Maturation of Human Dendritic Cells upon delicery of cGAMP
Author
Banasik, Agnieszka Janina
Term
4. term
Publication year
2018
Submitted on
2018-05-31
Pages
44
Abstract
Kræfteimmunoterapi sigter mod at aktivere kroppens eget forsvar, hvor dendritceller spiller en nøglerolle i at starte T-celle-responser. Denne afhandling undersøger, om cGAMP—et signalmolekyle dannet af cGAS ved påvisning af cytosolisk DNA og kendt for at aktivere STING—kan modne humane monocytafledte dendritceller (moDCs) in vitro. Celledød blev induceret i L929-celler med kombinationer af TNF + cycloheximid eller TNF + zVAD, og både døde celler og deres supernatant blev brugt til at stimulere umodne moDCs. Modning blev vurderet ved opregulering af overflademarkørerne CD80, CD86 og HLA-DR ved flowcytometri. Tilsætning af caspasehæmmeren zVAD-fmk udløste også DC-aktivering, hvilket peger på et bidrag fra nekroptotiske celler. Desuden viste forsøget, at cGAMP alene kan modne moDCs og gør det mere potent end andre DAMPs (fx ATP og urinsyrekrystaller) ved samme molare koncentration. Funktionelt førte kokultur af PBMCs med cGAMP-modnede DCs til PBMC-proliferation og forhøjede niveauer af cytokinerne IL-10, IL-1β og IFNγ målt med MSD-multiplex. Samlet peger resultaterne på, at cGAMP kan fungere som et faresignal, der modner humane dendritceller, muligvis frigivet fra nekroptotiske celler, og understøtter videre udforskning i en immunterapikontekst.
Cancer immunotherapy aims to enlist the body’s own defenses, with dendritic cells (DCs) playing a key role in initiating T cell responses. This thesis examines whether cGAMP—produced by cGAS upon sensing cytosolic DNA and known to activate STING—can mature human monocyte-derived DCs (moDCs) in vitro. Cell death was induced in L929 cells using TNF plus cycloheximide or TNF plus zVAD, and both dead cells and their supernatants were used to stimulate immature moDCs. Maturation was assessed by upregulation of surface markers CD80, CD86 and HLA-DR by flow cytometry. Addition of the caspase inhibitor zVAD-fmk also triggered DC activation, implicating a contribution from necroptotic cells. Furthermore, cGAMP alone induced moDC maturation and did so more potently than other DAMPs (e.g., ATP and uric acid crystals) at equivalent molar concentrations. Functionally, co-culture of PBMCs with cGAMP-matured DCs led to PBMC proliferation and increased levels of IL-10, IL-1β and IFNγ measured by an MSD multiplex assay. Together, these in vitro findings indicate that cGAMP can act as a danger signal that matures human DCs, potentially released by necroptotic cells, supporting further exploration in an immunotherapy context.
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