| 1. |
- Ding, Mei, et al.
(author)
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Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells
- 2021
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; , s. 16767-
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Journal article (peer-reviewed)abstract
- Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.
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| 2. |
- Gehrmann, Ulf
(author)
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Exosomes : the future of vaccination?
- 2011
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 3. |
- Johansson, Henrik J., et al.
(author)
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Extracellular nanovesicles released from the commensal yeast Malassezia sympodialis are enriched in allergens and interact with cells in human skin
- 2018
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In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Journal article (peer-reviewed)abstract
- 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 hostmicrobe 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.
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