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The cytokine Interleukin-4 (IL-4) plays a crucial role in the pathophysiology and progression of asthma and other atopic diseases. Its activities are signaled into the cells upon binding to and signaling through a shared receptor complex composed of the subunits IL-4Rα and common γc. Another cytokine, Interleukin-13 shares many functions with IL-4. This can be explained by the fact that both, IL-4 and IL-13, can signal via a shared receptor complex comprising the IL-4R and the IL-13R1 subunit. Therefore, the IL-4Rα receptor subunit has become a highly promising drug target, since it mediates IL-4 and IL-13 responses and blocking IL-4Rα will abrogate IL-4 as well as IL-13 effector functions. Currently, an IL-4 based mutein (Pitrakinra), acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. This work describes the generation and production of biologically active IL-4 muteins, which contain a single additional engineered cysteine. The introduction of a free thiol group allows site-specific chemical modification. The muteins were expressed in E. coli in insoluble form, refolded and purified. The thiol group of the mutein was protected as mixed disulfide with the tripeptide glutathione. A first attempt to chemically reduce the engineered cysteine residue failed, because the three native disulfide bonds of IL-4 exhibit a similar reactivity and chemical reduction of the native disulfide resulted in full deactivation and precipitation of the IL-4 protein. Therefore, an enzymatic approach was developed which specifically reduces the mixed disulfide bonds with an attached glutathion moiety and thus leaves the native structurally essential disulfide bonds unaltered. For optimization, four different IL-4 cysteine muteins with four cysteine residues introduced at positions close to the IL-4Rα binding site were tested and their reduction rates by glutaredoxin was determined. The enzymatic reduction occured at different rates for all four muteins indicating that accessibility is an important influence and must be determined individually for each mutant protein. After optimization of the pH value and particularly the reaction time, all muteins could be prepared with the engineered thiol group being released in reasonable yield. The proteins exhibiting the free thiol group were then modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effects of these modifications at different positions on binding to IL-4R were measured employing SPR biosensor technology. In the second project of this study, foldamers, which represent a new class of stable, compactly folded biomolecules and can specifically interact with proteins and nucleic acids, were examined to identify their potential as new drugs to interfere with IL-4 activities. Fragment-based drug discovery offers great promise for providing new starting points for drug discovery and facilitates the lead optimization. As foldamers equipped with a thiol-group for tethering could not to be produced; only the effect of foldamers present in a synthesized foldamer library on the binding to IL-4R could be tested. Two libraries containing different foldamers based on aromatic amide were synthesized by Michael Grotz and Dr. Michael Deligny and tested in our lab for their capability to disrupt the ligand-receptor interaction of IL-4 and its receptor IL-4Rα [ECD] using surface plasmon resonance technology. None of the studied foldamers could specifically inhibit the IL-4/IL-4Rα interaction. Some foldamers showed non-specific binding. The study presented here shows the design and production of a potentially new type of IL-4 antagonists, which employ site-specific chemical modification to exert their antagonistic function. / Das Zytokin Interleukin-4 (IL-4) spielt eine entscheidende Rolle in der Entstehung und Pathophysiologie von Asthma und anderen atopischen Krankheiten. Seine Aktivitäten können in die Zelle durch die Bindung an einen Rezeptorkomplex übertragen werden, welcher aus den Untereinheiten IL-4Rα und γc besteht. Interleukin-13 (IL-13), ein verwandtes Zytokin, und IL-4 besitzen viele gemeinsame Funktionen. Das kann dadurch erklärt werden, dass IL-4 wie auch IL-13 ihre Signale über einen gemeinsamen Rezeptorkomplex übertragen können, der aus der IL-4R und der IL-13R1 Untereinheit besteht. Die IL-4R Untereinheit ist ein vielversprechendes Zielmolekül für die Entwicklung von Pharmaka, da sie IL-4 und IL-13 Reaktionen vermittelt. Durch Blockieren von IL-4R werden die Aktivitäten von IL-4 sowie IL-13 unterdrückt. Ein IL-4 basiertes Doppelmutein (Pitrakinra), welches als Gegenspieler zu IL-4 und IL-13 Rezeptoren fungiert, befindet sich derzeit in der klinischen Entwicklung. In dieser Arbeit wird die Bildung und Produktion von biologisch aktiven IL-4 Muteinen mit einem einzelnen zusätzlich eingefügten Cysteinrest beschrieben. Die Einführung einer freien Thiol-Gruppe ermöglicht ortsspezifische chemische Modifizierungen. Ein „Tethering“ Ansatz sollte dann auch eine sehr schwach Bindung von thiol-reaktiven Verbindungen an IL-4 messbar machen. Die Muteine wurden in unlöslicher Form in E. coli exprimiert, zurückgefaltet und auf gereinigt. Dabei wurde die Thiolgruppe des Muteins als Disulfid mit dem Tripeptid Glutathion geschützt. Erste Versuche gezielt den eingeführten Cysteinrest selektiv chemisch zu reduzieren schlugen fehl, da die drei proteineigenen Disulfidbrücken von IL-4 eine ähnliche Reaktivität zeigten, und die Reduktion zur vollständigen Desaktivierung und Fällung des IL-4 Proteins führte. Daher wurde ein enzymatischer Ansatz entwickelt, der gezielt die Disulfidbrücke zum Glutathionrest reduziert und die proteineigenen strukturell essentiellen Disulfidbrücken unverändert lässt. Zur Optimierung wurden vier verschiedene IL-4 Cystein-Muteine mit Cysteinresten an verschiedenen Positionen nahe der IL-4Rα Bindungsstelle getestet und die Reduktionsgeschwindigkeit in Gegenwart von Glutaredoxin bestimmt. Die enzymatische Reduktion verlief für alle vier Muteine mit verschiedenen Geschwindigkeiten. Dies deutet darauf hin, dass die Zugänglichkeit der Disulfidgruppe einen wichtigen Einfluss besitzt. Die Reduktionsbedingungen mussten daher für jedes Mutein neu bestimmt werden. Nach Optimierung des pH Wertes und insbesondere der Reaktionszeit konnten alle Muteine mit einer freien Thiolgruppe in angemessener Ausbeute erhalten werden. Die Proteine mit jeweils einer freien Thiolgruppe wurden daraufhin mit N-Ethylmaleinimid (NEM) oder Maleimido-PEG modifiziert. Die Effekte der Modifizierung an verschiedenen Positionen des IL-4 auf die Bindung an IL-4R wurden mit Hilfe der SPR-Spektroskopie (Oberflächen Plasmon Resonanz Spektroskopie) gemessen. Im zweiten Teil dieser Arbeit wurde die Interaktion von Foldameren mit der IL-4Ra Rezeptorkette untersucht. Foldamere stellen eine neue Klasse von stabilen, kompakt gefalteten Biomolekülen dar, die möglicherweise spezifisch mit Proteinen und Nukleinsäuren wechselwirken können. Es sollten Vorversuche durchgeführt werden um zu sondieren, ob aus Foldameren Hemmstoffe für IL-4 und IL-13 entwickelt werden können. Da Foldamere mit einer Thiolgruppe zur Anbindung (Tethering) an IL-4 nicht hergestellt werden konnten, wurden zunächst nur nichtreaktive Foldamare aus einer synthetisierten Foldamer-Bibliothek getestet. Zwei Bibliotheken mit verschiedenen auf aromatischen Amiden basierenden Foldameren wurden von Michael Grotz und Dr. Michael Deligny synthetisiert und von mir mit Hilfe der SPR Spektroskopie auf ihre Fähigkeit getestet, die Ligand-Rezeptor Wechselwirkung von IL-4 und derIL-4Rα Rezeptoruntereinheit zu unterbinden. Keines der untersuchten Foldamere konnte die IL-4/IL-4Rα Wechselwirkung spezifisch hemmen. Einige Foldamere zeigten eine unspezifische Bindung. Die hier dargestellten Studien zeigen das Design und die Herstellung eines potentiell neuen Typs von Gegenspieler zu IL-4, welcher ortsspezifische chemische Modifikationen ausnutzt um seine antagonistische Funktion zu erfüllen.

Obesity and type 2 diabetes are the most prevalent metabolic diseases affecting over 50% of people in the western world. Although the pathogenesis of type 2 diabetes is not fully understood, growing evidence links this disease to a state of chronic inflammation, which occurs in metabolically active tissue such as the liver, adipose tissue and skeletal muscle and results in the secretion of inflammatory cytokines, of which interleukin-6 (IL-6) is one. It is generally accepted that elevations in the plasma and/or tissue of this family of cytokines have a negative effect on whole body glucose homeostasis. While there is compelling evidence for the negative effects of resistin and TNF-á on insulin sensitivity, the role of IL-6 in the etiology of insulin resistance is not fully understood. The notion of negative effects of IL-6 in metabolic processes is further confounded by the marked elevations of IL-6 which occur in conjunction with the beneficial activity of exercise. We firstly sought to examine the effect of the lipolytic hormone adrenaline on IL-6 expression and release in order to establish whether IL-6 acts independently of adrenaline in the regulation of fat metabolism. Reporting the absence of an effect of adrenaline on IL-6, we then investigated the role of IL-6 on metabolic processes in humans at rest and during exercise in circumstances where lipolysis was inhibited. Marked increases in IL-6 circulating protein and tissue gene expression were observed with exercise and further so with fatty acid suppression. In a mouse model of IL-6 depletion marked insulin sensitivity was observed, which was reversed with IL-6 treatment. In a mouse model with normal endogenous IL-6 levels IL-6 treatment also impaired glucose tolerance. Contrastingly, in a rat model both chronic and acute IL-6 treatment improved glucose tolerance In summary, studies from this thesis suggest that, rather than being causally related to insulin resistance, the cytokine IL-6 increases lipolysis, fat oxidation, and glucose metabolism in insulin sensitive tissues in humans. This does not appear to be the case in the mouse, where contrasting actions are observed, perhaps due to differences in the reliance of various parameters for metabolic processes between the species.

Type IV secretion systems have been identified in several human pathogens including Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila. These systems are responsible for the translocation of virulence proteins and/or DNA, thereby playing an important role in the pathogenesis of infection and plasticity of genomes. Burkholderia cenocepacia is an important opportunistic human pathogen, particularly in persons with cystic fibrosis (CF). Respiratory tract infection by B. cenocepacia in CF patients is often associated with a decline in respiratory function, and can result in acute systemic infection. Burkholderia cenocepacia strain K56-2 is part of the epidemic and clinically problematic ET12 lineage. Two type IV secretion systems have been identified in this strain; one system is plasmid encoded (designated the Ptw type IV secretion system) whereas the other is chromosomally encoded (designated the VirB/D type IV secretion system) and shows homology to the Agrobacterium tumefaciens VirB/D4 type IV secretion system. It was determined that the plasmid encoded Ptw system is a chimeric type IV secretion system composed of VirB/D4-like elements and F-specific subunits. More recently, it was found that this system translocates a protein effector (PtwE1) that is cytotoxic to plant cells. It was also determined that the positively charged C-terminal region of PtwE1 is important for translocation via the Ptw type IV secretion system. Strains of the epidemic B. cenocepacia PHDC lineage contain only a chromosomal VirB/D4-like type IV secretion system (designated BcVirB/D); and a putative effector protein associated with this system has been identified that has C-terminal transport signal and sequences different from the effectors of the Ptw type IV secretion system. It has also been shown that a competing plasmid substrate and a plasmid fertility inhibition factor act to render B. cenocepacia of the PHDC lineage incapable of expressing a plant phenotype. Thus, three type IV secretion systems have been identified in epidemic B. cenocepacia lineages. From two of these, an effector has been identified that has cytotoxic effects on eukaryotic cells, and at least one of these type IV secretion systems is able to translocate DNA substrates.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.

Dendritic cells (DCs) modified by some immunomodulatory genes can stimulate a strong antitumor immunity and improve the treatment of tumor cells on the condition that the sources of tumor-associated antigens (TAAs) are available. IL-6, a pleotropic cytokine, has been found to inhibit CD4+25+ regulatory T (Treg)-cell-mediated immune suppression and decrease activation-induced cell death (AICD) without interfering the process of T-cell activation. To enhance DC-based cancer vaccine, we engineered DCs to express transgene IL-6.<p> We constructed a fiber-modified recombinant adenovirus vector AdVIL-6 expressing IL-6, infected DCs with AdVIL-6, and then investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express IL-6 transgene. We demonstrated that DCs infected with the recombinant adenovirus AdVIL-6 induced DC maturation by up-regulation of the expression of MHC class U (Iab), CD40, CD54 and CD80 expression. We also demonstrated that vaccination of OVA-pulsed AdVIL-6-infected DCs (DCOVA/AdVIL-6) was able to stimulate a stronger OVA-specific effector CD8+ cytotoxic T lymphocyte (CTL) response than vaccination with the control virus AdVpLpA-infected DCs (DCOVA/AdVpLpA). More importantly, vaccination of mice with DCOVA/AdVpLpA could protect 100% mice from intravenous (i.v.) challenge of a low dose (0.5~105 cells per mouse, 8/8 mice protected) of OVA-expressing BL6-10OVA tumor cells, but only 63% mice from i.v. challenge of a high dose (1~105 cells per mouse, 5/8 mice protected) of BL6-10OVA tumor cells. However, vaccination of DCOVA/AdVIL-6 induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of BL6-10OVA tumor cells.<p> To study the immune mechanism underlying the result of IL-6 engineered-DC vaccine, we generated the DCOVA/AdVIL-6-activated OTI CD8+ T cells and DCOVA/AdVpLpA-activated OTI CD8+ T cells. We demonstrated that DCOVA/AdVIL-6-activated CD8+ T cells displayed a higher level of CD62L, FasL and perforin than DCOVA/AdVpLpA-activated CD8+ T cells. DCOVA/AdVIL-6-activated CD8+ T cells had a prolonged T cell survival after they were transferred into C57BL/6 mice. Furthermore, the results of the animal study showed that 100% of mice bearing OVA-expressing EG7 tumors (8mm in diameter, 8 mice per group) were tumor-free after they were i.v. treated with DCOVA/AdVIL-6-activated CD8+ T cells (2~106 cells per mouse). However, the control DCOVA/AdVpLpA-activated CD8+ T cells failed in eradication of EG7 tumors in all 8/8 mice.<p> Taken together, Adenovirus-mediated IL-6 transgene engineered DC vaccine stimulates efficient CD8+ T cell responses and antitumor immunity via enhanced T cell cytotoxicity and prolonged T cell survival. DCs engineered to express IL-6 by adenovirus-mediated IL-6 gene transfer may offer a new strategy in production of DC cancer vaccines.

For the treatment of cancer, immunotherapy has some inherent advantages over other treatment modalities: disseminated disease can be eradicated due to the systemic nature of immunity, the immune system is effective against a wide range of targets, long-term memory can offer added protection against disease relapse, immunotherapy should be relatively non-toxic, and it can be synergistically combined with other treatment platforms such as radiation and chemotherapy. Type 1 immune responses are thought to be superior for the treatment of cancer and, as the quintessential Th1 polarizing cytokine, interleukin-12 (IL-12) holds much promise; however, optimal therapeutic protocols have yet to be developed and clinical results have fallen short of this promise. The in vivo IL-12 experiments described here highlight a characteristic of cellular therapy that has not previously been appreciated. That is, the effect of cell-mediated cytokine delivery on the immediate microenvironment and how that affects the immune response initiated. This observation has implications for the clinical application of IL-12 therapy but may also prove to be an important consideration when studying other immunostimulants. I have herein developed a novel in vitro assay system that I have used to dissect the cellular responses to IL-12 and to identify the signals that are required for activation of a cluster of differentiation 4 (CD4)+ effector population that affects leukaemia cell clearance both in vitro and in vivo. This work, and the future studies proposed, will expand our understanding of the potential of IL-12 immunotherapy and enhance our ability to manipulate therapeutic conditions to favour the desired response. Moreover, the in vitro assay system offers a method for further characterization of CD4+ effector cells and the development of protocols to initiate their potent anticancer activity.

Immunity is known to be sexually dimorphic. This dimorphism may be attributed to the action of different hormones. Aluminum is a component of several vaccines and acts as an adjuvant of immunogenicity. The activation of the Nalp3 inflammasome plays a role in aluminum’s adjuvancy. Estrogen affects immune cells by regulating the expression of genes involved in immune-related mechanisms; such as the modulation of cytokine secretion. We hypothesized that estrogen modulates the aluminum-induced secretion of IL-1β and IL-18. Using an ex vivo mouse macrophage model this study examined: (i) the effects of estrogen on Nalp3 inflammasome genes expression and (ii) the estrogen receptor involved in the modulation of these genes. Our results indicate that estrogen up-regulates Nalp3 gene expression via ERα/β heterodimerization, and caspase-1 activity may be indirectly modulated due to the up-regulation of SPI-6 via ERβ.

Cytotoxic CD8 T lymphocytes kill virus-infected cells and are critical for viral clearance from the body. Cytokines, particularly those sharing the common gamma receptor chain (gamma c), play a key role in this cytotoxic function as well as in the growth, differentiation and homeostasis of CD8 T lymphocytes. In order to exert these biological effects, cytokine-dependent signal transduction via the Janus kinase (Jak) / Signal Transducers and Activators of Transcription (STAT) pathway, the phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways is required. In HIV infection however, the CD8 T lymphocytes become defective and are characterized by impaired cytotoxicity, altered differentiation patterns, and increased susceptibility to apoptosis. I hypothesized that impaired cytokine responsiveness resulting from defects in cytokine-dependent signal transduction contributes to the CD8 T cell impairment observed in HIV+ patients. I investigated the activation of the Jak/STAT signaling pathway to cytokines in CD8 T cells from HIV+ patients. Interestingly, these cells were responsive to IL-2, IL-4, IL-10, IL-15, and IL-21 at the level of their respective STAT activation. However, impairment of the IL-7 / IL-7Ralpha signaling axis was identified and characterized by a defect in STAT5 signaling. The impaired STAT5 activation correlated with a low IL-7Ralpha surface expression. The expanded population of IL- 7Ralphalow-expressing CD8 T cells, found particularly in viremic HIV+ patients, expressed higher levels of the transcriptional repressor Growth Factor Independent-1 (GFI1) compared to their IL-7Ralphahigh counterparts. This prompted further investigations into the role of GFI1 in IL-7Ralpha regulation in primary human CD8 T cells as a model. Though silencing of GFI1 did not modulate basal IL-7Ralpha expression, exogenous overexpression negatively regulated IL-7Ra surface levels. The gc cytokines, IL-2, IL-4, IL-7, and IL-15, but not IL-21, were found to efficiently suppress IL-7Ralpha expression however, only IL-4 simultaneously upregulated GFI1 expression. RNA interference studies targeting GFI1 in IL-4 stimulated CD8 T cells established a specific role for GFI1 in sustaining the suppression of IL-7Ralpha expression. Furthermore, transient downregulation of GFI1 in CD8 T cells subjected to IL- 4-dependent proliferation reduced their proliferative capacity. Other functions identified for GFI1 were in the suppression of CXCR4 and Bax expression in CD8 T cells. Studies aimed at identifying the signal transduction pathways responsible for regulating GFI1 and IL-7Ralpha expression revealed that IL-4-mediated downregulation of IL-7Ralpha expression required activation of the Jak/STAT and the PI3K pathways. On the other hand, IL-4-induced upregulation of GFI1 expression was mediated via the PI3K pathway. The JNK and P38 MAPK pathways appeared to be important as regulators of basal IL-7Ralpha expression levels, but had no statistically significant effects on GFI1 expression. To conclude, these studies have clarified the important biological effects of GFI1 in mature human CD8 T lymphocytes. Furthermore, exposure to IL-4 may generate CD8 T cell populations with an exhausted phenotype similar to those found in chronically-infected HIV+ patients, characterized by reduced cytotoxic activity and increased IL-4 production. Thus, the IL-4 study model may prove valuable for investigating the activity of human CD8 T cells in such chronic diseases and those characterized by a type 2 cytokine profile.