We investigated the role of Fas ligand in the development of anti-tumor immunity. The LSA tumor specific cytotoxic T lymphocyte (CTL) clone, PE-9, expressed both Fas and Fas ligand. This CTL clone upregulated Fas and Fas ligand expression upon activation through the T-cell receptor and induced apoptosis in Fas+, LSA tumor cells using the FasL-based pathway. However, LSA and EL-4 tumor cells constitutively expressed Fas ligand and killed Fas+ PE-9 CTLs and Fas+, but not Fas-negative (Fas-) activated T cells and thymocytes. These data suggested that T cells and cancer cells can kill each other and that cancer cells may use Fas ligand to evade the action of the immune T cells. In addition to the expression of membrane-bound form, FasL+ LSA and EL-4 tumor cells produced a soluble form of Fas ligand when they grew in vivo and in vitro. Serum from EL-4 or LSA-bearing wild type mice contained significant levels of Fas ligand. The soluble FasL induced apoptosis in liver and thymus of C57BL/6 wild type (Fas+) mice, but not C57BL/6 lpr/lpr (Fas-) mice. The detection of apoptosis in the liver of C57BL/6 gld/gld (FasL-defective) mice suggested that the source of Fas ligand found in the sera of EL-4 or LSA-bearing mice was from the tumor cells rather than the host cells. CTL or NK cells used FasL-based apoptosis to kill the target cells when activated. To this end, we tested whether constitutive expression of Fas on tumor cells generate enhanced anti-tumor immunity. IL-2 or poly-I-C induced/ activated NK/LAK cells displayed higher cytotoxicity against L1210 Fas+, but not L1210 Fas- tumor cells. Furthermore, growth of L1210 Fas+, but not Fas- tumor, in vivo, generated Fas-specific cytotoxic T lymphocytes. Therefore, mice bearing L1210 Fas+ tumor cells survived for a longer time than mice bearing L1210 Fas- tumor cells. To determine the role of the Fas, FasL, and perforin in the initiation of tumor, C57BL/6 +/+ (FasL+, Fas+), C57BL/6 lpr/lpr (Fas-), C57BL/6 gld/gld (FasL-), and perforin knock-out (PKO) (FasL+, Fas+, but perforin-deficient) mice were injected with methylcholanthrane (MCA). Tumor development in lpr or gld mice was faster and uncontrollable, compared to C57BL/6 (wild-type) and PKO mice. However, wild-type and PKO mice showed delayed tumor appearence and were able to suppress tumor growth. In addition to the deficiency of Fas or FasL, high levels of TGF-b and IL-10 expression detected in lpr and gld mice were also responsible for the early tumor development. Together these data suggested that interactions between Fas and Fas ligand, expressed on immune cells and tumor cells, play an important role in the generation of anti-tumor immunity. Tumor cells use FasL to evade the action of the immune system, and upregulation of FasL makes T cells more cytolytic. Tumor growth may depend on the number of cancer cells vs. the number of cancer specific T cells. / Ph. D.

No description available.

Cytotoxic T lymphocytes (CTL) are differentiated CD8+ T cells that eliminate virally infected cells and tumor cells. CTL lyse target cells by at least two distinct mechanisms: degranulation of cytolytic molecules and cell surface expression of Fas ligand (FasL), which induces apoptosis of Fas-expressing target cells. In addition to their defense function, these two cytolytic mechanisms also play crucial roles in homeostatic regulation and contribute to pathogenesis in many different model systems. To fully exploit killer cells in tumor and virus elimination, or dampen the immune response in, for example, autoimmune diseases, it is essential to understand the mechanisms that CTL employ to destroy target cells. In contrast to the well-characterized degranulation mechanism, the regulation of FasL expression on the CTL cell surface remains elusive and even controversial. The prevailing model at the time I initiated my studies was that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. In this thesis, I revealed for the first time that there are two waves of FasL cell surface expression upon target cell engagement, which are differentially regulated by TCR signaling and perform distinct roles in CTL mediated responses. I demonstrated that CTL degranulation and FasL lytic mechanisms are fully independent with respect to stored component localization and regulation. Finally, based on cell fractionation and imaging studies, I suggested that FasL is stored in a recycling endosome associated compartment, which is located in a special niche between the ER and mitochondria and uses a novel microtubule-independent secretory mechanism to translocate to the cell surface. Together, these findings provide important insight into the regulation and role of FasL in CTL mediated responses. / Immunology

No description available.

Fas Ligand (FasL) binds to the Fas receptor to induce apoptosis or activate other signaling pathways. FasL can also transduce "reverse signals" and thus participate in bidirectional signaling. The FasL intracellular domain contains consensus sequences for phosphorylation and a proline rich protein interaction domain. This latter region of FasL has previously been implicated in FasL reverse signaling and regulation of FasL surface expression. In this report, we sought to identify novel FasL interacting proteins to help understand signaling through and trafficking of this death factor. Using mass spectrometry, we identified sorting nexin 18, adaptin beta, Grb2, PACSIN2 and PACSIN3 as FasL interacting proteins. RNAi mediated knockdown of Grb2 significantly reduced the surface expression of FasL and increased its expression intracellularly. Our data show that Grb2 controls the subcellular localization of FasL. All other proteins identified in our screen could be classified as trafficking-associated proteins, highlighting the complex regulation of the surface expression of this death factor.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 82-102).

Zugl.: Stuttgart, Univ., Diss., 2006

Regensburg, Univ., Diss., 2010.

Die Stimulation des CD95-Todesrezeptors durch seinen natürlichen membranständigen Li-ganden CD95L führt zur kontextabhängigen Aktivierung von sowohl apoptotischen als auch nicht-apoptotischen Signalwegen. Durch Proteolyse wird aus dem membranständigen CD95L löslicher trimerer CD95L freigesetzt. Die Bindung von löslichem trimerem CD95L an CD95 ist nicht ausreichend, um die CD95-Signaltransduktion effizient zu stimulieren. Die Fähigkeit von löslichen CD95L-Trimeren CD95-vermittelte Signalwege robust zu aktivieren kann jedoch durch Oligomerisierung und artifizielle Immobilisierung an eine Oberfläche drastisch gesteigert werden. In dieser Arbeit wurde zunächst bestätigt, dass nur oligomere CD95L-Varianten, die z.B. durch Antikörpervernetzung von N-terminal getaggten rekombinanten CD95L-Varianten oder durch eine gentechnisch erzwungene Hexamerisierung von CD95L-Molekülen erhalten wur-den, in der Lage sind, effizient apoptotische und nicht-apoptotische Signalwege zu aktivieren. Ferner zeigte sich dann, dass die Bindung von löslichen CD95L-Trimeren nicht ausreichend ist, um die Translokation von CD95-Molekülen in detergenzunlösliche „Lipid Raft“- Membrandomänen zu stimulieren. Die „Lipid Raft“-Translokation ist ein zentrales Ereignis bei der CD95-Aktivierung und vor allem für die Induktion der Apoptose bedeutsam. Dabei ist ein selbstverstärkender Prozess aus Caspase-8-Aktivierung und „Lipid Raft“-Assoziation des CD95 von Bedeutung. Um die Interaktion von CD95 und CD95L mit Hilfe von hoch sensitiven zellulären Bindungs-studien analysieren zu können, wurden in dieser Arbeit desweiteren CD95L-Fusionsproteine entwickelt und hergestellt, an welche N-terminal eine Gaussia princeps Luziferase (GpL)- Reporterdomäne gekoppelt ist. So konnte mit den GpL-CD95L-Fusionsproteinen gezeigt werden, dass die Oligomerisierung von CD95L-Trimeren keinen Effekt auf die Ligandenbele-gung des CD95 hat. Dies spricht dafür, dass die höhere spezifische Aktivität von oligomeri-sierten CD95L-Trimeren nicht auf einer Aviditäts-vermittelten Zunahme der apparenten Affi-nität beruht, sondern dies deutet darauf hin, dass die sekundäre Aggregation von sich initial bildenden trimeren CD95L-CD95-Komplexen eine entscheidende Rolle in der CD95-Aktivierung spielt. Durch Scatchard-Analysen zeigte sich ferner, dass trimerer CD95L mit mindestens zwei zellulären Bindungsstellen unterschiedlicher Affinität interagiert. Bindungs-studien mit löslichen monomeren und trimeren GpL-CD95-Rezeptoren an membranständigen CD95L, als auch Inhibitionsstudien ergaben, dass trimerer CD95 weitaus besser an CD95L bindet. Dies legt nahe, dass es sich bei den zuvor beobachteten hoch- und niederaffinen Bindungsstellen für CD95L um monomere bzw. prä-assemblierte CD95-Moleküle handelt. Die GpL-CD95L-Fusionsproteine wurden auch genutzt, um die CD95-Translokation in „Lipid Rafts“ zu analysieren. So wurde trimerer GpL-CD95L als „Tracer“ zur Markierung von inaktiven CD95-Molekülen eingesetzt. Nach Aktivierung der übrigen freien CD95-Moleküle mit hoch aktivem hexameren Fc-CD95L konnte eine Zunahme der inaktiven GpL-CD95L-markierten Rezeptoren in „Lipid Rafts“ beobachtet werden. Offensichtlich stimulieren also aktivierte CD95-Moleküle in „trans“ die Ko-Translokation inaktiver CD95-Rezeptoren in „Lipid Rafts“. Dies bestätigte sich auch in Experimenten mit Transfektanten, die einen chimären CD40-CD95-Rezeptor exprimieren. Letzterer ist nach Stimulation mit CD40L in der Lage, intrazellu-läre CD95-vermittelte Signalwege zu aktivieren. Die Aktivierung von CD95-assoziierten Sig-nalwegen durch Stimulation von endogenem CD95 in CD40-CD95-Transfektanten resultierte nun in der Ko-Translokation von unstimulierten CD40-CD95-Rezeptoren in „Lipid Rafts“. Vice versa zeigte sich die Ko-Translokation von endogenem CD95 nach spezifischer Aktivierung des chimären CD40-CD95-Rezeptors. Schlussendlich erwiesen sich eine funktionsfähige Todesdomäne und die Aktivierung der Caspase-8 als essentiell für die „Lipid Raft“-Assoziation von aktivierten CD95-Molekülen und auch für die durch diese Rezeptorspezies induzierte Ko-Translokation von inaktiven Rezeptoren in „Lipid Rafts“. / Membrane-bound CD95L activates the CD95 death receptor to induce context-dependent apoptotic and non-apoptotic signaling pathways. In contrast, soluble trimeric CD95L, which is released by proteolysis, is not sufficient to stimulate CD95-induced signaling. However, the ability of soluble CD95L trimers to activate robust CD95 mediated signaling pathways can be increased drastically by oligomerization and artificial immobilization on the cell surface. In this work, it has been confirmed that only the oligomeric CD95L-variants, produced by an-tibody crosslinking of N-terminal tagged recombinant CD95L-variants or by genetic engineer-ing-enforced formation of hexamers, are able to efficiently activate both apoptotic and non-apoptotic signaling pathways. Moreover, it has been shown that binding of soluble trimeric CD95L is not sufficient to stimulate translocation of CD95 molecules to the “lipid raft”-containing compartment of the cell membrane. This translocation of CD95 to “lipid rafts” is a pivotal event in CD95 activation and mainly meaningful, especially for induction of apoptosis. Thereby an auto-amplification-loop of caspase-8 activation and association of CD95 with “lipid rafts” is of importance. To analyze CD95-CD95L interactions, highly sensitive cellular binding studies using CD95L fusion proteins linked to the N-terminal Gaussia princeps luciferase (GpL) have been per-formed. With GpL-CD95L fusion proteins it has been demonstrated that oligomerization of CD95L trimers has no major effect on CD95 occupancy. Therefore higher specific activity of oligomerized CD95L trimers is not related to an avidity-driven increase in apparent affinity. This suggests that a process of secondary aggregation of the initially formed trimeric CD95L-CD95 complexes is crucial for CD95 activation. Furthermore, the data obtained from scat-chard analysis showed that trimeric CD95L interacts with at least two binding sites of different affinity. This was further examined by performing binding studies of soluble monomeric and trimeric GpL-CD95 receptors to membrane-bound CD95L and neutralization assays. It was observed that trimeric CD95 receptor can bind to CD95L much better. These results suggest that the high and low affinity binding sites concern to monomeric or rather pre-assembled CD95 molecules. Moreover, GpL-CD95L fusion proteins have been employed to analyze translocation of CD95 to “lipid rafts”. In these experiments, GpL-CD95L trimers were applied to “mark” inactive CD95 molecules. Upon activation of the remaining free CD95 molecules using highly active Fc-CD95L, an increased association of these inactive receptors with “lipid rafts” was observed. Apparently activated CD95 molecules stimulate in “trans” the co-translocation of inactive CD95 receptors to “lipid rafts”. This has also been confirmed in experiments with transfectants expressing chimeric CD40-CD95 receptors. These chimeric receptors are able to activate CD95-mediated signaling pathways after stimulation with CD40L. After stimulation of endogenous CD95 in CD40-CD95 transfectants the unstimulated chimeric CD40-CD95 receptors co-translocated to “lipid rafts”. Conversely, activation of CD95-associated pathways by specific stimulation of chimeric CD40-CD95 receptors resulted in co-translocation of the endogenous CD95. In conclusion, it has been shown that a functional death domain and caspase-8 activation turned out to be essential for both “lipid raft” association of signaling-active CD95 molecules and co-translocation of inactive CD95 receptors induced by active receptor species.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.