Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on July 8, 2010). Includes bibliographical references.

Erlangen-Nürnberg, Universiẗat, Diss., 2008.

The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed. / Die Plasmamembran gehört zu den am meisten untersuchten, gleichzeitig aber auch zu den komplexesten, vielfältigsten und am wenigsten verstandenen biologischen Strukturen. Ihre Funktion wird nicht nur durch die molekulare Zusammensetzung bestimmt, sondern auch durch die räumliche Anordnung ihrer Bestandteile. Selbst nach Jahrzehnten intensiver Forschung und der Veröffentlichung dutzender Membranmodelle und Theorien bleibt die genaue strukturelle Organisation der Plasmamembran ein Rätsel. Moderne Bildgebungsverfahren wie etwa die hochauflösende Fluoreszenzmikroskopie gehören mittlerweile zu den effizientesten Techniken der Lebenswissenschaften und werden immer öfter verwendet, um die räumliche Anordnung als auch die Anzahl von Biomolekülen in fixierten und lebenden Zellen zu studieren. Im Rahmen dieser Arbeit wurde die hochauflösende Mikroskopie-Methode dSTORM (direct stochastic optical reconstruction microscopy) angewendet, um die räumliche Verteilung von Membranmolekülen mit annähernd molekularer Auflösung zu untersuchen. Schwerpunkte dieser Arbeit sind dabei verschiedene Präparations- und Färbemethoden für die mikroskopische Untersuchung von Zellmembranen sowie lokalisationsbasierte quantitative Analysemethoden von Membranmolekülen. Eine Voraussetzung für die räumliche als auch quantitative Analyse von Membranmolekülen ist die Vermeidung von Photoschalt-Artefakten in rekonstruierten Lokalisationsmikroskopie-Bildern. Um dies genauer zu demonstrieren, wurden die Auswirkungen von Anregungsintensität, Markierungsdichte und verändertem Photoschalten auf die räumliche Verteilung von Proteinen der Plasma- und Mitochondrienmembran in dSTORM-Bildern analysiert. Es wird gezeigt, dass eine dicht markierte Plasmamembran in Kombination mit ungeeigneten Photoschaltraten zu artifiziellen Clustern in der Membran führt. Es sind vor Allem oft die Projektionen dreidimensionaler Membranstrukturen wie etwa Mikrovilli und Vesikel dafür verantwortlich, dass lokale Unterschiede in der Lokalisationsdichte entstehen, wodurch unter Umständen Bildartefakte generiert werden können. Darüber hinaus werden alternative Mikroskopie-Methoden und Möglichkeiten, Artefakte in Einzelmolekül-Lokalisationsmikroskopie-Bildern zu verhindern, präsentiert und ausführlich diskutiert. Ein weiteres zentrales Thema dieser Arbeit ist die räumliche Anordnung von glykosylierten Membranmolekülen. Es wird demonstriert, wie ein bioorthogonales chemisches Reportersystem bestehend aus modifizierten Monosacchariden und organischen Fluorophoren für die spezifische Markierung von Membran-assoziierten Glykoproteinen und –lipiden eingesetzt werden kann. Mittels dSTORM wird gezeigt, dass die Verteilung von Glykanen in der Plasmamembran unterschiedlicher Zelllinien homogen und frei von Clustern ist. Des Weiteren zeigt eine quantitative Analyse, dass sich in etwa fünf Millionen Glykane auf einer einzigen Zelle befinden. Die Ergebnisse demonstrieren, dass die Kombination aus metabolisch markierten Zielmolekülen, Click-Chemie und Einzelmolekül-Lokalisationsmikroskopie effizient genutzt werden kann, um Glykokonjugate auf Zelloberflächen zu untersuchen. In einem dritten Projekt wurde dSTORM zur Untersuchung von Rezeptormolekülen auf Krebszellen verwendet. Die Expression dieser Oberflächenproteine ist so gering, dass sich nur wenige Moleküle auf einer Zelle befinden, die jedoch als Zielmoleküle in der personalisierten Immuntherapie dienen könnten. Dafür wurden primäre Tumorzellen aus dem Knochenmark von Patienten, die am Multiplen Myelom erkrankt sind, auf die Expression des CD19-Oberflächenproteins als potentielles Ziel für CAR-modifizierte T-Zellen (chimeric antigen receptor) untersucht. Es wird gezeigt, dass sich, abhängig vom untersuchten Patienten, auf einer Zelle 60 bis 1600 CD19-Moleküle befinden. Funktionale in-vitro-Experimente demonstrieren, dass weniger als 100 CD19 Moleküle ausreichen, um CD19-CAR-T-Zellen zu aktivieren. Diese Ergebnisse werden mit Durchflusszytometrie-Daten verglichen und die wichtige Rolle von Lebendzellfärbung und geeigneten Kontrollexperimenten wird diskutiert.

C3d is a sub-fragment of the C3 component of the complement system.  Covalent binding of multiple C3ds to antigen reduces the activation threshold of cognate B lymphocytes by one thousand fold through co-ligation of the BCR and complement receptor 2 (CR2/CD21). Reverse transcriptase polymerase chain reaction (RT-PCR), revealed that, in cattle, four distinct complement receptors are produced from the Cr2 gene by alternative splicing. Cattle express two major variants of the Cr2 gene representing homologues of murine CR1 and CR2, each of which are expressed in both a long and a short form. Expression of CR1 was detected in splenocytes but not in splenic mononuclear cells or monocyte derived macrophages. CR2 was detected only on IgM+ blood cells and unsorted splenocytes but not in CD3+ cells, CD14+ cells or neutrophils. Additionally, the coding sequence of CD19, the CR2 co-signaling molecule was found.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / HematogÃnias sÃo cÃlulas jovens normais da medula Ãssea responsÃveis pela produÃÃo das cÃlulas de linhagem B do sistema imunolÃgico. A leucemia linfoblÃstica aguda de cÃlulas precursoras B representa um dos tipos de transformaÃÃo neoplÃsica das hematogÃnias. Devido a alta similaridade do ponto de vista citolÃgico dos dois tipos celulares, à possÃvel haver erros de interpretaÃÃo na anÃlise citolÃgica, fazendo-se necessÃrio em algumas circunstÃncias o uso de tÃcnicas complementares diagnÃsticas para diferenciar as cÃlulas benignas das malignas. O uso de marcadores imunolÃgicos atravÃs de anticorpos monoclonais marcados com fluorescÃncia tem grande aplicabilidade nos laboratÃrios especializados como rotina no estudo das leucemias. Os antÃgenos CD10 e CD19 estÃo expressos em ambos os tipos celulares de forma que se faz necessÃria uma extensÃo no uso de outros marcadores para caracterizaÃÃo da natureza benigna ou maligna das cÃlulas. Testou-se possÃveis diferenÃas nas curvas de expressÃo de CD10 e CD19 dos dois tipos celulares. Foram colhidas 36 amostras de medula Ãssea de pacientes pediÃtricos nÃo neoplÃsicos como grupo controle. A idade variou de 24 dias de vida a 15 anos com uma mÃdia de 5 anos. Foram colhidas tambÃm 39 amostras de pacientes portadores de LLA de linhagem B por ocasiÃo do diagnÃstico. A idade variou de 4 meses a 14 anos com uma mÃdia de 6,6 anos. Analisou-se as diferenÃas nas distribuiÃÃes quanto a intensidade de fluorescÃncia pelas mÃdias, desvios-padrÃo, coeficientes de variaÃÃo, coeficientes de inclinaÃÃo e coeficiente de curtose para os dois marcadores CD10 e CD19 nos dois grupos. Os valores individuais de cada amostra foram comparados com os intervalos gerados pelos valores do grupo controle com os seguintes respectivos resultados de sensibilidade e especificidade: 89,7% e 75% para um cut-off de mÃdia+2DP; 79,5% e 100% para mÃdia+2,5DP; e 71,8% e 100% para mÃdia+3DP. ConclusÃo: A expressÃo de CD10 e CD19 em blastos e hematogÃnias à diferente podendo ser de utilidade prÃtica na distinÃÃo entre os dois tipos celulares. / Hematogones are normal immature cells from bone marrow that are responsible for the production of the immune systemâs B cell lineage. The acute lymphoblastic leukemia (ALL) of precursors B cells represents one type of neoplastic transformation of hematogones. Due to their high similarity there are risks of erroneous interpretation consequently making it necessary use to complementary diagnostic techniques. The CD10 and CD19 antigens are expressed on both types of cells so, it is necessary use other monoclonal antibodies to identify malign or benign nature. In attempt to avoid the use of different antibodies we investigate possible differences in the expression of CD10 and CD19 in both cell types. We collected 36 samples of bone marrow from non-neoplastic patients as a control group. The age raged from 0 to 15 years with an average of 5 years. It was also collected 39 samples from patients with ALL of B cells. The age ranged from 0 to 14 years with an average of 6.6 years. We analyzed the differences between the fluorescence intensity concerning average, standard deviation, variation, inclination and kurtosis coefficients for the two markers. The individual values of each sample were compared with the intervals generated by the values of the control group: MEÂ2SD; MEÂ2.5SD and MEÂ3SD. It was possible to distinguish the groups with 89.7% and 75%; 79.5% and 100% and 71.8% e 100% of sensibility and specificity, respectively for the intervals. In conclusion, the expression of CD10 and CD19 antigens on blasts and hematogones is significantly different and may be useful in the differentiation of both cell types

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

Il est connu que les lymphocytes B (LB) ont un rôle pathogène dans de nombreuses maladies auto-immunes. Afin de comprendre comment la perte de tolérance B survient dans le lupus érythémateux systémique, maladie auto-immune prototypique, nous avons tout d’abord développé, dans un modèle murin, une méthode de marquage des LB pathogènes en cytométrie en flux. D’autre part, nous avons trouvé qu’un défaut de réponse au TLR9 est une caractéristique unique des LB de patients lupiques et est associée à une diminution d’expression du complexe CD19/CD21 à la surface des LB. Ces anomalies pourraient favoriser la survie des LB autoréactifs, et permettre la survenue de troubles auto-immuns. Dans un second temps nous avons exploré les fonctions d’une population lymphocytaire B méconnue: les LB thymiques humains. Nos résultats appuient le rôle tolérogène de ces LB, avec notamment l’expression, par 5% d’entre eux, de la protéine AIRE (AutoImmune REgulator), qui joue un rôle majeur dans la sélection négative des lymphocytes T. L’ensemble de ces données montrent la dichotomie fonctionnelle des LB et nous rappelle la complexité des processus régissant la tolérance et son maintien. / It is well known that B cells play pathogenic roles in a variety of autoimmune diseases. In order to understand how B tolerance breakdown occurs during systemic lupus erythematosus (SLE), a prototypical autoimmune disease, we first developed a method to identify autoreactive B cells by flow cytometry in a mouse model. In addition we found that a defective in vitro response to TLR9 agonists is a specific feature of SLE B cells and is associated with a CD19/CD21 complex downregulation. These abnormal functions in SLE B cells may contribute to B cell tolerance breakdown in these patients. But B-cell involvement in autoimmune diseases should not lead us to forget the functional diversity of B cells. This prompted us, in a second project, to analyze the tolerogenic role of a B cell population considered as simple bystanders of thymopoiesis: the human thymic B cells. Our results support the tolerogenic role of thymic B cells, as 5% of them express AIRE (AutoImmune REgulator), which plays a major role in cells in T cells negative selection. All these data underline the functional dichotomy of B cells and remind us the complexity of the processes governing tolerance and its maintenance.

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

Human T cells may be genetically modified to express targeted chimeric antigen receptors (CARs). We have previously demonstrated that T cells modified to express a CAR specific to the B cell tumor antigen CD19, termed 19-28z, successfully eradicate systemic human CD19+ tumors in SCID-Beige mice. While these results are encouraging, this xenogeneic tumor model fails to address potential limitations of this therapeutic approach in the clinical setting wherein these modified T cells encounter a hostile tumor microenvironment. Specifically, these models fail to address potential effector T cell inhibition mediated by endogenous regulatory T cells (Tregs). To investigate the role of inhibitory Tregs, we initially assessed the in vitro function of CAR-modified T cells in the context of Tregs. We found that CD19-targeted T cell proliferation and cytotoxicity were inhibited by purified natural Tregs. To further assess the role of these Tregs in vivo, we isolated and genetically modified Tregs to express the CD19-targeted 19z1 CAR. We verified specific trafficking of targeted Tregs to CD19+ tumors in vivo, and demonstrate that 19z1 Tregs wholly inhibit anti-tumor function of subsequently injected 19-28z effector T cells even at low Treg to effector T cell ratios (1:8). In order to overcome this limitation, we assessed whether the addition of a pro-inflammatory cytokine in vitro could overcome Treg inhibition. Indeed, the addition of exogenous IL-12 mediated resistance of 19-28z T cells to Treg inhibition. In light of this data we generated a bicistronic retroviral vector containing both the 19-28z CAR as well as the murine IL-12 fusion gene (19-28z IRES IL-12). Significantly, we found that 19-28z/IL-12+ T cells when compared to 19-28z+ T cells exhibited enhanced proliferation in vitro as well as resistance to Treg mediated inhibition. Finally, we demonstrate that 19-28z/IL-12+ T cells overcome Treg inhibition in vivo in our SCID-Beige Treg tumor model. In conclusion, tumor targeted T cells modified to express IL-12 demonstrate significantly enhanced in vivo anti-tumor efficacy in the presence of Tregs that are similarly targeted to the site of tumor. These results validate utilization of IL-12 secreting tumor targeted T cells in future clinical trials.

To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.