Stuttgart, Univ., Diss., 2001.

CD4+ T-Helferzellen (Th) entwickeln sich zu Th1 und Th2 Zellen, die nach ihrer Funktion und Zytokinexpression eingeteilt werden. Die differentielle Induktion von Th Zellen, die Th1 oder Th2 Zytokine exprimieren, ist der Schlüssel zur Regulation von Immunantworten bei Infektionskrankheiten, Allergien und Autoimmunerkrankungen. Daher können stabil exprimierte Oberflächenmoleküle, die spezifisch für die funktionell unterschiedlichen Th Zellen sind, von besonderer Bedeutung für die Analyse und selektive funktionelle Modulation von Th Subtypen sein und erlauben es neue therapeutische Strategien für die Behandlung von allergischen und Autoimmunerkrankungen zu etablieren. TIM-3 wurde kürzlich identifiziert als ein Molekül, welches selektiv auf der Oberfläche von Th1 Zellen exprimiert wird und welches möglicherweise eine Rolle bei der Induktion von Autoimmunerkrankungen spielt. Um monoklonale Antikörper gegen humanes TIM-3 zu produzieren, wurde die humane TIM-3 cDNA von in vitro generierten dendritischen Zellen kloniert. Der extrazelluläre Teil des Gens wurde in den prokaryotischen Expressionsvektor pQE100S insertiert und in E.coli BL21(DE3) exprimiert. Die gesamte codierende Sequenz wurde in den eukaryotischen Expressionsvektor pIRES2EGFP subkloniert und auf der Oberfläche von Säugetierzellen exprimiert. Stabile Transfektanten der CHO-K1 und HEK293 Zelllinie wurde etabliert. Die Balb/c Mäuse wurden mit löslichem und unlöslichem rekombinanten humanem TIM-3 sowie mit stabilen Transfektanten für humanes TIM-3 immunisiert. Milzzellen dieser Tiere wurden mit der Myelomzelllinie P3 X 63 Ag8.653 fusioniert. Die entwickelten Hybridome wurden im ELISA und mittels FACS auf Spezifität gegen humanes TIM-3 hin untersucht. Ein Klon der generierten Hybridome war positiv im ELISA zeigte jedoch kein Signal gegen TIM-3 auf der Oberfläche von Zellen. / CD4+ T helper (Th) cells develop into effector Th1 and Th2 cells, which are frequently categorized according to their function and cytokine expression. The differential induction of Th cells expressing Th1 or Th2 cytokines is key to the regulation of immune responses by infectious diseases, allergies and autoimmnune diseases. Thus, stably expressed surface molecules, significant for functionally different types of Th cells could be of utmost importance for the analysis and selective functional modulation of Th subsets and provide new therapeutic strategies for the trestment of allergic or autoimmune diseases. TIM-3 was recently identified as a molecule that is selectively expressed on the surface of Th1 cells and that may have a role in the induction of autoimmune disease. To produce monoclonal antibody of human TIM-3, the human TIM-3 cDNA was cloned from in vitro generated dendritic cells. The extracellular domain of human TIM-3 was inserted into prokaryotic expression vector pQE100S and expressed in E.coli BL21(DE3). The whole coding region was subcloned into eukaryotic expression vector pIRES2EGFP and expressed on the surface of mammalian cells. The stable transfectants of CHO-K1 and HEK-293 cell line was established. The BALB/c mice were immunized with soluble and insoluble recombinant human TIM-3 and also with stable transfectants. Splenocytes were fused with P3 X 63 Ag8.653 myeloma cells. The generated hybridomas were tested in ELISA and FACS for specificity against human TIM-3. A generated clone was positive in ELISA but did not respond to the TIM-3 molecule on the cell surface.

The availability of several methods to unambiguously mark individual cells has strongly fostered the understanding of clonal developments in hematopoiesis and other stem cell driven regenerative tissues. While cellular barcoding is the method of choice for experimental studies, patients that underwent gene therapy carry a unique insertional mark within the transplanted cells originating from the integration of the retroviral vector. Close monitoring of such patients allows accessing their clonal dynamics, however, the early detection of events that predict monoclonal conversion and potentially the onset of leukemia are beneficial for treatment. We developed a simple mathematical model of a self-stabilizing hematopoietic stem cell population to generate a wide range of possible clonal developments, reproducing typical, experimentally and clinically observed scenarios. We use the resulting model scenarios to suggest and test a set of statistical measures that should allow for an interpretation and classification of relevant clonal dynamics. Apart from the assessment of several established diversity indices we suggest a measure that quantifies the extension to which the increase in the size of one clone is attributed to the total loss in the size of all other clones. By evaluating the change in relative clone sizes between consecutive measurements, the suggested measure, referred to as maximum relative clonal expansion (mRCE), proves to be highly sensitive in the detection of rapidly expanding cell clones prior to their dominant manifestation. This predictive potential places the mRCE as a suitable means for the early recognition of leukemogenesis especially in gene therapy patients that are closely monitored. Our model based approach illustrates how simulation studies can actively support the design and evaluation of preclinical strategies for the analysis and risk evaluation of clonal developments.

No description available.

The availability of several methods to unambiguously mark individual cells has strongly fostered the understanding of clonal developments in hematopoiesis and other stem cell driven regenerative tissues. While cellular barcoding is the method of choice for experimental studies, patients that underwent gene therapy carry a unique insertional mark within the transplanted cells originating from the integration of the retroviral vector. Close monitoring of such patients allows accessing their clonal dynamics, however, the early detection of events that predict monoclonal conversion and potentially the onset of leukemia are beneficial for treatment. We developed a simple mathematical model of a self-stabilizing hematopoietic stem cell population to generate a wide range of possible clonal developments, reproducing typical, experimentally and clinically observed scenarios. We use the resulting model scenarios to suggest and test a set of statistical measures that should allow for an interpretation and classification of relevant clonal dynamics. Apart from the assessment of several established diversity indices we suggest a measure that quantifies the extension to which the increase in the size of one clone is attributed to the total loss in the size of all other clones. By evaluating the change in relative clone sizes between consecutive measurements, the suggested measure, referred to as maximum relative clonal expansion (mRCE), proves to be highly sensitive in the detection of rapidly expanding cell clones prior to their dominant manifestation. This predictive potential places the mRCE as a suitable means for the early recognition of leukemogenesis especially in gene therapy patients that are closely monitored. Our model based approach illustrates how simulation studies can actively support the design and evaluation of preclinical strategies for the analysis and risk evaluation of clonal developments.