T-Zellepitope von viralen und tumor-assoziierten Antigenen: Induktion und Analyse von antigen-spezifischen T Zellen

T cells are important effectors in the defense of human pathogens entering the organism. CD8+ T cells recognize peptides which are presented by MHC class I molecules and lyse cells which are infected by virus or intracellular pathogens. Moreover, they are able to destroy cancer cells. CD4+ T cells recognize peptides from exogenous proteins acquired by endocytosis or from internalized plasma membrane proteins which are presented on MHC class II. CD4+ T cells play an important role in the defense of bacteria and activation of the humoral immune response. The human cytomegalovirus (HCMV) is a cause of morbidity and mortality in immunocompromised persons. In contrast, in immunocompetent individuals HCMV is successfully controlled by specific CD8+ and CD4+ T cell responses. One aim of this thesis was the analysis of CD8+ and CD4+ T cell responses against HCMV to identify T cell epitopes which could be used for the development of a vaccine against the virus. Ex vivo analyses were performed with blood of healthy donors of different HLA-types using recombinant peptide/MHC I complexes and/or intracellular cytokine staining to characterize T cell epitopes of HCMV. New T cell epitopes of several HLA restrictions could be identified. Furthermore, some were analysed with respect to immunodominant features and hierarchies among T cell epitopes identified. Furthermore, in vitro experiments were used to identify new T cell epitopes, e.g. stimulation of PBMCs from healthy donors with peptide loaded dendritic cells (DCs). Knowledge of T cell epitopes and their immundominant features can be useful for the generation of antigen-specific T cells for adoptive immunotherapy or for the development of a peptide-based vaccine against HCMV. A further aim of this thesis was the induction and analysis of CD8+ and CD4+ T cell responses against tumor-associated antigens. MHC class II ligands identified by mass spectrometric methods were used to perform in vitro stimulations. For CD8+ T cell stimulation, artificial antigen presenting cells (aAPCs) were used, for generation of antigen specific CD4+ T cells, DCs were loaded with peptides and used as APCs. Several new helper T cell epitopes from different tumor-associated antigens were identified.Zur Abwehr humanpathogener Organismen sind T-Zellen unerlässlich. CD8+ T-Zellen erkennen Peptide, die auf MHC Klasse I präsentiert werden und stellen Abwehrreaktionen gegen Viren und intrazelluläre Erreger, aber auch gegen Krebs, bereit. CD4+ T-Zellen erkennen MHC Klasse II-präsentierte Peptide, die aus dem Inneren von Vesikeln stammen und spielen somit eine Rolle bei der Abwehr von Bakterien, aber auch bei der Anregung des humoralen Immunsystems über Aktivierung von B-Zellen. Das humane Cytomegalievirus (HCMV) verursacht bei Patienten mit geschädigtem oder nicht voll ausgebildetem Immunsystem häufig schwere bis tödlich verlaufende Infektionen. Bei immunkompetenten Individuen hingegen wird das Virus erfolgreich durch spezifische CD8+ und CD4+ T-Zellen kontrolliert. Ein Ziel dieser Arbeit war es, CD8+ und CD4+ T-Zellantworten gegen das humane Cytomegalievirus (HCMV) zu untersuchen, um T-Zellepitope für mögliche Impfstoffe zu identifizieren. Zur Charakterisierung von T-Zellepitopen aus HCMV wurden zum einen ex vivo-Analysen in gesunden Spendern verschiedener HLA-Typisierung unter Verwendung rekombinanter Peptid/MHC I-Komplexe oder intrazellulärer Zytokinfärbung durchgeführt. Neue T-Zellepitope zahlreicher HLA-Restriktionen konnten identifiziert werden. Einige wurden zusätzlich hinsichtlich ihrer Immundominanz oder Hierarchie untereinander beleuchtet. Auch in vitro-Experimente, beipielsweise die Stimulation von gesunden Spender-PBMCs mit Peptid-beladenen dendritischen Zellen (DCs), trugen zur Identifizierung neuer T-Zellepitope bei. Dieses Wissen um T-Zellepitope und ihre immundominanten Eigenschaften kann nützlich sein für die Generierung antigen-spezifischer T-Zellen für adoptive Immuntherapien oder für die Entwicklung einer Peptid-basierenden HCMV-Vakzine. Neben viralen T-Zellantworten war ebenso die Induktion und Analyse von CD8+ und CD4+ T-Zellantworten gegen tumorassoziierte Antigene Bestandteil dieser Arbeit. Mittels massenspektrometrischer Verfahren identifizierte MHC Klasse II-Liganden wurden für in vitro Stimulationen eingesetzt. Artifizielle Antigen-präsentierende Zellen wurden als Werkzeuge für die Stimulation von Antigen-spezifischen CD8+ T-Zellen genutzt, während CD4+ T-Zellen mittels Peptid-beladener dendritischer Zellen stimuliert wurden. Dies führte zur Identifizierung neuer T-Zellepitope aus verschiedenen Tumor-assoziierten Antigenen

Human CD4+ T Cell Epitopes from Vaccinia Virus Induced by Vaccination or Infection

Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4+ T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4+ T cell responses have been poorly characterized, and CD4+ T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens

An Expanded Self-Antigen Peptidome Is Carried by the Human Lymph As Compared to the Plasma

The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing.A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration.The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance

Identification of novel CD8+ T cell epitopes in human herpesvirus 6B U11 and U90

Human herpesvirus 6B (HHV6B) infects over 90% of the population, and normally establishes a latent infection, where episodes of reactivation are asymptomatic. However, in immunocompromised patients HHV6B reactivation is associated with high morbidity and mortality. Cellular immunotherapy has been utilised against other herpesvirus in immunocompromised settings. However, limited information on the immune response against HHV6B has hampered the development of immunotherapy for HHV6B-driven disease. In this study we have analysed the cellular immune response against four HHV6B antigens in a panel of 30 healthy donors. We show that the base-line level of T cell reactivity in peripheral blood is very low to undetectable. A short-term reactivation step enabled expansion of T cell responses, and all donors responded to at least 1 antigen, but more commonly 3 or 4. A hierarchy of immunogenicity was determined with antigens U90 and U54 being co-dominant, followed by U11 and U39. Putative CD8+ T cell epitopes were mapped to U90 and U11, predicted to be presented in the context of HLA-A1, A29, B39 and C6. T cells reactive against these novel epitopes were able to recognise virus-infected cells. Our data is supportive of the application and on-going development of T cell immunotherapy against HHV6B-driven disease in the immunocompromised host

Identification and HLA-Tetramer-Validation of Human CD4(+) and CD8(+) T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy

Precision mouse models with expanded tropism for human pathogens

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics

Towards Novel Process Designs for Enzymatic and Chemoenzymatic Syntheses of Nitrogen-based Heterocycles

Nastke A. Towards Novel Process Designs for Enzymatic and Chemoenzymatic Syntheses of Nitrogen-based Heterocycles. Bielefeld: Universität Bielefeld; 2023

Biotransformations with Imine Reductases: Design of a Practical Process Avoiding an Extractive Work-Up by Entrapment of Water and Enzymes in an Immobilized Phase

Nastke A, Gröger H. Biotransformations with Imine Reductases: Design of a Practical Process Avoiding an Extractive Work-Up by Entrapment of Water and Enzymes in an Immobilized Phase. European Journal of Organic Chemistry. 2023.A process concept for the asymmetric biocatalytic reduction of heterocyclic imines addressing the efficiency of the reaction as well as downstream-processing steps was studied by utilizing a "heterogenized aqueous phase", which contains the needed enzymes and cofactor within a superabsorber (polyacrylate) network, for the biotransformation. The immobilized biocatalytic system, which comprises an imine reductase (IRED), NADPH and an alcohol dehydrogenase for cofactor-recycling, enables to run the reaction in pure organic medium. Thus, instead of an extractive work-up as typical solution for biotransformations in aqueous medium, which, however, can be tedious due formation of emulsions, this type of IRED-catalyzed process leads to a simplified work-up consisting only of a decantation of the liquid organic reaction medium with the product from the heterogenized aqueous biocatalyst system. Exemplified for the (R)-enantioselective reduction of 1-methyl-3,4-dihydroisoquinoline by the IRED of Streptomyces viridochromogenes as a model reaction, a process was developed leading to 98 % conversion, 88 % yield and >99 % ee at a substrate concentration of 40 mM

Differenzierende und vergleichende Analysen über ausländische Studierende und WissenschaftlerInnen an Hochschulen in Deutschland

Im Mittelpunkt dieser Untersuchung stehen die Entwicklung und die Verteilung ausländischer Hochschulakteure. Dabei werden sowohl die Gruppe der Studierenden als auch das gesamte wissenschaftliche Personal und die Gruppe der ProfessorInnen in den Blick genommen. Es wird eine differenzierte Betrachtung und Analyse der Entwicklung zwischen 2005 und 2012 durchgeführt. Zunächst wird die zunehmende Relevanz der Internationalisierung für die Hochschulen verdeutlicht. Dabei geht es neben der Definition der Internationalisierung darum, zu beschreiben, welche unterschiedlichen Komponenten die Internationalisierung der Hochschulen umfasst. Zudem geht es um die Relevanz, die ausländischen Hochschulakteuren im Kontext der Internationalisierungsbestrebungen zukommt. Darauf aufbauend wird die Verteilung der ausländischen Studierenden auf die Bundesländer, die Hochschularten und die Fächergruppen im deutschen Hochschulwesen beschrieben und über den Ausländeranteil ermittelt, welchen Grad der Internationalität einzelne Bundesländer, Hochschularten und Fächergruppen anhand des Indikators Staatsangehörigkeit aufweisen. Darüber hinaus wird beschrieben, aus welchen Weltregionen ausländische Studierende stammen. Analog zur Gruppe der Studierenden findet eine Analyse für die Gruppe des wissenschaftlichen Personals mit ausländischer Staatsangehörigkeit und die Gruppen der ausländischen ProfessorInnen statt. Dies bildet die Grundlage zur Untersuchung der Gemeinsamkeiten und Unterschiede zwischen den Studierenden, dem wissenschaftlichen Personal und den ProfessorInnen sowohl im Hinblick auf die Entwicklung als auch durch den Vergleich der Ausländeranteile in den Bundesländern, Hochschularten und den Fächergruppen. Darüber hinaus werden Unterschiede bei der weltregionalen Herkunft der drei Gruppen analysiert. (DIPF/Autor