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Seit fast 30 Jahren wird das Immunpeptidom durch Elution von Peptiden aus HLA-Molekülen analysiert. Weltweit nutzen mittlerweile mehrere Institute und Unternehmen diese Methode für ein breites Spektrum an Untersuchungen, die von der simplen Identifizierung von HLA-Peptidmotiven für verschiedene Organismen bis hin zum Nachweis kryptischer krankheitsspezifischer Peptide reichen. Die Immunpeptidomik ist populärer denn je, seit sich die Medikamentenentwicklung in den letzten Jahren auf die positive Modulation des Immunsystems fokussiert hat. Die Zulassung der ersten Checkpoint-Antikörper leitete die Ära der Immuntherapie ein und spezifische Immuntherapien mit weniger Nebenwirkungen stehen nun im Blickpunkt. Das Anwendungsspektrum der Immunpeptidomik ist mittlerweile breit gefächert, dennoch enthält das Immunpeptidom immer noch eine große Fülle von Informationen, die darauf warten, entschlüsselt zu werden. Aktuell ist die Immunpeptidomik darin eingeschränkt, dass die große Anzahl von Peptiden, mit unterschiedlichen Affinitäten und Stabilitäten der Peptid-HLA-Komplexe, nicht optimal erfasst werden kann und daher unter anderem nur begrenzte Wiederfindungsraten möglich sind. Zu Beginn dieser Doktorarbeit gab es ungelöste Fragestellungen auf dem Gebiet der Immunpeptidomik, die in dieser Arbeit untersucht werden sollten: Ist es möglich, die Immunpeptidomik zu validieren und diese zuverlässig für klinische Studien und die Medikamentenentwicklung einzusetzen? Gibt es heute eine zuverlässige Methode zur Identifizierung von Peptidmotiven für Peptid-präsentierende MHC-Klasse-I-Allotypen, dem Grundstein für Epitopvorhersagen und Wirkstoffidentifizierungen? Ist es möglich, Peptide zur Klassifizierung von HLA-Allotypen oder zur Unterscheidung zwischen gesundem und bösartigem Gewebe zu verwenden? Können tumorspezifische Peptide mit dieser Omik-Technologie zuverlässig charakterisiert werden? In dieser Doktorarbeit wurde die immunpeptidomische Methode validiert, um die Zuverlässigkeit der LC-MS/MS-Peptid-Identifizierung zu gewährleisten, und es wurden alle erforderlichen Parameter der Europäischen Arzneimittel-Agentur und U. S. Food and Drug Administration untersucht. Darüber hinaus wurde ein aktualisiertes Protokoll für die Identifizierung von MHC-Liganden, die Entschlüsselung von Peptidmotiven und die Generierung von Matrizen für die Epitopvorhersage erstellt, das sowohl für monoallele Zellen als auch für multiallele Gewebe verwendet werden kann. Schließlich wurde eine Methode entwickelt, um allotypische Peptide zu identifizieren, die eine HLA-Typisierung ermöglichen. Diese Peptide können auch als interner Standard für die semi-quantitative Untersuchung der Tumorspezifität von Peptiden verwendet werden. Diese Methode wurde erfolgreich implementiert, um gewebe- und dignitätsspezifische Muster im Immunpeptidom zu identifizieren und die Dignität von immunpeptidomischen Proben zu bestimmen. For almost 30 years now, the immunopeptidome has been analyzed by eluting peptides from HLA molecules. This method has already been established in several institutes and companies worldwide and is now used for a wide range of investigations from the simple identification of HLA peptide motifs for different organisms to the detection of cryptic disease-specific peptides. The field of immunopeptidomics is more popular than ever as drug development has focused on the positive modulation of the immune system in recent years. Since the approval of the first checkpoint antibodies, the era of immunotherapy has been running and specific immunotherapies with fewer side effects are in the focus. There is a wide range of applications, yet, the immunopeptidome still contains a great wealth of information waiting to be deciphered. Currently, immunopeptidomics is limited in the identification of the large number of peptides with different affinities and stabilities of the peptide-HLA complexes. Therefore, amongst many other factors, only limited recovery rates are possible. When this doctoral thesis started, there were several unresolved questions in the field of immunopeptidomics that should be approached in this thesis: Is it possible to validate immunopeptidomics and use it reliably for clinical studies and drug development? Is there nowadays a reliable method to identify the peptide motif for peptide presenting MHC class I allotypes, the cornerstone for epitope predictions or active substance identification? Is it possible to use peptides to classify HLA allotypes or differentiate between healthy and malignant tissue? Can tumor-specific peptides be reliably characterized with this omic technology? In this doctoral thesis the immunopeptidomic method was validated to ensure the reliability of LC-MS/MS peptide identification and all required parameters of the European Medicines Agency (EMA) and Food and Drug Administration (FDA) were investigated. In addition, an updated protocol for the identification of MHC ligands, deconvolution of peptide motifs and generation of matrices for epitope prediction was established, which can be used for monoallelic cells as well as multiallelic tissue. Finally, a method was developed to identify allotypic peptides that allow HLA typing. These peptides can also be used as an internal standard for semi-quantitative investigation of the tumor specificity of peptides. The developed method was also successfully implemented to identify tissue and dignity specific patterns in the immunopeptidome and to determine the dignity of immunopeptidomic samples.

Biomaterials play an increasing role in clinical applications and regenerative medicine. A perfectly designed biomaterial should restore the function of damaged tissue without triggering an undesirable immune response, initiate self-regeneration of the surrounding tissue and gradually degrade after implantation. The immune system is well recognized to play a major role in influencing the biocompatibility of implanted medical devices. To obtain a better understanding of the effects of biomaterials on the immune response, we have developed a highly sensitive novel test system capable of examining changes in the immune system by biomaterial. Here, we evaluated for the first time the immunopeptidome, a highly sensitive system that reflects cancer transformation, virus or drug influences and passes these cellular changes directly to T cells, as a test system to examine the effects of contact with materials. Since monocytes are one of the first immune cells reacting to biomaterials, we have tested the influence of different materials on the immunopeptidome of the monocytic THP-1 cell line. The tested materials included stainless steel, aluminum, zinc, high-density polyethylene, polyurethane films containing zinc diethyldithiocarbamate, copper, and zinc sulfate. The incubation with all material types resulted in significantly modulated peptides in the immunopeptidome, which were material-associated. The magnitude of induced changes in the immunopeptidome after the stimulation appeared comparable to that of bacterial lipopolysaccharides (LPS). The source proteins of many detected peptides are associated with cytotoxicity, fibrosis, autoimmunity, inflammation, and cellular stress. Considering all tested materials, it was found that the LPS-induced cytotoxicity-, inflammation- and cellular stress-associated HLA class I peptides were mainly induced by aluminum, whereas HLA class II peptides were mainly induced by stainless steel. These findings provide the first insights into the effects of biomaterials on the immunopeptidome. A more thorough understanding of these effects may enable the design of more biocompatible implant materials using in vitro models in future. Such efforts will provide a deeper understanding of possible immune responses induced by biomaterials such as fibrosis, inflammation, cytotoxicity, and autoimmune reactions.

The potency of viral vector-based vaccines depends on their ability to induce strong transgene-specific immune response without triggering anti-vector immunity. Previously, Orf virus (ORFV, Parapoxvirus) strain D1701-V was reported as a novel vector mediating protection against viral infections. The short-lived ORFV-specific immune response and the absence of virus neutralizing antibodies enables repeated immunizations and enhancement of humoral immune responses against the inserted antigens. However, only limited information exists about the D1701-V induced cellular immunity. In this study we employed major histocompatibility complex (MHC) ligandomics and immunogenicity analysis to identify ORFV-specific epitopes. Using liquid chromatography-tandem mass spectrometry we detected 36 ORFV-derived MHC I peptides, originating from various proteins. Stimulated splenocytes from ORFV-immunized mice did not exhibit specific CD8+ T cell responses against the tested peptides. In contrast, immunization with ovalbumin-expressing ORFV recombinant elicited strong SIINFEKL-specific CD8+ T lymphocyte response. In conclusion, our data indicate that cellular immunity to the ORFV vector is negligible, while strong CD8+ T cell response is induced against the inserted transgene. These results further emphasize the ORFV strain D1701-V as an attractive vector for vaccine development. Moreover, the presented experiments describe prerequisites for the selection of T cell epitopes exploitable for generation of ORFV-based vaccines by reverse genetics.

n this work, we report on the prototyping of thin-film silicon tandem solar cells with periodic light-trapping textures. Our approach combines an industrial applicable nano-imprint process, for the fabrication of advanced light management concepts, with state-of-the-art thin film silicon solar cell technology. In a joint experimental effort between project partners of the European thin-film silicon project “Fast-Track”, we demonstrate that optimal periodic light-trapping nano-textures at the front-side of a multi-junction solar cell enhance the power conversion efficiency of the device in comparison to solar cells with conventional textured TCO (transparent conductive oxide) front contacts from 11.7% to 12.1%. This improved power conversion efficiency is mainly driven by an enhancement of the short-circuit current density through the use of periodic light-trapping nano-textures.

Etwa die Hälfte aller Solarzellen weltweit wird aus blockerstarrtem Silizium hergestellt. Derartige Blöcke weisen in ihren Außenbereichen eine verringerte Diffusionslänge der Minoritätsladungsträger auf. Um die Ursache dafür im Fall des bodennahen Bereichs zu bestimmen wurden zwei Spezialblöcke (ein Block mit reduzierter Bor-Dotierung und ein Block mit Phosphor-Dotierung) - u. a. mittels DLTS und FTIR - auf Kristalldefekte untersucht. Zusätzlich zu Dotierelementen (B, P, Al, As) wurden im Bodenbereich folgende Defekte nachgewiesen: Metalle: Fe, Cr Sauerstoffhaltige Defekte: Interstitieller Sauerstoff, Thermische Donatoren (TD), O1, O2 Stickstoffhaltige Defekte: NN-Paar, NNO-Komplex, Shallow Thermal Donors (STD) Ausgedehnte Defekte: Versetzungen, Ausscheidungen, Korngrenzen. Die Verteilung der flachen Donatoren (P, TD, STD, As) und Akzeptoren (B, Al) bestimmt den Widerstandsverlauf im bodennahen Bereich des Phosphor dotierten Spezialblocks. Das dortige Diffusionslängenprofil kann im Rahmen der Shockley-Read-Hall-Statistik erst durch eine Erhöhung des Minoritätseinfangquerschnitts für das Cr-Niveau (Faktor 5) bzw. für das STD-Niveau (Faktor 10) nachgezeichnet werden. Eisen, Versetzungen und Korngrenzen haben hier keinen wesentlichen Einfluss. In den untersten Millimetern des Spezialblocks müssen weitere Defekte hinzukommen, die die Diffusionslänge zusätzlich reduzieren; Thermische Donatoren und O1 und eventuell Ausscheidungen kommen dazu in Frage. Die sinngemäße Übertragung der Konzentrationsverläufe aus den beiden Spezialblöcken auf einen Block mit einer produktionsüblichen Dotierung ([B]≈1016/cm3) ergibt, dass in diesem Fall verschiedene Defekte (TD, STD, CrB und FeB) einen Beitrag zur Diffusionslängenreduktion im bodennahen Blockbereich liefern.

Funder: Deutschen Konsortium für Translationale Krebsforschung; funder-id: http://dx.doi.org/10.13039/501100012353, Funder: Natural and Medical Sciences Institute (D), Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek’s disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek’s disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek’s disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek’s disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek’s disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek’s disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek’s disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek’s disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans., This study shows that chicken MHC class II molecules present peptides from only a handful of the more than 100 genes of the oncogenic herpesvirus Marek’s disease virus, explaining the strong genetic association of chicken MHC with resistance and susceptibility to this and other economically-important pathogens.