The miRNA Biogenesis Machinery Modulates Lineage Commitment During αβ T Cell Development

This thesis investigates the role of the Dicer RNA endonuclease in promoting CD4 and CD8 lineage commitment in developing αβ T lymphocytes. Our laboratory has previously shown that Dicer promotes survival of developing thymocytes undergoing antigen receptor induced DNA double strand breaks. During the course of these studies we made the unexpected observation that mice with T cell specific Dicer inactivation and transgenic expression of the pro-survival molecule BCL2 (LckCre;Eμ;BCL2;Dicerflox/flox) contain a population of aberrant CD4+CD8+ peripheral lymphocytes of unknown origin. I have utilized molecular biology, cellular immunology, and complex mouse genetic models to examine the developmental history and fate of these aberrant CD4+CD8+ cells. My results indicate that aberrant peripheral CD4+CD8+ cells arise from impaired co-receptor silencing of either CD4 or CD8 in MHCI or MHCII-restricted T cells, respectively. Initiation of co-receptor silencing is impaired during thymic development, whereas maintenance of co-receptor silencing in mature T cells does not appear to be defective in the absence of Dicer. Aberrant CD4+CD8+ cells from mice expressing either MHCI or MHCII-restricted TCR transgenes exhibit reduced expression of the master transcriptional regulators Runx3 and ThPOK, which promote commitment to the CD8 and CD4 lineages, respectively. Thus, lineage commitment is impaired in the absence of Dicer. Impaired co-receptor silencing was also observed in developing T cells lacking the RNA endonuclease Drosha, suggesting that miRNAs mediate appropriate co-receptor silencing and lineage commitment during αβ T cell development. These results identify a novel role for the miRNA biogenesis machinery in promoting appropriate lineage commitment during αβ T cell development, and indicate that thymic egress and lineage commitment are separable genetic programs during T cell development

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

The miRNA biogenesis machinery modulates lineage commitment during αβ T cell development

This thesis investigates the role of the Dicer RNA endonuclease in promoting CD4 and CD8 lineage commitment in developing αβ T lymphocytes. Our laboratory has previously shown that Dicer promotes survival of developing thymocytes undergoing antigen receptor induced DNA double strand breaks. During the course of these studies we made the unexpected observation that mice with T cell specific Dicer inactivation and transgenic expression of the pro-survival molecule BCL2 (LckCre;Eµ;BCL2;Dicerflox/flox) contain a population of aberrant CD4+CD8+ peripheral lymphocytes of unknown origin. I have utilized molecular biology, cellular immunology, and complex mouse genetic models to examine the developmental history and fate of these aberrant CD4+CD8+ cells. My results indicate that aberrant peripheral CD4+CD8 + cells arise from impaired co-receptor silencing of either CD4 or CD8 in MHCI or MHCII-restricted T cells, respectively. Initiation of co-receptor silencing is impaired during thymic development, whereas maintenance of co-receptor silencing in mature T cells does not appear to be defective in the absence of Dicer. Aberrant CD4+CD8+ cells from mice expressing either MHCI or MHCII-restricted TCR transgenes exhibit reduced expression of the master transcriptional regulators Runx3 and ThPOK, which promote commitment to the CD8 and CD4 lineages, respectively. Thus, lineage commitment is impaired in the absence of Dicer. Impaired co-receptor silencing was also observed in developing T cells lacking the RNA endonuclease Drosha, suggesting that miRNAs mediate appropriate co-receptor silencing and lineage commitment during αβ T cell development. These results identify a novel role for the miRNA biogenesis machinery in promoting appropriate lineage commitment during αβ T cell development, and indicate that thymic egress and lineage commitment are separable genetic programs during T cell development

Cholesterol induced heart valve inflammation and injury: efficacy of cholesterol lowering treatment

BACKGROUND: Heart valves often undergo a degenerative process leading to mechanical dysfunction that requires valve replacement. This process has been compared with atherosclerosis because of shared pathology and risk factors. In this study, we aimed to elucidate the role of inflammation triggered by cholesterol infiltration and cholesterol crystals formation causing mechanical and biochemical injury in heart valves. METHODS: Human and atherosclerotic rabbit heart valves were evaluated. New Zealand White male rabbits were fed an enriched cholesterol diet alone or with simvastatin and ezetimibe simultaneous or after 6 months of initiating cholesterol diet. Inflammation was measured using C-reactive protein (CRP) and RAM 11 of tissue macrophage content. Cholesterol crystal presence and content in valves was evaluated using scanning electron microscopy. RESULTS: Cholesterol diet alone induced cholesterol infiltration of valves with associated increased inflammation. Tissue cholesterol, CRP levels and RAM 11 were significantly lower in simvastatin and ezetimibe rabbit groups compared with cholesterol diet alone. However, the treatment was effective only when initiated with a cholesterol diet but not after lipid infiltration in valves. Aortic valve cholesterol content was significantly greater than all other cardiac valves. Extensive amounts of cholesterol crystals were noted in rabbit valves on cholesterol diet and in diseased human valves. CONCLUSIONS: Prevention of valve infiltration with cholesterol and reduced inflammation by simvastatin and ezetimibe was effective only when given during the initiation of high cholesterol diet but was not effective when given following infiltration of cholesterol into the valve matrix

CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells.

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies

CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells.

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies