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Im Rahmen der vorliegenden Arbeit wurde der Einfluss von MV auf die Expression und Nutzung verschiedener Rezeptoren auf Dendritischen Zellen (DC) untersucht. Dazu wurden DC in vitro aus Monozyten gewonnen und mit IL- 4 und GM-CSF ausdifferenziert. In der Arbeit wurden das Wildtypvirus WTF und der Vakzinestamm ED eingesetzt. Im ersten Teil der Arbeit wurde die Expression der Chemokinrezeptoren 5 (CCR5) und 7 (CCR7) auf MV-infizierten DC-Kulturen sowie das Migrationsverhalten der DC untersucht. Die Expression von CCR5 ist abhängig vom Reifungszustand der DC. Während unreife DC CCR5 exprimieren, verringert sich die Expression im Zuge der Ausreifung, während die Expression von CCR7 induziert wird. In der vorliegenden Arbeit konnte gezeigt werden, dass die MV-Infektion einer DC-Kultur die Expression von CCR5 nicht beeinflusst, obwohl die Expression anderer charakteristischer Reifungsmarker erhöht wird. Weiterhin konnte die Expression von CCR7 durch eine Infektion der DC-Kulturen mit MV nicht induziert werden. Die Ergebnisse der durchgeführten durchflusszytometrischen Analysen zur Rezeptorexpression wurden in Chemotaxis-Assays eingehender untersucht. DC aus MV-infizierten Kulturen zeigten trotz anhaltender CCR5-Expression nur eine geringe Migration in Richtung des CCR5-Liganden CCL-3. Aufgrund der fehlenden CCR7- Expression konnte erwartungsgemäß keine Migration der eingesetzten DC gegenüber dem CCR7-Liganden CCL-19 beobachtet werden. Weiterhin konnten Unterschiede im induzierten Chemokinmuster in MV-infizierten DCKulturen im Vergleich zu LPS-ausgereiften oder mit einer Mockpräparation behandelten DC nachgewiesen werden. Kurz nach der Infektion konnte in MVinfizierten DC-Kulturen eine reduzierte Menge CXCL-8 und CCL-20 sowohl auf mRNA Ebene als auch auf Proteinebene detektiert werden. In weiteren Experimenten konnte eine verminderte Migration von T-Zellen auf Zusammenfassung 152 Zellkulturüberstände aus infizierten DC-Kulturen im Vergleich zu Überständen aus LPS-behandelten DC-Kulturen festgestellt werden. Im zweiten Teil der vorgelegten Arbeit konnte MV als Ligand für das DCspezifische Oberflächenmolekül DC-SIGN (dendritic cell-specific intercellular adhesion molecule (ICAM)-3 grabbing non-integrin) identifiziert werden. Dabei konnte gezeigt werden, dass beide verwendeten Virusstämme an DC-SIGN binden können, DC-SIGN jedoch keinen Eintrittsrezeptor für MV darstellt. Weitere Analysen zeigten, dass DC-SIGN die Infektion, besonders bei geringen Viruskonzentrationen, verstärkt. In erster Linie zeigt die MV-Infektion unreifer DC die Bedeutung von DC-SIGN als Bindungsrezeptor für MV. Die hohe Expression von DC-SIGN auf unreifen DC ermöglicht eine effiziente effektive Infektion dieser Zellen. Auf reifen DC ist der Einfluss von DC-SIGN auf die Effektivität der Infektion der DC deutlich verringert. Im dritten Teil dieser Arbeit wurden die Kontaktzeiten zwischen DC und TZellen in dreidimensionalen Kollagenmatrices untersucht. Dabei wurde eine in etwa doppelt so lange Kontaktzeit zwischen MV-infizierten DC und durch Oxidative Mitogenese veränderten T-Zellen gegenüber LPS-behandelten DC bzw. MV-infizierten und nicht-modifizierten T-Zellen festgestellt. In den parallel durchgeführten Proliferationstests wurde eine reduzierte Proliferation der TZellen beobachtet, die mit MV-infizierten DC kokultiviert wurden. Im Gegensatz zu den kürzeren Kontakten zwischen LPS-behandelten DC und modifizierten TZellen waren die Kontakte zwischen MV-infizierten DC und modifizierten TZellen nicht-stimulatorisch.
Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.
Background: Stimulation of CD40 can augment anti-cancer T cell immune responses by triggering effective activation and maturation of antigen-presenting cells (APCs). Although CD40 agonists have clinical activity in humans, the associated systemic activation of the immune system triggers dose-limiting side-effects.
Methods: To increase the tumor selectivity of CD40 agonist-based therapies, we developed an approach in which soluble trimeric CD40L (sCD40L) is genetically fused to tumor targeting antibody fragments, yielding scFv: CD40L fusion proteins. We hypothesized that scFv: CD40L fusion proteins would have reduced CD40 agonist activity similar to sCD40L but will be converted to a highly agonistic membrane CD40L-like form of CD40L upon anchoring to cell surface exposed antigen via the scFv domain.
Results: Targeted delivery of CD40L to the carcinoma marker EpCAM on carcinoma cells induced dose-dependent paracrine maturation of DCs similar to 20-fold more effective than a non-targeted control scFv: CD40L fusion protein. Similarly, targeted delivery of CD40L to the B cell leukemia marker CD20 induced effective paracrine maturation of DCs. Of note, the CD20-selective delivery of CD40L also triggered loss of cell viability in certain B cell leukemic cell lines as a result of CD20-induced apoptosis.
Conclusions: Targeted delivery of CD40L to cancer cells is a promising strategy that may help to trigger cancer-localized activation of CD40 and can be modified to exert additional anti-cancer activity via the targeting domain.
Abstract
Background
HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases.
Results
As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde.
Conclusion
The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G.
Atherosclerosis is accepted to be a chronic inflammatory disease of the arterial vessel wall. Several cellular subsets of the immune system are involved in its initiation and progression, such as monocytes, macrophages, T and B cells. Recent research has demonstrated that dendritic cells (DCs) contribute to atherosclerosis, too. DCs are defined by their ability to sense and phagocyte antigens, to migrate and to prime other immune cells, such as T cells. Although all DCs share these functional characteristics, they are heterogeneous with respect to phenotype and origin. Several markers have been used to describe DCs in different lymphoid and non-lymphoid organs; however, none of them has proven to be unambiguous. The expression of surface molecules is highly variable depending on the state of activation and the surrounding tissue. Furthermore, DCs in the aorta or the atherosclerotic plaque can be derived from designated precursor cells or from monocytes. In addition, DCs share both their marker expression and their functional characteristics with other myeloid cells like monocytes and macrophages. The repertoire of aortic DCs in healthy and atherosclerotic mice has just recently started to be explored, but yet there is no systemic study available, which describes the aortic DC compartment. Because it is conceivable that distinct aortic DC subsets exert dedicated functions, a detailed description of vascular DCs is required. The first part of this thesis characterizes DC subsets in healthy and atherosclerotic mice. It describes a previously unrecognized DC subset and also sheds light on the origin of vascular DCs. In recent years, microRNAs (miRNAs) have been demonstrated to regulate several cellular functions, such as apoptosis, differentiation, development or proliferation. Although several cell types have been characterized extensively with regard to the miRNAs involved in their regulation, only few studies are available that focus on the role of miRNAs in DCs. Because an improved understanding of the regulation of DC functions would allow for new therapeutic options, research on miRNAs in DCs is required. The second part of this thesis focuses on the role of the miRNA cluster miR- 17~92 in DCs by exploring its functions in healthy and atherosclerotic mice. This thesis clearly demonstrates for the first time an anti-inflammatory and atheroprotective role for the miR17-92 cluster. A model for its mechanism is suggested.
The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAM\(^{low}\) CD103\(^{-}\) XCR1\(^{-}\) CD172a\(^{+}\) CD11b\(^{+}\) cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL‐10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4\(^{-/-}\) mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT‐II cells induced proliferation and IFN‐γ production that was similar to GM‐CSF‐generated BM‐DC and higher than Flt3L‐generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP‐derived ESAM\(^{low}\) cDC2 (cDC2B) development and survival in vitro.
Thymus-derived natural Foxp3\(^{+}\) CD4\(^{+}\) regulatory T cells (nTregs) play a key role in maintaining immune tolerance and preventing autoimmune disease. Several studies indicate that dendritic cells (DCs) are critically involved in the maintenance and proliferation of nTregs. However, the mechanisms how DCs manage to keep the peripheral pool at constant levels remain poorly understood. Here, we describe that the NF-κB/Rel family transcription factor RelB controls the frequencies of steady-state migratory DCs (ssmDCs) in peripheral lymph nodes and their numbers control peripheral nTreg homeostasis. DC-specific RelB depletion was investigated in CD11c-Cre × RelB\(^{fl/fl}\) mice (RelB\(^{DCko}\)), which showed normal frequencies of resident DCs in lymph nodes and spleen while the subsets of CD103\(^{-}\) Langerin\(^{-}\) dermal DCs (dDCs) and Langerhans cells but not CD103\(^{+}\) Langerin\(^{+}\) dDC of the ssmDCs in skin-draining lymph nodes were increased. Enhanced frequencies and proliferation rates were also observed for nTregs and a small population of CD4\(^{+}\) CD44\(^{high}\) CD25\(^{low}\) memory-like T cells (Tml). Interestingly, only the Tml but not DCs showed an increase in IL-2-producing capacity in lymph nodes of RelB\(^{DCko}\) mice. Blocking of IL-2 in vivo reduced the frequency of nTregs but increased the Tml frequencies, followed by a recovery of nTregs. Taken together, by employing RelB\(^{DCko}\) mice with increased frequencies of ssmDCs our data indicate a critical role for specific ssmDC subsets for the peripheral nTreg and IL-2\(^{+}\) Tml frequencies during homeostasis.
The diploid, polymorphic yeast Candida albicans is one of the most important human pathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within the human host for a long time. However, alterations in the host environment can render C. albicans virulent. In this review, we describe the immunological cross-talk between C. albicans and the human innate immune system. We give an overview in form of pairs of human defense strategies including immunological mechanisms as well as general stressors such as nutrient limitation, pH, fever etc. and the corresponding fungal response and evasion mechanisms. Furthermore, Computational Systems Biology approaches to model and investigate these complex interactions are highlighted with a special focus on game-theoretical methods and agent-based models. An outlook on interesting questions to be tackled by Systems Biology regarding entangled defense and evasion mechanisms is given.
Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction.
Background
Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.
Methodology/Principal
Findings Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.
Conclusions
Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.