A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / In this study we examine the utility of using CD8+ T cell hybridomas to measure the ability of bone marrow dendritic cells (BMDCs) to internalize cancer proteins and display them to cytotoxic T cells, a process termed cross‐presentation. We test the ability of a newly generated T cell hybridoma called BUSA14 to detect cross‐presentation of the melanoma antigen gp100. BUSA14 produces a dose‐dependent response to human and mouse gp100 peptides. However, cross‐presentation of gp100 by BMDCs using SK‐MEL‐28 human melanoma cell lysates or direct MHC class I‐restricted presentation by B16 murine melanoma cells was not detected. Both SKMEL‐28 and B16 cells express gp100 protein by immunoblot, and gp100 as a membrane bound protein may be concentrated by cell fractionation techniques. We validated our crosspresentation assay with another T cell hybridoma B3Z to detect cross‐presentation of the model antigen ovalbumin. Lastly, we determined that although BUSA14 expresses the coreceptor CD8, BUSA14 lacks CD3 expression, which likely impairs the ability of this hybridoma to respond to engagement of the T cell receptor and contributes to the inability to detect presentation of native gp100 protein. To resolve these issues, we plan to use primary gp100‐specific T cells from pmel mice expressing the same T cell receptor as the BUSA14 hybridoma to detect presentation of gp100 protein. Ultimately, we plan to evaluate the requirements for cross‐presentation of gp100, including a role for gamma‐interferon‐inducible lysosomal thiol reductase (GILT), a disulfide bond reducing enzyme.

No description available.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this thesis was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants, stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this thesis was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants, stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

The discovery of immune checkpoints and the subsequent development of pharmacological immune checkpoint inhibitors generated excitement and optimism that researchers had finally found the key to inducing complete remission of tumors. Inhibitors targeting the PD-1/PD-L1 signaling axis, though still facing limitations with widespread utility, have proven to be beneficial in multiple types of cancerous tissues. While some patients experience dramatic results, immune checkpoint inhibitors are not the panacea that was initially hoped. Why anti-PD-1/anti-PD-L1 therapies often come up short might be that the underlying regulators of PD-L1 expression or other molecular regulators of immune suppression are not being addressed by the pharmacological immunomodulators. One protein often active in the tumor microenvironment (TME) is involved in both these possibilities: the aryl hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor that can suppress immune activation as well as drive cancer progression by regulating transcription of a myriad of different genes within both malignant and immune cells. While AhR activation often correlates with poor prognosis in a variety of cancers, how AhR activation affects functional characteristics within and between malignant cells and immune cells in the TME is not fully understood. This is especially true in lung cancer, one of the most common and deadliest malignancies, which develops in an organ with many tissue-resident immune cells and opportunities to encounter exogenous pathogens—and environmental AhR agonists. To this end, we sought to elucidate the interactions between the AhR and its immunomodulatory target genes, PD-L1 and IDO1, in malignant lung adenocarcinoma (LUAD) cells and to determine how these interactions influence immunosuppression in the greater TME. The following work describes how AhR activation in LUAD cells results in the upregulation of two immune checkpoints, PD-L1 and IDO, in the malignant cell. We showed that AhR expression in CMT167 LUAD cells led to rapid tumor growth following subcutaneous or orthotopic transplantation whereas AhR deletion resulted in slower proliferation and up to complete rejection of tumor cells. AhR knockout also resulted in greater infiltration of CD3+ T cells. T cells from CMT167AhR-KO tumors were characterized by expression of granzyme B and upregulation of genes involved in activated and cytotoxic immune response, while T cells from CMT167WT tumors expressed higher levels of genes associated with T cell exhaustion and non-Th1 phenotypes. In addition, we developed a novel orthotopic model that will allow us to better understand how immune checkpoints from tumor cells modulate the lung TME by tracing the interactions of both myeloid and lymphoid immune cells with malignant cells. Further, in human LUAD cells, we implicated the AhR in the control of a long non-coding RNA shown to promote PD-L1 expression in the presence of IFNγ. Together, these results implicate AhR as a regulator of immune suppression in LUAD through multiple mechanisms and support it as a therapeutic target in the quest to improve immunotherapies.

L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles. / The injection of immuno-competent cells into a histo-incompatible host can result in the development of Graft-versus-Host disease (GVHD). GVHD is the most significant barrier to a more widespread use of allogeneic hematopoietic cell transplantation (AHCT), a potent treatment for several diseases. A better understanding of the pathophysiological underpinnings of GVHD would facilitate the design of rational approaches to treat and prevent this complication of AHCT. Gamma-interferon (IFN-γ) and Transforming Growth Factor-beta (TGF-β) are master cytokines of immunity and have a role in the function and homeostasis of transplanted cells. Using a murine model, we show that IFN-γ curtails lympho-hamatopoitic reconstitution in a dose-dependent fashion by increasing apoptosis and by limiting donor cell proliferation. TGF-β is an immunosuppressive cytokine that controls immune cells through multiple signaling pathways. The relative contribution of these pathways in AHCT is unknown. We specifically studied the role of one of these pathways by transplanting SMAD3 deficient cells (SMAD3-KO) in histo-incompatible hosts. SMAD3 is a key mediator of the so-called canonical TGF-β signaling pathway. Although SMAD3-KO donor mice are healthy, the injection of SMAD3-KO cells leads to severe GVHD in the hosts, characterized by intestinal involvement associated with Th1 skewing and massive granulocyte infiltration. These findings hint at a crucial role for SMAD3 in CD4 T-cell and myeloid cell biology. We then focalized on the role of SMAD3 in CD4 T cells knowing that SMAD3 is active in tolerant, resting CD4 T cells. We found that SMAD3 was rapidly inactivated upon T cell activation, suggesting that SMAD3 inactivation was functionally important to break the state of tolerance. Our cDNA microarray experiments show that indeed, SMAD3 regulates the transcript levels of multiple genes known to be involved in T cell tolerance and in biological processes plausibly related to immune tolerance.

Das Ubiquitin-Proteasom-System sorgt in eukaryontischen Zellen für einen kontrollierten Abbau von Proteinen. Das 20S Proteasom ist als Multikatalytischer Protease Komplex der zentrale Bestandteil dieses Systems. In der vorliegenden Arbeit konnte gezeigt werden, dass sich gereinigtes 20S Proteasom aus HeLa-Zellen chromatographisch in Subtypen auftrennen lässt, die sich strukturell und in ihrer proteolytischen Aktivität unterscheiden. Nach Induktion der Zellen mit gamma-Interferon (gamma-IFN) werden Immuno-Proteasomen gebildet und es kommt zu einer Veränderung des Subtypen-Musters und der Aktivitäten. Unter dem Einfluss von gamma-IFN bilden sich hauptsächlich Mischkomplexe mit sowohl konstitutiven als auch Immuno-Untereinheiten. Weiterhin konnte gezeigt werden, dass in den Zellkompartimenten Cytoplasma, Zellkern und Microsomen von HeLaS3-Zellen unterschiedliche 20S Proteasom-Subtypen vorkommen. Dies war unter anderem auf eine unterschiedliche Glykosylierung einzelner proteasomaler Untereinheiten zurückzuführen. Die genaue Kenntnis von Struktur und Funktion der 20S Proteasom-Subtypen ist im Hinblick auf neue diagnostische und therapeutische Ansätze in der Humanmedizin von großem Interesse. / The Ubiquitin-proteasome system is responsible for the regulated protein degradation in eucaryotic cells. The 20S proteasome is as a multicatalytic protease the central complex of these system. This study has shown that it is possible to separate 20S proteasome subtypes from HeLa cells by chromatography. 20s proteasome subtypes differ in structure and proteolytic activity. The subtype-pattern and the activity are significantly changed after an induction of the cells with gamma-Interferon (gamma-IFN) under formation of immuno proteasomes. After gamma-IFN induction mainly mixed complexes have been formed with both constitutive and immuno subunits. Further it has been shown that in cell compartements cytoplasm, microsomes and nucleus of HeLaS3 cells different 20S proteasome subtypes are located. Among other things glycosylation of some subunits is responsible for that phenomenon. With regard to new strategies in diagnostic and therapy of human diseases the exactly knowledge of structure and function of the proteasome subtypes is a case of interest.

Psoriasis ist eine komplexe entzündliche Erkrankung der Haut. Charakteristisch ist eine dichte Infiltration von T-Lymphozyten und eine Hyperproliferation der Epithelschicht. Heutzutage belegen die Ergebnisse vieler experimteller Studien, daß Psoriasis eine T-Zell-induzierte Erkrankung ist. Die Spezifität der T-Zell-stimulierenden Antigene ist noch unbekannt. Voraussetzung für die Charakterisierung psoriatischer T-Zellen ist die Isolation von T-Zell-Klonen aus psoriatischen Plaques, deren Restimulation in vitro qualitativ und quantitativ erfaßbar ist. Hierfür haben wir einen hochsensiblen gamma-Interferon Elispot-Assay etabiert, der die Aktivität der T-Zellen aus Hautplaques erfaßt. Zudem zeigen wir, daß man aktivierte T-Zell-Klone mittels CD25-Markierung isolieren und anschließend klonieren kann. Unsere Ergebnisse können als eine Grundlage für weitere Versuche dienen, die die Spezifität von T-Zell-Klonen aus psoriatischen Plaques charakterisieren sollen. / Psoriasis is a complex inflammatory disease of the skin characterized by a dense infiltration of T-lymphocytes and a hyperproliferation of the epithelial layer. A host of experimental and clinical data suggest that psoriasis is a T cell mediated disorder. The nature of T-cell-stimulating antigens is still unknown. One way to identify putative antigen(s) is the definition of T-cell-receptor specifities using randomized combinatorial peptide libraries. This requires the isolation and expansion of T cell clones from psoriatic plaques in vitro. Therefore we established a gamma-interferon Elispot-assay which allows quantification of the frequency of activated plaque-derived T cells in vitro. In addition, we show that activated T cell clones can be sorted via CD25 and cloned. The expanded clones can also be restimulated by autologous cells. Our results should be useful in the design of experiments aiming at a systematic analysis of the specifity of T cell clones present in psoriatic plaques.

L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles. / The injection of immuno-competent cells into a histo-incompatible host can result in the development of Graft-versus-Host disease (GVHD). GVHD is the most significant barrier to a more widespread use of allogeneic hematopoietic cell transplantation (AHCT), a potent treatment for several diseases. A better understanding of the pathophysiological underpinnings of GVHD would facilitate the design of rational approaches to treat and prevent this complication of AHCT. Gamma-interferon (IFN-γ) and Transforming Growth Factor-beta (TGF-β) are master cytokines of immunity and have a role in the function and homeostasis of transplanted cells. Using a murine model, we show that IFN-γ curtails lympho-hamatopoitic reconstitution in a dose-dependent fashion by increasing apoptosis and by limiting donor cell proliferation. TGF-β is an immunosuppressive cytokine that controls immune cells through multiple signaling pathways. The relative contribution of these pathways in AHCT is unknown. We specifically studied the role of one of these pathways by transplanting SMAD3 deficient cells (SMAD3-KO) in histo-incompatible hosts. SMAD3 is a key mediator of the so-called canonical TGF-β signaling pathway. Although SMAD3-KO donor mice are healthy, the injection of SMAD3-KO cells leads to severe GVHD in the hosts, characterized by intestinal involvement associated with Th1 skewing and massive granulocyte infiltration. These findings hint at a crucial role for SMAD3 in CD4 T-cell and myeloid cell biology. We then focalized on the role of SMAD3 in CD4 T cells knowing that SMAD3 is active in tolerant, resting CD4 T cells. We found that SMAD3 was rapidly inactivated upon T cell activation, suggesting that SMAD3 inactivation was functionally important to break the state of tolerance. Our cDNA microarray experiments show that indeed, SMAD3 regulates the transcript levels of multiple genes known to be involved in T cell tolerance and in biological processes plausibly related to immune tolerance.

Members of the important disease causing bacterial generas, Chlamydia and Chlamydophila, are characterised by a complex developmental cycle which is comprehensively described by microscopy. The inability to use standard genetic techniques for this obligate intracellular bacterium, however, means that significant gaps in our understanding of the molecular mechanisms used to control growth and development of Chlamydia still exist. The current study investigated the function of bacterial guanosine triphosphatases (GTPases), components of the organism's limited signal transduction arsenal, in regulatory control of the chlamydial development cycle. Initial analysis of the gene transcription of chlamydial GTPases and other predicted signal transduction genes using real time RT-PCR, in a Chlamydophila pneumoniae A-03 tryptophan depletion model of persistence, revealed significant differential expression of genes in response to the addition of interferon gamma (IFN-γ). Predicted chlamydial GTPase encoding genes, ychF, yhbZ and yphC, associated with ribosome function amongst other processes were strongly up-regulated, while hflX was down-regulated in the persistent cultures. Analysis of an additional model of Cp. pneumoniae persistence, induced by limitation of host cell iron, revealed that ychF, yhbZ and yphC were also up-regulated in the persistent cultures. This study provided the most comprehensive analysis of Cp. pneumoniae gene transcription to date and suggest that chlamydial GTPases serve a role in generation of the persistent chlamydial phenotype. Cloning and expression of Cp. pneumoniae and Cp. abortus yhbZ, including demonstration of in vitro GTPase activity, indicates that this chlamydial gene encodes a member of the universally conserved and essential bacterial Obg subfamily of GTPases. Evidence is building that members of this latter family of bacterial GTPases are important regulators of bacterial growth and morphological differentiation in developmentally complex bacteria. Over-expression of chlamydial YhbZ subfamily GTPases in Escherichia coli revealed inhibition of bacterial growth and disruption of cell division and chromosome functions leading to the generation of elongated cells with limited chromosome segregation, as described for Obg subfamily members from E. coli and other bacteria. Although more analysis is required, we suggest a novel mechanism of chlamydial Obg GTPase regulation involving sensing of host cell GTP/GDP pools to control secondary differentiation of reticulate bodies (RBs) back to elementary bodies (EBs). Analysis of the chlamydial complement of bacterial GTPases was extended to HflX, a previously uncharacterised and only predicted GTPase conserved in bacteria. HflX sequence analysis revealed conservation of G motifs responsible for nucleotide binding and hydrolysis (G1, G3, G4) and protein interaction (G2), although the latter was unique to HflX subfamily GTPases. Recombinant Cp. pneumoniae HflX displays GTPase activity with nucleotide specificity for GTP. We tested Cp. pneumoniae HflX function by over-expression in E. coli which led to inhibition of growth in E. coli and elongation of cells with normal chromosome partitioning. This phenotype was the probable result of disruption of a stage in cell division subsequent to chromosome segregation. This present study provides the first evidence to show that bacterial HflX is a GTPase and suggests a regulatory role in bacterial cell cycle control.