The aim of my project was to examine the extent to which endogenous expression of a largely renal-specific antigen influences the repertoire in adulthood of autoreactive T cells specific to that antigen. The renal-specific antigen, human α3(IV)NC1, is the target of autoimmune attack in Goodpasture’s disease. This protein was expressed and purified in recombinant (using bacterial and mammalian cell expression systems) and purified in native (extracted from human tissue) forms. Transgenic mice were generated that express HLA-DR15 (associated with Goodpasture’s disease) as their sole MHC class II molecule, and for which α3(IV)NC1 can be endogenous or exogenous. The CD4 T cell responses of these mice were then tested following immunisation with α3(IV)NC1. In mice with endogenous expression of α3(IV)NC1 there were no consistent detectable proliferative T cell responses to any α3(IV)NC1 peptides in a set of overlapping peptides representative of the entire sequence. In the mice lacking endogenous α3(IV)NC1 there were consistent responses to the peptide α3(IV)NC1 136-150. This contains part of the peptide recognised by the most abundant autoreactive T cells in patients with acute Goodpasture’s disease. Therefore, the T cell responses seen in man to an endogenous (auto)antigen have similar fine specificity to those seen in mice responding to the same protein as a foreign antigen. This is surprising as one might expect self-tolerance in man to be most secure to such dominantly presented and immunogenic (in HLA DR15 mice) self peptides. However, recent work suggests that the peptide most commonly presented in humans is normally destroyed during antigen processing, giving a possible explanation for the lack of tolerance. Future work should study why tolerance is ineffective to this particular peptide, whether tolerance can be reinforced, these questions could be addressed using a transgenic mouse model that develops Goodpasture-like pathology. In addition, how processing is defective in Goodpasture’s disease could be explored by making antigen presenting hybridomas from patient samples or from the transgenic mouse line described within this thesis.

Dendritische Zellen (DCs) und Makrophagen sind spezialisierte antigenpräsentierende Zellen, die eigene und fremde Antigene prozessieren und mittels Haupthistokompatibilitätsmoleküle, humane Leukozytenantige (HLA) im Menschen, T-Zellen präsentieren, um Toleranzen zu induzieren oder T-Zell-vermittelte Immunantworten zu initiieren. Abhängig von ihrer Differenzierung haben sie spezifische Phänotypen und Funktionen undunterschiedliche Interaktionen mit Pathogenen, in dieser Arbeit durch Leishmania donovani (LD) repräsentiert, welche in Phagolysosomen der Makrophagen propagieren. Der Einfluss der Differenzierungszustände und von intrazelluläre Infektionen auf die Antigenprozessierung und -präsentation waren weitgehend undefiniert. Um hier Einblick zu gewinnen, haben wir die HLA-I-präsentierten Selbstpeptidome von menschlichen unreifen und reifen DCs, die aus der MUTZ3-Zelllinie generiert wurden, und LD-infizierte bzw. nicht-infizierte aus der THP1-Zelllinie generierte Makrophagen mittels Flüssigchromatographie-Tandem-Massenspektrometrie (LC-MS/MS), sowie die Proteasom-Zusammensetzung per RT-PCR und die HLA-Expression und Aktivierungszustände der Zellen per Durchflusszytometrie analysiert und verglichen. Wir fanden, dass die HLA-I-Selbstpeptidome der Zellen heterogen und individualisiert waren, von Nonapeptiden dominiert wurden und ähnliche HLA-Bindungsaffinitäten und Ankerreste aufwiesen. Sie stammten aus Quellenproteinen aus fast allen subzellulären Lokalisationen und mit unterschiedlichen zellulären Funktionen in ähnlichen Anteilen und schlossen Tumor-assoziierter Antigene (TAAs) ein. Die Persistenz der LD hatte keinen Einfluß auf den Aktivierungszustand der Makrophagen, verursachte aber eine weitgehende Veränderungen des Peptidoms, der HLA-Bindungsaffinitäten und Ankerreste, der Quellproteine einschließlich TAAs und der HLA- und Proteasom-Expression. / Dendritic cells (DCs) and macrophages are specialized antigen presenting cells that process self and foreign antigens and present them to T cells via major histocompatibility complex molecules, human leukocyte antigens (HLA) in humans, for induction of tolerance or initiation of T cell-mediated immune responses. Related to differentiation state, they have specific phenotypes and functions, and varied interactions with pathogens herein exemplified by Leishmania donovani (LD) that parasitize macrophages and propagate within their phagolysosomes. The impact of the differentiation state and intracellular infection on antigen processing and presentation by HLA class I remained undefined. To gain insight, we analyzed and compared the HLA-I self peptidomes of MUTZ3 cell line-derived human immature and mature DCs, and THP1 cell line-derived LD-infected and none-infected macrophages by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as proteasome compositions by quantitative RT-PCR, and HLA expression and cell activation states by flow cytometry. We found that the HLA I-presented self-peptidomes of the cells in the different states were heterogeneous and individualized, dominated by nonapeptides with similar HLA binding affinities and anchor residues. They were sampled from source proteins of almost all subcellular locations and from proteins involved in various cellular functions in similar proportion including tumour-associated antigens (TAAs). The persistence of LD within the macrophage, did not affect macrophage activation. However, its impact was observed in self-peptidome heterogeneity, HLA binding affinities, anchor residue preferences, source protein peptide sampling (including TAAs) and HLA and proteasome expression.