An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be... Show moreAn exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fc gamma receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation. Show less
In the current thesis, we provide novel insights in antigen uptake, storage, processing, and sustained cross-presentation mechanisms in dendritic cells (DCs) in vitro and in vivo. We have studied... Show moreIn the current thesis, we provide novel insights in antigen uptake, storage, processing, and sustained cross-presentation mechanisms in dendritic cells (DCs) in vitro and in vivo. We have studied antigen handling functions by dendritic cells in three different antigen delivery routes: antibody targeting involving Fcγ receptors and complement factor C1q, C-type lectin receptor targeting, and toll-like receptor ligand targeting systems. Our data highlights that antigen storage in specialized compartments in DCs, despite the chosen uptake route, is beneficial for prolonged antigen cross-presentation by DCs and sustained T cell activation. Further in vivo studies in different antigen presenting cell (APC) subsets confirmed the presence of antigen storage compartments by isolating APC subsets after in vivo antigen uptake. Besides, we revealed a dominant role of C1q in antigen-antibody immune complex uptake and cross-presentation in vivo in contrast to the crucial role of Fcγ receptors in vitro. Furthermore, we demonstrated that autophagosomes have a negative impact on the storage of antigen in those specialized compartments and thereby affecting DC cross-presentation efficiency. With the current studies, we unraveled some mechanics of antigen processing in DCs which contribute to future vaccine designs against diseases such as cancer. Show less
Thiem, K.; Hoeke, G.; Zhou, E.C.; Hijmans, A.; Houben, T.; Boels, M.G.; ... ; Diepen, J.A. van 2019
Background: C-type lectin receptors, including Dectin-2, are pattern recognition receptors on monocytes and macrophages that mainly recognize sugars and sugar-like structures present on fungi.... Show moreBackground: C-type lectin receptors, including Dectin-2, are pattern recognition receptors on monocytes and macrophages that mainly recognize sugars and sugar-like structures present on fungi. Activation of C-type lectin receptors induces downstream CARD9 signalling, leading to the production of cytokines. We hypothesized that under hyperglycaemic conditions, as is the case in diabetes mellitus, glycosylated protein (sugar-like) structures activate C-type lectin receptors, leading to immune cell activation and increased atherosclerosis development. Methods: Low-density lipoprotein receptor-deficient mice were lethally irradiated and transplanted with bone marrow from control wild-type, Dectin-2(-/-) or Card9(-/-) mice. After 6 weeks of recovery, mice received streptozotocin injections (50 mg/g BW; 5 days) to induce hyperglycaemia. After an additional 2 weeks, mice were fed a Western-type diet (0.1% cholesterol) for 10 weeks. Results and Conclusion: Deletion of haematopoietic Dectin-2 reduced the number of circulating Ly6C(hi) monocytes, increased pro-inflammatory cytokine production, but did not affect atherosclerosis development. Deletion of haematopoietic CARD9 tended to reduce macrophage and collagen content in atherosclerotic lesions, again without influencing the lesion size. Deletion of haematopoietic Dectin-2 did not influence atherosclerosis development under hyperglycaemic conditions, despite some minor effects on inflammation. Deletion of haematopoietic CARD9 induced minor alterations in plaque composition under hyperglycaemic conditions, without affecting lesion size. Show less
