4 and C4 when incubated with the intermediate leukotriene A4. Exosomes could also induce granulocyte migration, which increased when incubated with the substrate AA. Finally, we found that the commensal yeast Malassezia sympodialis secreted nanovesicles that carry M. sympodialis allergens and induced significantly higher IL-4 responses in peripheral blood mononuclear cells (PBMC) of AE patients sensitized to the yeast than in PBMC of healthy controls (HC). Nanovesicles induced TNF-α production in PBMC of both groups. Further, we find that exosomes from monocyte-derived DCs, co-cultured with M. sympodialis induce significantly higher IL-4 and TNF-α responses than exosomes from unstimulated DCs in PBMC of both AE patients and HC. This suggests a role for nanovesicles in the allergic immune response. In summary, we have identified three new pathways, which might be exploited to induce more potent immune responses to exosomes. Including B cell epitopes and CD1d ligands as well as exploiting the chemoattractive capacity of exosomes when designing future exosomal vaccines might increase the efficacy in a clinical setting. The finding that immunogenic nanovesicles are produced by M. sympodialis highlights novel host microbe interactions in AE and emphasizes the immunostimulatory potential of exosomes also in humans

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

Soziale Herkunft von Kindern und Jugendlichen als Barriere für den Zugang zum Sport – Theoriebasierte Erklärungen und empirische Erkenntnisse

Gehrmann S. Soziale Herkunft von Kindern und Jugendlichen als Barriere für den Zugang zum Sport – Theoriebasierte Erklärungen und empirische Erkenntnisse. In: Gebken U, Pfitzner M, Wiesche D, eds. Grenzen und Entgrenzungen sportpädagogischen Handelns. Abstractband. Essen: Universität Duisburg-Essen; 2024: 55-56

A MALT1 inhibitor suppresses human myeloid DC, effector T-cell and B-cell responses and retains Th1/regulatory T-cell homeostasis.

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or T-cell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-ɣ) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-ɣ, in a mouse model of acute T-cell activation, and long-term treatment did not lead to an increase in IFN-ɣ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases

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

Early diagnostic BioMARKers in exacerbations of chronic obstructive pulmonary disease: protocol of the exploratory, prospective, longitudinal, single-centre, observational MARKED study

Introduction Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) play a pivotal role in the burden and progressive course of chronic obstructive pulmonary disease (COPD). As such, disease management is predominantly based on the prevention of these episodes of acute worsening of respiratory symptoms. However, to date, personalised prediction and early and accurate diagnosis of AECOPD remain unsuccessful. Therefore, the current study was designed to explore which frequently measured biomarkers can predict an AECOPD and/or respiratory infection in patients with COPD. Moreover, the study aims to increase our understanding of the heterogeneity of AECOPD as well as the role of microbial composition and hostmicrobiome interactions to elucidate new disease biology in COPD.Methods and analysis The ‘Early diagnostic BioMARKers in Exacerbations of COPD’ study is an exploratory, prospective, longitudinal, single-centre, observational study with 8-week follow-up enrolling up to 150 patients with COPD admitted to inpatient pulmonary rehabilitation at Ciro (Horn, the Netherlands). Respiratory symptoms, vitals, spirometry and nasopharyngeal, venous blood, spontaneous sputum and stool samples will be frequently collected for exploratory biomarker analysis, longitudinal characterisation of AECOPD (ie, clinical, functional and microbial) and to identify host–microbiome interactions. Genomic sequencing will be performed to identify mutations associated with increased risk of AECOPD and microbial infections. Predictors of time-to-first AECOPD will be modelled using Cox proportional hazards’ regression. Multiomic analyses will provide a novel integration tool to generate predictive models and testable hypotheses about disease causation and predictors of disease progression.Ethics and dissemination This protocol was approved by the Medical Research Ethics Committees United (MEC-U), Nieuwegein, the Netherlands (NL71364.100.19).Trial registration number NCT05315674

Human lung conventional dendritic cells orchestrate lymphoid neogenesis during chronic obstructive pulmonary disease

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined. Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD. Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21(+)CXCL13(+)(IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs. Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21(+)CXCL13(+) Tfh-like cells. Importantly, the capacity to induce IL-21(+)Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX4OL (OX4O ligand)-OX4O axis reduced Tfh-cell induction by control lung cDC2s. Conclusions: In COPD lungs, we found lung EBI2(+) (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21(+) Tfh-like cells, suggesting an involvement of these cells in TLO formation

Toll-like receptor 4 engagement on dendritic cells restrains phago-lysosome fusion and promotes cross-presentation of antigens

The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross- presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens