Medizinische Klinik und Poliklinik II
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In acute graft-versus-host disease (GVHD) alloreactive donor T cells selectively damage skin, liver, and the gastrointestinal tract while other organs are rarely affected. The mechanism of this selective target tissue infiltration is not well understood. We investigated the importance of alloantigen expression for the selective organ manifestation by examining spatiotemporal changes of cellular and molecular events after allogeneic hematopoietic cell transplantation (allo-HCT). To accomplish this we established a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized protocols for antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) and T cell responses in Peyer’s patches following allo-HCT. In addition, we proofed that LSFM is suitable to map individual T cell subsets after HCT and detected rare cellular events. We employed this versatile technique to study the role of alloantigen expression for the selective organ manifestation after allo-HCT. Therefore, we used a T cell receptor (TCR) transgenic mouse model of GVHD that targets a single peptide antigen and thereby mimics a major histocompatibility complex (MHC)-matched single antigen mismatched (miHAg-mismatched) HCT. We transplanted TCR transgenic (OT-I) T cells into myeloablatively conditioned hosts that either express the peptide antigen ovalbumin ubiquitously (βa-Ova) or selectively in the pancreas (RIP-mOva), an organ that is normally not affected by acute GVHD. Of note, at day+6 after HCT we observed that OT-I T cell infiltration occurred in an alloantigen dependent manner. In βa-Ova recipients, where antigen was ubiquitously expressed, OT-I T cells infiltrated all organs and were not restricted to gastrointestinal tract, liver, and skin. In RIP-mOva recipients, where cognate antigen was only expressed in the pancreas, OT-I T cells selectively infiltrated this organ that is usually spared in acute GVHD. In conditioned RIP-mOva the transfer of 100 OT-I T cells sufficed to effectively infiltrate and destroy pancreatic islets resulting in 100% mortality. By employing intact tissue LSFM in RIP-mOva recipients, we identified very low numbers of initial islet infiltrating T cells on day+4 after HCT followed by a massive T cell migration to the pancreas within the following 24 hours. This suggested an effective mechanism of effector T cell recruitment to the tissue of alloantigen expression after initial antigen specific T cell encounter. In chimeras that either expressed the model antigen ovalbumin selectively in hematopoietic or in parenchymal cells only, transplanted OT-I T cells infiltrated target tissues irrespective of which compartment expressed the alloantigen. As IFN-γ could be detected in the serum of transplanted ovalbumin expressing recipients (βa-Ova, βa-Ova-chimeras and RIP-mOva) at day+6 after HCT, we hypothesized that this cytokine may be functionally involved in antigen specific OT-I T cell mediated pathology. In vitro activated OT-I T cells responded with the production of IFN-γ upon antigen re-encounter suggesting that IFN-γ might be relevant in the alloantigen dependent organ infiltration of antigen specific CD8+ T cell infiltration after HCT. Based on these data we propose that alloantigen expression plays an important role in organ specific T cell infiltration during acute GVHD and that initial alloreactive T cells recognizing the cognate antigen propagate a vicious cycle of enhanced T cell recruitment that subsequently culminates in the exacerbation of tissue restricted GVHD.
Although the field of fungal infections advanced tremendously, diagnosis of invasive pulmonary aspergillosis (IPA) in immunocompromised patients continues to be a challenge. Since IPA is a multifactorial disease, investigation from different aspects may provide new insights, helpful for improving IPA diagnosis. This work aimed to characterize the human immune response to Aspergillus fumigatus in a multilevel manner to identify characteristic molecular candidates and risk factors indicating IPA, which may in the future support already established diagnostic assays. We combined in vitro studies using myeloid cells infected with A. fumigatus and longitudinal case-control studies investigating patients post allogeneic stem cell transplantation (alloSCT) suffering from IPA and their match controls.
Characteristic miRNA and mRNA signatures indicating A. fumigatus-infected monocyte-derived dendritic cells (moDCs) demonstrated the potential to differentiate between A. fumigatus and Escherichia coli infection. Transcriptome and protein profiling of alloSCT patients suffering from IPA and their matched controls revealed a distinctive IPA signature consisting of MMP1 induction and LGAL2 repression in combination with elevated IL-8 and caspase-3 levels. Both, in vitro and case-control studies, suggested cytokines, matrix-metallopeptidases and galectins are important in the immune response to A. fumigatus. Identified IPA characteristic molecular candidates are involved in numerous processes, thus a combination of these in a distinctive signature may increase the specificity. Finally, low monocyte counts, severe GvHD of the gut (grade ≥ 2) and etanercept administration were significantly associated with IPA diagnosis post alloSCT. Etanercept in monocyte-derived macrophages (MDM) infected with A. fumigatus downregulates genes involved in the NF-κB and TNF-α pathway and affects the secretion of CXCL10.
Taken together, identified characteristic molecular signatures and risk factors indicating IPA may in the future in combination with established fungal biomarkers overcome current diagnostic challenges and help to establish tailored antifungal therapy. Therefore, further multicentre studies are encouraged to evaluate reported findings.
Mesenteric lymph nodes (mLNs) are sentinel sites of enteral immunosurveillance and immune homeostasis. Immune cells from the gastrointestinal tract (GIT) are constantly recruited to the mLNs in steady-state and under inflammatory conditions resulting in the induction of tolerance and immune cells activation, respectively. Surgical dissection and transplantation of lymph nodes (LN) is a technique that has supported seminal work to study LN function and is useful to investigate resident stromal and endothelial cell biology and their cellular interactions in experimental disease models. Here, we provide a detailed protocol of syngeneic mLN transplantation and report assays to analyze effective mLN engraftment in congenic recipients. Transplanted mLNs allow to study T cell activation and proliferation in preclinical mouse models. Donor mLNs proved viable and functional after surgical transplantation and regenerated blood and lymphatic vessels. Immune cells from the host completely colonized the transplanted mLNs within 7-8 weeks after the surgical intervention. After allogeneic hematopoietic cell transplantation (allo-HCT), adoptively transferred allogeneic CD4+ T cells from FVB/N (H-2q) mice homed to the transplanted mLNs in C57BL/6 (H-2b) recipients during the initiation phase of acute graft-versus-host disease (aGvHD). These CD4+ T cells retained full proliferative capacity and upregulated effector and gut homing molecules comparable to those in mLNs from unmanipulated wild-type recipients. Wild type mLNs transplanted into MHCII deficient syngeneic hosts sufficed to activate alloreactive T cells upon allogeneic hematopoietic cell transplantation, even in the absence of MHCII+ CD11c+ myeloid cells. These data support that orthotopically transplanted mLNs maintain physiological functions after transplantation. The technique of LN transplantation can be applied to study migratory and resident cell compartment interactions in mLNs as well as immune reactions from and to the gut under inflammatory and non-inflammatory conditions.
Diverse roles of B cells in the pathophysiology of rheumatoid arthritis are now well established. B cells contribute to autoimmunity by producing autoantibodies, processing autoantigen and the production of different cytokines which are involved in the inflammatory cascade. Therefore approaches to target B lymphocytes directly or indirectly are developed for clinical practice to treat autoimmune diseases including rheumatoid arthritis. Transient B cell depletion by rituximab (anti-CD20 antibody) has gained prime importance in recent years. Meanwhile anti-CD20 mediated transient B cell depletion therapy is now used with clinical efficiency in the treatment of patients with rheumatoid arthritis. Rituximab induces noteworthy changes in the homeostasis of peripheral B cell subpopulations during the repletion phase with emerging immature B cells in peripheral blood followed by normalization of the naïve B cell pool and a longterm delay in memory B cell subsets in patients with rheumatoid arthritis. Particularly IgD+CD27+ memory B cells repopulate very slowly during B cell regeneration. In a prospective clinical study, our laboratory has shown that the overall number of memory B cells correlates well to the duration of clinical response to rituximab. Little is known about the particular molecular changes in the memory B cell repertoire after rituximab therapy. To better understand peripheral memory B cell subsets, we explored in detail the somatic mutational frequency and pattern of Ig-VH3 gene rearrangements by using a single B cell sorting technique followed by nested PCR before and up to 6 years after rituximab therapy in 18 RA patients. We compared rituximab inflicted dynamics of mutational acquisition to memory B cell repopulation in 4 healthy donors and 6 non RA patients undergoing high dose chemotherapy followed by autologous or allogeneic stem cell transplantation (SCT). Firstly we analyzed the peripheral composition of memory B cell subsets. The phenotypic analysis of peripheral pre-switch (IgD+CD27+) and post-switch (IgD-CD27+) memory B cells did not reveal any quantitative differences in RA patients prior to B cell depletion therapy compared to healthy donors. However extending those studies in directly analysing the B cell immunoglobulin receptor from individual B cells of RA patients and healthy controls brought interesting results. Pre-switched and post-switched memory B cells showed a highly significant difference in the amount of mutations/sequence. The population of IgD+CD27+ memory B cells is comprised of non-mutated, low and highly mutated (median= 9 mutations/ sequence) rearranged Ig receptors whereas the IgD-CD27+ memory B cell compartment shows quite uniformly highly mutated (median 18 mutations/ sequence) sequences indicating a significant difference between these two groups (mutational frequencies 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Profound changes were noted in the re-emerging pre-switch memory B cells (IgD+/ CD27+) after transient B cell depletion with rituximab. These cells showed over a time period of 6 years after treatment with rituximab significantly delayed acquisition of mutations in Ig receptors on the single B cell level. One year after a single course of rituximab 84% of single repopulating IgD+/CD27+ B cells were unmutated and no highly mutated Ig-VH gene rearrangements were found(P=0.0001). Over time increasing numbers of mutations could be detected i-e 7.8% during 2nd year of regeneration (P=0.0001), 14% after 4 years (n=2). Nevertheless even 6 years after rituximab, VH mutations in IgD+ memory B cells were still reduced with 27% highly mutated sequences compared to 52% pre therapy(P=0.0001). Post-therapy analysis of CDR3 length of regenerated IgD+ memory B cells revealed increased CDR3 length which also correlates well with elevated number of non-mutated VH gene rearrangements observed during repletion phase. In comparison patients undergoing high dose chemotherapy followed by allogeneic stem cell transplantation repopulated IgD+ memory cells earlier with higher numbers of mutations in IgD+ memory B cells. One year after transplantation Ig receptors showed already 22% highly mutated and 42 % unmutated VH rearrangements. These findings indicated that anti-CD20 mediated B cell depletion seems not only to delay the production of pre-switch memory B cells but also significantly affects the acquisition of mutations in the IgD+ memory B cell pool. In contrary to the mutational pattern of IgD+ memory B cells after rituximab class switched memory B cells repopulate in the periphery with quantitatively normal mutations in their Ig receptors. Although the numeric replenishment of these recirculating class-switched memory B cells was also reduced after rituximab, we found no delay in quantitative acquisition of mutations also an increased proportion of IgA expressing B cells in this memory B cell subset was detected. Our data showed that post-therapy mutational targeting in RGYW/WRCY motifs were significantly increased as compared with that of pre-treatment (27% before rituximab vs. 43% after therapy, P=0.0003) indicating that affinity maturation may operate differently in class-switched memory B cells before and after B cell depletion. These results indicate a normal development process with an unimpaired mechanism of mutational acquisition in class-switched memory B cells. These data argue for different requirements to undergo somatic hypermutations in IgD+ memory B cells in comparison to class switched memory B cells. To conclude, our work has demonstrated for the first time a delayed acquisition of somatic hypermutations at single Ig receptor VH gene rearrangements of IgD+ memory B cells in comparison to class-switched memory B cells. These results demonstrate that IgD+ memory B cells are particularly susceptible to anti-CD20 treatment in patients with rheumatoid arthritis. In addition antigenic pressure and/or selection are substantially reduced by rituximab therapy which is basically not seen in the class-switched memory compartment. These data are in line with the hypothesis that IgD+ memory B cells have distinct requirements for activating their mutational machinery compared to class-switched memory B cells which recover normal mutations during regeneration phase. The results have implications in understanding the pathophysiology of memory B cell in rheumatoid arthritis and may be helpful in designing new targeted therapies.
After priming in Peyer's patches (PPs) and mesenteric lymph nodes (mLN) T- cells infiltrate the intestine through lymphatic draining and homing through the bloodstream. However, we found that in mouse models of acute graft-versus-host disease (GvHD), a subset of alloreactive T-cells directly migrates from PPs to the adjacent intestinal lamina propria (LP), bypassing the normal lymphatic drainage and vascular trafficking routes. Notably, this direct migration occurred in irradiated and unirradiated GvHD models, indicating that irradiation is not a prerequisite for this observed behavior.
Next, we established a method termed serial intravascular staining (SIVS) in mouse models to systematically investigate the trafficking and migration of donor T- cells in the early stages of acute GvHD initiation. We found that the direct migration of T-cells from PPs to LP resulted in faster recruitment of cells after allogeneic hematopoietic cell transplantation (allo-HCT). These directly migrating T-cells were found to be in an activated and proliferative state, exhibiting a TH1/TH17-like phenotype and producing cytokines such as IFN-γ and TNF-α. Furthermore, we observed that the directly migrating alloreactive T-cells expressed specific integrins (α4+, αE+) and chemokine receptors (CxCR3+, CCR5+, and CCR9+). Surprisingly, blocking these integrins and chemokine-coupled receptors did not hinder the direct migration of T- cells from PPs to LP, suggesting the involvement of alternative mechanisms. Previous experiments ruled out the involvement of S1PR1 and topographical features of macrophages, leading us to hypothesize that mediators of cytoskeleton reorganization, such as Coro1a, Dock2, or Cdc42, may play a role in this unique migration process.
Additionally, we observed that directly migrating T-cells created a local inflammatory microenvironment, which attracts circulating T-cells. Histological analysis confirmed that alloreactive PPs-derived T-cells and bloodborne T-cells colocalized. We employed two experimental approaches, including either photoconversion of T-cells in PPs or direct transfer of activated T-cells into the vasculature, to demonstrate this colocalization. We hypothesize that cytokines released by migrating T-cells, such as IFN-γ and TNF-α, may play a role in recruiting T-cells from the vasculature, as inhibiting chemokine-coupled receptors did not impair recruitment.
Articular cartilage defects represent one of the most challenging clinical problem for orthopedic surgeons and cartilage damage after trauma can result in debilitating joint pain, functional impairment and in the long-term development of osteoarthritis. The lateral cartilage-cartilage integration is crucial for the long-term success and to prevent further tissue degeneration. Tissue adhesives and sealants are becoming increasingly more popular and can be a beneficial approach in fostering tissue integration, particularly in tissues like cartilage where alternative techniques, such as suturing, would instead introduce further damage. However, adhesive materials still require optimization regarding the maximization of adhesion strength on the one hand and long-term tissue integration on the other hand. In vitro models can be a valuable support in the investigation of potential candidates and their functional mechanisms. For the conducted experiments within this work, an in vitro disc/ring model obtained from porcine articular cartilage tissue was established. In addition to qualitative evaluation of regeneration, this model facilitates the implementation of biomechanical tests to quantify cartilage integration strength. Construct harvesting for histology and other evaluation methods could be standardized and is ethically less questionable compared to in vivo testing. The opportunity of cell culture technique application for the in vitro model allowed a better understanding of cartilage integration processes.
Tissue bonding requires chemical or physical interaction of the adhesive material and the substrate. Adhesive hydrogels can bind to the defect interface and simultaneously fill the gap of irregularly shaped defect voids. Fibrin gels are derived from the physiological blood-clot formation and are clinically applied for wound closure. Within this work, comparisons of different fibrin glue formulations with the commercial BioGlue® were assessed, which highlighted the need for good biocompatibility when applied on cartilage tissue in order to achieve satisfying long-term integration. Fibrin gel formulations can be adapted with regard to their long-term stability and when applied on cartilage disc/ring constructs improved integrative repair is observable. The kinetic of repairing processes was investigated in fibrin-treated cartilage composites as part of this work. After three days in vitro cultivation, deposited extracellular matrix (ECM) was obvious at the glued interface that increased further over time. Interfacial cell invasion from the surrounding native cartilage was detected from day ten of tissue culture. The ECM formation relies on molecular factors, e.g., as was shown representatively for ascorbic acid, and contributes to increasing integration strengths over time. The experiments performed with fibrin revealed that the treatment with a biocompatible adhesive that allows cartilage neosynthesis favors lateral cartilage integration in the long term. However, fibrin has limited immediate bonding strength, which is disadvantageous for use on articular cartilage that is subject to high mechanical stress. The continuing aim of this thesis was to further develop adhesive mechanisms and new adhesive hydrogels that retain the positive properties of fibrin but have an increased immediate bonding strength.
Two different photochemical approaches with the advantage of on-demand bonding were tested. Such treatment potentially eases the application for the professional user. First, an UV light induced crosslinking mechanism was transferred to fibrin glue to provide additional bonding strength. For this, the cartilage surface was functionalized with highly reactive light-sensitive diazirine groups, which allowed additional covalent bonds to the fibrin matrix and thus increased the adhesive strength. However, the disadvantages of this approach were the multi-step bonding reactions, the need for enzymatic pretreatment of the cartilage, expensive reagents, potential UV-light damage, and potential toxicity hazards. Due to the mentioned disadvantages, no further experiments, including long-term culture, were carried out. A second photosensitive approach focused on blue light induced crosslinking of fibrinogen (RuFib) via a photoinitiator molecule instead of using thrombin as a crosslinking mediator like in normal fibrin glue. The used ruthenium complex allowed inter- and intramolecular dityrosine binding of fibrinogen molecules. The advantage of this method is a one-step curing of fibrinogen via visible light that further achieved higher adhesive strengths than fibrin. In contrast to diazirine functionalization of cartilage, the ruthenium complex is of less toxicological concern. However, after in vitro cultivation of the disc/ring constructs, there was a decrease in integration strength. Compared to fibrin, a reduced cartilage synthesis was observed at the defect. It is also disadvantageous that a direct adjustment of the adhesive can only be made via protein concentration, since fibrinogen is a natural protein that has a fixed number of tyrosine binding sites without chemical modification.
An additional cartilage adhesive was developed that is based on a mussel-inspired adhesive mechanism in which reactivity to a variety of substrates is enabled via free DOPA amino acids. DOPA-based adhesion is known to function in moist environments, a major advantage for application on water-rich cartilage tissue surrounded by synovial liquid. Reactive DOPA groups were synthetically attached to a polymer, here POx, to allow easy chemical modifiability, e.g. insertion of hydrolyzable ester motifs for tunable degradation. The possibility of preparing an adhesive hybrid hydrogel of POx in combination with fibrinogen led to good cell compatibility as was similarly observed with fibrin, but with increased immediate adhesive strength. Degradation could be adjusted by the amount of ester linkages on the POx and a direct influence of degradation rates on the development of integration in the in vitro model could be shown.
Hydrogels are well suited to fill defect gaps and immediate integration can be achieved via adhesive properties. The results obtained show that for the success of long-term integration, a good ability of the adhesive to take up synthesized ECM components and cells to enable regeneration is required. The degradation kinetics of the adhesive must match the remodeling process to avoid intermediate loss of integration power and to allow long-term firm adhesion to the native tissue.
Hydrogels are not only important as adhesives for smaller lesions, but also for filling large defect volumes and populating them with cells to produce tissue engineered cartilage. Many different hydrogel types suitable for cartilage synthesis are reported in the literature. A long-term stable fibrin formulation was tested in this work not only as an adhesive but also as a bulk hydrogel construct. Agarose is also a material widely used in cartilage tissue engineering that has shown good cartilage neosynthesis and was included in integration assessment. In addition, a synthetic hyaluronic acid-based hydrogel (HA SH/P(AGE/G)) was used. The disc/ring construct was adapted for such experiments and the inner lumen of the cartilage ring was filled with the respective hydrogel. In contrast to agarose, fibrin and HA-SH/P(AGE/G) gels have a crosslink mechanism that led to immediate bonding upon contact with cartilage during curing. The enhanced cartilage neosynthesis in agarose compared to the other hydrogel types resulted in improved integration during in vitro culture. This shows that for the long-term success of a treatment, remodeling of the hydrogel into functional cartilage tissue is a very high priority. In order to successfully treat larger cartilage defects with hydrogels, new materials with these properties in combination with chemical modifiability and a direct adhesion mechanism are one of the most promising approaches.
Die invasive Aspergillose (IA) z¨ ahlt zu den seltenen, bei immunsupprimierten Patienten
jedoch mit einer hohen Letalit¨ at verbundenen Infektionskrankheiten. Sie wird, wie alle
Aspergillosen, durch den humanpathogenen Schimmelpilz Aspergillus fumigatus ausgel¨ ost.
Bis heute ist die oft nicht effektive Therapie einer IA mit hohen Nebenwirkungen und
Kosten verbunden. Die Entwicklung von Pathogen-spezifischen Immuntherapien soll durch
die Forschung im Bereich der immunologischen Infektionsbiologie vorangetrieben werden.
Fur neuen Erkenntnisse wird die Interaktion von humanen Immunzellen mit ¨ A. fumigatus
analysiert.
In der vorliegenden Studie wurde mit dendritischen Zellen (DCs) gearbeitet, da diese
Pilzmorphologien von A. fumigatus phagozytieren k¨ onnen und uber Antigenpr ¨ ¨ asentation
das adaptive Immunsystem aktivieren. Es wurden aus humanen Monozyten differenzierte DCs (moDCs) verwendet, mit welchen viele Forschergruppen aufgrund ihrer verfugbar ¨
großen Anzahl arbeiten. Allerdings dauert die Generierung von moDCs funf Tage. Aus ¨
dem peripheren Blut entstammende CD1c-positive myeloide DCs (mDCs) oder CD303-
positive plasmazytoide DCs (pDCs) k¨ onnen dagegen direkt nach der Isolation verwendet
werden. Die beiden DC-Populationen werden aus verschiedenen Vorl¨ auferzellen des h¨ amatopoetischen Systems im Knochenmark gebildet. Ihr Ph¨ anotyp und ihre Immunfunktionen
unterscheiden sich untereinander und auch von denen der moDCs.
In Interaktionsstudien konnte analysiert werden, dass die drei verwendeten DC-Subtypen
(moDCs, mDCs, pDCs) unterschiedlich auf A. fumigatus reagieren. moDCs und mDCs reiften in direktem Kontakt zu Aspergillus, sie sekretierten ein relativ ¨ ahnliches Zytokinprofil
und exprimierten die bekannten Aspergillus-Rezeptoren Dectin-1, TLR2 und TLR4. Im
Kontrast dazu verblieben pDCs trotz Aspergillus-Kontakt unreif und sekretierten nahezu
keine Zytokine. Da moDCs und mDCs eine Immunreaktion auf den Pilz zeigten, wurden
sie mit verschiedenen Aspergillus-Antigenen beladen und n¨ aher untersucht.
Bevor verschiedene Aspergillus-Antigene zur Beladung der DCs eingesetzt werden konnten, wurden diese analysiert und aufgereinigt. Hierfur wurde ein routinierter Arbeitsprozess ¨
etabliert. Zwei der vier verfugbaren Proteinantigene waren mit Endotoxin kontaminiert. ¨
Da schon geringe Mengen an Endotoxinen auf DCs einen stimulatorischen Effekt ausubten, ¨
wurden die Proteine mittels Affinit¨ atschromatografie von den verunreinigenden Endotoxinen befreit.
In den Stimulationsexperimenten wirkten die beiden Proteinantigene CcpA und SHMT
immunogen auf moDCs und mDCs. CcpA induzierte eine st¨ arkere Maturierung und Zytokinfreisetzung als SHMT. Auff¨ allig war, dass mDCs im Vergleich zu moDCs die Expression
von MHC Klasse II st¨ arker erh¨ ohten und mehr IL18 freisetzten. Die mit CcpA oder SHMT
beladenen moDCs und mDCs aktivierten autologe T-Zellen zur IFNg Sekretion und zur
Proliferation. Zudem wurden durch die beiden Proteine Aspergillus-spezifische, CD154-
positive T-Zellen induziert. Diese Aspergillus-spezifische Immunogenit¨ at von CcpA und
SHMT macht die beiden Proteine zu interessanten Kandidaten fur einen Vakzinierungs- ¨
Cocktail einer DC-Immuntherapie. Aspergillus Lysat induzierte als weiteres Antigen eine
T-Zell Immunantwort mit CD154-positiven T-Zellen. Zudem war die Proliferation und
IFNg Sekretion von T-Zellen induziert, obwohl moDCs und mDCs nicht reiften und nur
wenige Zytokine sekretierten. Die beiden Aspergillus-Proteine CpcB und fg-gap induzierten die Reifung und Zytokinsekretion von moDCs und mDCs nicht. Demzufolge sind CpcB
und fg-gap fur eine DC-Immuntherapie nicht empfehlenswert. ¨
Ein Vakzinierungs-Cocktail enth¨ alt in der Regel Adjuvantien, welche die Immunreaktion
verst¨ arken. Adjuvante Effekte auf moDCs konnten die getesteten Aspergillus-RezeptorLiganden Zymosan, Pam3CSK4, LPS ultrapur und R848 ausl¨ osen. Hyalurons¨ aure konnte keine Verbesserung der Reifung oder Vitalit¨ at von moDCs und mDCs bewirken. Die
Antigen-bedingte Reifung der DCs fur n ¨ ¨ otige Restimulationen w¨ ahrend der Therapie konnte mittels einer tiefgekuhlten Lagerung in CryoStor Einfriermedium stabil beibehalten ¨
werden.
Die beiden immunogenen Aspergillus-Proteine, die adjuvanten Rezeptor-Agonisten und
die stabile Lagerung in CryoStor k¨ onnen als elementare Grundsteine fur einen Vakzinierungs- ¨
Cocktail einer anti-fungalen DC-Immuntherapie mit moDCs oder mDCs angesehen werden.
Polymorphonuclear neutrophils (PMNs) are phagocytic cells of the innate immune system that efficiently kill bacteria. However, they also have regulatory effects on other immune cells and contribute to immunosuppression in cancer, which worsens the outcome. In particular, this has been demonstrated for a subset of granulocytic cells called myeloid- derived suppressor cells (MDSCs), but its distinction from PMNs is controversial. Most authors have explored the suppressive effects of MDSCs on T cells, but recent data suggest that NK cells are also affected. NK cells are crucial for the combat of tumor cells, in particular leukemic cells. There is hardly data available on the interaction between NK cells and suppressive granulocytic cells. Therefore, the aim of this thesis was to explore the effects of MDSCs and PMNs on the NK cell function against the leukemia cell line K562.
In co-culture experiments, I demonstrate that granulocytic MDSCs and PMNs had similar effects on NK cell function and homeostasis. On the one hand, they positively influenced the survival and maturation of NK cells. On the other, they inhibited the activation, cytotoxicity and cytokine production of NK cells, both IFNγ and TNFα, in response to K562 target cells. Furthermore, I show a down-regulation of the activating receptor NKp30 on NK cells in the presence of MDSCs or PMNs, which may form part of the underlying suppressive mechanisms.
However, there is also evidence for the involvement of other molecules. Further investigations are needed to confirm a relevant suppression of NK cells by granulocytic cells in cancer patients, and to identify therapeutic targets. The recognition that regular PMNs have similar effects on NK cells as MDSCs could simplify future experiments, since MDSCs are heterogeneous and laborious to isolate and identify.
NKcells and granulocytes are among the first immune cells to reconstitute after hematopoietic stem cell transplantation, and NK cells may be particularly exposed to suppressive effects of granulocytes this scenario. Modulating these suppressive effects of granulocytes on NK cells therapeutically may yield a better NK cell function and an improved cancer prognosis.

Wilms tumor protein 1 (WT1) is a suitable target to develop an immunotherapeutic approach against high risk acute myeloid leukemia (AML), particularly their relapse after allogeneic hematopoietic stem cell transplantation (HSCT). As an intracellular protein traversing between nucleus and cytoplasm, recombinant expression of WT1 is difficult. Therefore, an induction of WT1-specific T-cell responses is mostly based on peptide vaccination as well as dendritic cell (DC) electroporation with mRNA encoding full-length protein to mount WT1-derived peptide variations presented to T cells. Alternatively, the WT1 peptide presentation could be broadened by forcing receptor-mediated endocytosis of DCs.
In this study, antibody fusion proteins consisting of an antibody specific to the human DEC205 endocytic receptor and various fragments of WT1 (anti-hDEC205-WT1) were generated for a potential DC-targeted recombinant WT1 vaccine. Anti-hDEC205-WT1 antibody fusion proteins containing full-length or major parts of WT1 were not efficiently expressed and secreted due to their poor solubility and secretory capacity. However, small fragment-containing variants: anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were obtained in good yields.
Since three of these fusion proteins contain the most of the known immunogenic epitopes in their sequences, the anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were tested for their T-cell stimulatory capacities. Mature monocyte-derived DCs loaded with anti-hDEC205-WT191-138 could induce ex vivo T-cell responses in 12 of 16 blood samples collected from either healthy or HSC transplanted individuals compared to included controls (P < 0.01). Furthermore, these T cells could kill WT1-overexpressing THP-1 leukemia cells in vitro after expansion.
In conclusion, alongside proving the difficulty in expression and purification of intracellular WT1 as a vaccine protein, our results from this work introduce an alternative therapeutic vaccine approach to improve an anti-leukemia immune response in the context of allogeneic HSCT and potentially beyond.
The human pathogen Aspergillus (A.) fumigatus is a fungal mold that can cause severe infections in immunocompromised hosts. Pathogen recognition and immune cell cross-talk are essential for clearing fungal infections efficiently. Immune cell interactions in particular may enhance individual cell activation and cytotoxicity towards invading pathogens.
This study analyzed the reciprocal cell activation of natural killer (NK) cells and monocyte-derived dendritic cells (moDCs) after stimulation with A. fumigatus cell wall fractions and whole-cell lysates. Furthermore, the impact of the on moDCs expressed fungal receptors Dectin-1 and TLR-2 on NK cell activation was analyzed. Stimulation of moDCs with ligands for Dectin-1 and TLR-2 and transfer of soluble factors on autologous NK cells showed that moDCs could induce NK cell activation solely by secreting factors. In summary, both cell types could induce reciprocal cell activation if the stimulated cell type recognized fungal morphologies and ligands. However, moDCs displayed a broader set of A. fumigatus receptors and, therefore, could induce NK cell activation when those were not activated by the stimulus directly.
Consequently, new fungal receptors should be identified on NK cells. The NK cell characterization marker CD56 was reduced detected in flow cytometry after fungal co-culture. Notably, this decreased detection was not associated with NK cell apoptosis, protein degradation, internalization, or secretion of CD56 molecules. CD56 was shown to tightly attach to hyphal structures, followed by its concentration at the NK-A. fumigatus interaction site. Actin polymerization was necessary for CD56 relocalization, as pre-treatment of NK cells with actin-inhibitory reagents abolished CD56 binding to the fungus. Blocking of CD56 suppressed fungal mediated NK cell activation and secretion of the immune-recruiting chemokines MIP-1α, MIP-1β, and RANTES, concluding that CD56 is functionally involved in fungal recognition by NK cells.
CD56 binding to fungal hyphae was inhibited in NK cells obtained from patients during immune-suppressing therapy after allogeneic stem cell transplantation (alloSCT). Additionally, reduced binding of CD56 correlated with decreased actin polymerization of reconstituting NK cells challenged with the fungus. The immune-suppressing therapy with corticosteroids negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES in NK cells after fungal stimulation ex vivo. Similar results were obtained when NK cells from healthy donors were treated with corticosteroids prior to fungal co-culture. Thus, corticosteroids were identified to have detrimental effects on NK cell function during infection with A. fumigatus.