Regulation von T-Zell Apoptose und T-Zell Aktivierung durch kostimulatorische Signale

0\. Front page and table of contents 1 1\. Introduction 13 2\. Material 30 3\. Methods 33 4\. Results 41 5\. Discussion 63 6\. References 74 7\. Appendix 94 8\. Summary / Zusammenfassung 96Costimulation is essential for the induction of T lymphocyte proliferation and inhibition of activation-induced cell death (AICD). While signalling pathways activated through the costimulatory molecule CD28 are well defined, less is known about the molecular events induced by alternative costimulators. 4-1BB/CD137, a costimulatory member of the tumour necrosis factor receptor family, plays an important role during late primary T cell stimulation and restimulation. In the first part of my thesis, I demonstrate for the first time that inhibition of activation-induced cell death by exposure to the 4-1BB ligand involves up-regulation of the anti-apoptotic protein c-FLIPS. Inhibition of AICD by 4-1BB ligation and up-regulation of c-FLIPS and Bcl-xL were abolished by blocking the phosphatidylinositol 3 kinase or the AKT/protein kinase B, which also mediate CD28-induced inhibition of activation-induced cell death. My findings, therefore, demonstrate that costimulatory molecules, although belonging to different protein families and participating in distinct upstream signalling pathways, employ common downstream signalling pathways. These insights deepen our understanding of 4 -1BB-mediated signalling, bring them in relation to the well-investigated CD28-mediated signalling and thus help to elucidate molecular biological mechanisms of costimulation. The second part of my thesis focuses on tumour vaccination by the use of gene-modified cancer cells that provide costimulatory signals. Such cells have been successfully applied in preclinical animal models and are currently evaluated in a variety of clinical settings. In previous work, we demonstrated the efficacy of CD80 to promote tumour immunity in syngeneic murine models and to prevent deletion of activated T cells by AICD. In clinical trials, tumour cell vaccines are generally inactivated to avoid transfer of live tumour cells. Previous data indicated, however, that inactivation of tumour cells by lethal ionising irradiation abrogates tumour vaccination by CD80 expressing cells. Here, I compare living and irradiated allogeneic tumour cells regarding their capacity to induce T cell effector functions and their propensity to interfere with T cell AICD in vitro. Both, lethally irradiated and non-irradiated CD80 expressing tumour cells facilitated T cell proliferation, tumour cell lysis and interfered with AICD to a similar extent. In contrast, necrotic CD80 expressing tumour cells failed to costimulate T cell effector functions. These findings suggest that tumour cell vaccines should be inactivated by techniques that induce necrosis as little as possible. Taken together, my results broaden our understanding of T cell costimulation and the cell biological mechanisms of tumour cell vaccination. A thorough understanding of the mechanisms involved in T cell activation can lead to the development of more effective immunotherapies.Kostimulation ist essentiell für die Aktivierung der T-Zell-Effektorfunktionen und die Inhibition des aktivierungsinduzierten Zelltods (AICD). Während die Signalwege, die durch das kostimulatorische Molekül CD28 aktiviert werden, eingehend untersucht sind, ist wenig bekannt über die molekularen Ereignisse, die durch alternative Kostimulatoren induziert werden. 4-1BB/CD137, ein kostimulatorisches Mitglied der Tumor-Nekrose-Faktor-Rezeptor-Familie, spielt eine wichtige Rolle bei der späten primären T-Zell-Stimulation und bei der Restimulation. Im ersten Teil meiner Dissertation zeige ich erstmalig, dass die Inhibition des AICD durch 4-1BB-Ligand mit Expression des anti- apoptotischen Proteins c-FLIPS einhergeht. Die Inhibition des AICD und die Hochregulierung von c-FLIPS und Bcl-xL durch 4-1BB-Ligation wurden verhindert durch Hemmung der Phosphatidylinositol-3-Kinase oder AKT/Proteinkinase B, die auch bei der CD28-induzierten Inhibition des AICD beteiligt sind. Meine Ergebnisse verdeutlichen daher, daß diese Kostimulationsmoleküle, obwohl sie zu unterschiedlichen Superfamilien gehören, eine gemeinsame Signalendstrecke nutzen. Diese Ergebnisse tragen zum Verständnis der 4-1BB-vermittelten Signale bei, bringen sie in Relation zu den bereits gut untersuchten CD28-vermittelten Signalen und helfen so die molekularbiologischen Mechanismen der Kostimulation aufzuklären. Der zweite Teil meiner Dissertation beschäftigt sich mit Tumorvakzinierung durch genetisch modifizierte Krebszellen, die kostimulatorische Signale induzieren. Solche Zellen wurden bereits erfolgreich in präklinischen Tiermodellen eingesetzt und werden gegenwärtig in verschiedenen klinischen Anwendungen evaluiert. Es wurde in unserer Arbeitsgruppe bereits gezeigt, dass CD80 Tumorimmunität im syngenen Mausmodell erzeugen kann und dies die Deletion von aktivierten T-Zellen durch AICD verhindert. In klinischen Studien werden Tumorzellvakzine generell inaktiviert um die Übertragung von lebenden Tumorzellen auszuschließen. Frühere tierexperimentelle Arbeiten suggerieren, dass die Inaktivierung von CD80-exprimierenden Tumorzellen durch ionisierende Strahlung den Vakzinierungseffekt dieser Zellen aufhebt. Im Rahmen meiner Arbeit vergleiche ich lebende und bestrahlte allogene Tumorzellen bezüglich ihrer Fähigkeit, T -Zell-Effektorfunktionen hervorzurufen und T-Zell-AICD zu verhindern. Sowohl letal bestrahlte, als auch unbestrahlte CD80-exprimierende Tumorzellen induzieren in gleichem Umfang in vitro direkte T-Zell-Aktivierung mit T-Zell- Proliferation, Tumorzell-Lyse und Hemmung des AICD. Hingegen sind nekrotische, CD80-exprimierende Tumorzellen nicht in der Lage, T-Zellen direkt zu aktivieren. Diese Ergebnisse legen nahe, dass bei der Inaktivierung von Tumorzellen Verfahren gewählt werden sollten, die möglichst wenig Nekrose induzieren

Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway

Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-specific BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a single- vector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-deficient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wild-type HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x(L) and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not sufficient to confer sensitivity to Nbk

Targeting the epidermal growth factor receptor (HER) family by T cell receptor gene-modified T lymphocytes.

Human epidermal growth factor receptor 2 (HER2) has been successfully targeted as a breast cancer-associated antigen by various strategies. HER2 is also overexpressed in other solid tumors such as stomach cancer, as well as in hematological malignancies such as acute lymphoblastic leukemia. HER2-targeted therapies are currently under clinical investigation for a panel of malignancies. In this study, we isolated the T cell receptor (TCR) genes of a HER2-reactive allo-human leukocyte antigen-A2-restricted CTL clone and introduced the TCRalpha- and beta-chain genes into the retrovirus vector MP71. Murinization and codon optimization of the HER2-reactive TCR was required for efficient TCR expression in primary human T cells. The tumor recognition efficiency of HER2-TCR gene-modified T cells was similar to the parental CTL clone from which the TCR genes were isolated. The known cross-reactivity of the HER2-reactive TCR with HER3 and HER4 was retained when the TCR was transduced into primary T cells. Our results could contribute to the development of a TCR-based approach for the treatment of HER2-positive breast cancer, as well as of other malignancies expressing HER2, HER3, and/or HER4