Analyse molekularer Mechanismen der Immundominanz MHC Klasse I-präsentierter Antigene bei Humaner Cytomegalovirus-Infektion

Das Humane Cytomegalovirus (HCMV) ist ein Erreger von großer klinischer Relevanz. Die HCMV-Infektion, die insbesondere bei immunsupprimierten Patienten mit hoher Morbidität und Mortalität assoziiert ist, wird vorwiegend durch CD8+-zytotoxische T-Lymphozyten (CTL) kontrolliert. Das Tegumentprotein pp65 und das immediate early 1-Protein (IE1) waren als die dominanten CTL-Antigene bekannt. Ziel dieser Arbeit war es, die zur Immundominanz des pp65 führenden molekularen Mechanismen aufzuklären und die Grundlagen für die Analyse der IE1-spezifischen Immunantwort zu erarbeiten. Durch Peptidimmunisierung HLA-A2-transgener Mäuse wurden hochaffine pp65-spezifische CTL-Klone generiert. Für die Generierung ähnlicher CTL-Klone gegen IE1 konnte erstmals ein konserviertes HLA-A2-bindendes Peptid identifiziert werden. Mit Hilfe der pp65-spezifischen CTL-Klone konnte gezeigt werden, dass das durch Viruspartikel in die Zelle eingebrachte pp65 die Erkennung infizierter Zellen durch CD8+-CTL vermittelt. Durch den Nachweis der außergewöhnlichen Stabilität von pp65 in der Zelle gelang es, eine hohe metabolische Umsatzrate als eine Ursache von Immundominanz auszuschließen. Dagegen hob die Blockierung des CRM1-vermittelten nukleären Exportweges durch Zugabe von Hemmstoffen oder Zutransfektion kompetitiver Inhibitoren die Erkennung des pp65 nahezu auf. Hiermit wurde erstmalig eine Abhängigkeit der Präsentation eines immundominanten nukleären Proteins vom nukleozytoplasmatischen Transport nachgewiesen. Die Erkenntnisse dieser Arbeit stellen die Grundlage für die detaillierte Analyse der Zusammenhänge zwischen nukleärem Export und Antigenpräsentation dar.

Nanoparticles for efficient uptake by human dendritic cells and T lymphocytes to be used in adoptive immunotherapy

Dendritic cells (DCs) are the most potent cell type for capture, processing, and presentation of antigens. They are able to activate naïve T cells as well as to initiate memory T-cell immune responses. T lymphocytes are key elements in eliciting cellular immunity against bacteria and viruses as well as in the generation of anti-tumor and anti-leukemia immune responses. Because of their central position in the immunological network, specific manipulations of these cell types provide promising possibilities for novel immunotherapies. Nanoparticles (NP) that have just recently been investigated for use as carriers of drugs or imaging agents, are well suited for therapeutic applications in vitro and also in vivo since they can be addressed to cells with a high target specificity upon surface functionalization. As a first prerequisite, an efficient in vitro labeling of cells with NP has to be established. In this work we developed protocols allowing an effective loading of human monocyte-derived DCs and primary antigen-specific T cells with newly designed NP without affecting biological cell functions. Polystyrene NP that have been synthesized by the miniemulsion technique contained perylenmonoimide (PMI) as a fluorochrome, allowing the rapid determination of intracellular uptake by flow cytometry. To confirm intracellular localization, NP-loaded cells were analyzed by confocal laser scanning microscopy (cLSM) and transmission electron microscopy (TEM). Functional analyses of NP-loaded cells were performed by IFN-γ ELISPOT, 51Chromium-release, and 3H-thymidine proliferation assays. In the first part of this study, we observed strong labeling of DCs with amino-functionalized NP. Even after 8 days 95% of DCs had retained nanoparticles with a median fluorescence intensity of 67% compared to day 1. NP loading did not influence expression of cell surface molecules that are specific for mature DCs (mDCs) nor did it influence the immunostimulatory capacity of mDCs. This procedure did also not impair the capability of DCs for uptake, processing and presentation of viral antigens that has not been shown before for NP in DCs. In the second part of this work, the protocol was adapted to the very different conditions with T lymphocytes. We used leukemia-, tumor-, and allo-human leukocyte antigen (HLA) reactive CD8+ or CD4+ T cells as model systems. Our data showed that amino-functionalized NP were taken up very efficiently also by T lymphocytes, which usually had a lower capacity for NP incorporation compared to other cell types. In contrast to DCs, T cells released 70-90% of incorporated NP during the first 24 h, which points to the need to escape from intracellular uptake pathways before export to the outside can occur. Preliminary data with biodegradable nanocapsules (NC) revealed that encapsulated cargo molecules could, in principle, escape from the endolysosomal compartment after loading into T lymphocytes. T cell function was not influenced by NP load at low to intermediate concentrations of 25 to 150 μg/mL. Overall, our data suggest that NP and NC are promising tools for the delivery of drugs, antigens, and other molecules into DCs and T lymphocytes.