Development of regulatory T cells in the human thymus

The role of the immune system is to protect an organism against pathogens while maintaining tolerance against self. T cells are an essential component of the immune system and they develop in the thymus. The AIRE (autoimmune regulator) gene product plays an important role in T cell development, as it promotes expression of peripheral tissue antigens in the thymus. Developing T cells, thymocytes, which recognize self-antigens with high affinity are deleted. However, this deletion process is not perfect and not all autoreactive T cells are destroyed. When the distinction between self and non-self fails, tolerance breaks and the immune system attacks the host s own tissues. This results in autoimmunity. Regulatory T cells contribute to the maintenance of self-tolerance. They can actively suppress the function of autoreactive cells. Several populations of cells with regulatory properties have been described, but the best characterized population is the natural regulatory T cells (Treg cells), which develop in the thymus and express the transcription factor FOXP3. The thymic development of Treg cells in humans is the subject of this thesis. Thymocytes at different developmental stages were analyzed using flow cytometry. The CD4-CD8- double-negative (DN) thymocytes are the earliest T cell precursors in the T cell lineage. My results show that the Treg cell marker FOXP3 is up-regulated already in a subset of these DN thymocytes. FOXP3+ cells were also found among the more mature CD4+CD8+ double-positive (DP) cells and among the CD4+ and CD8+ single-positive (SP) thymocytes. The different developmental stages of the FOXP3+ thymocytes were isolated and their gene expression examined by quantitative PCR. T cell receptor (TCR) repertoire analysis was used to compare these different thymocyte populations. My data show that in humans commitment to the Treg cell lineage is an early event and suggest that the development of Treg cells follows a linear developmental pathway, FOXP3+ DN precursors evolving through the DP stage to become mature CD4+ Treg cells. Most T cells have only one kind of TCR on their cell surface, but a small fraction of cells expresses two different TCRs. My results show that the expression of two different TCRs is enriched among Treg cells. Furthermore, both receptors were capable of transmitting signals when bound by a ligand. By extrapolating flow cytometric data, it was estimated that the majority of peripheral blood Treg cells are indeed dual-specific. The high frequency of dual-specific cells among human Treg cells suggests that dual-specificity has a role in directing these cells to the Treg cell lineage. It is known that both genetic predisposition and environmental factors influence the development of autoimmunity. It is also known that the dysfunction or absence of Treg cells leads to the development of autoimmune manifestations. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare monogenic autoimmune disease, caused by mutations in the AIRE gene. In the absence of AIRE gene product, deletion of self-specific T cells is presumably disturbed and autoreactive T cells escape to the periphery. I examined whether Treg cells are also affected in APECED. I found that the frequency of FOXP3+ Treg cells and the level of FOXP3 expression were significantly lower in APECED patients than in controls. Additionally, when studied in cell cultures, the suppressive capacity of the patients' Treg cells was impaired. Additionally, repertoire analysis showed that the TCR repertoire of Treg cells was altered. These results suggest that AIRE contributes to the development of Treg cells in humans and the selection of Treg cells is impaired in APECED patients. In conclusion, my thesis elucidates the developmental pathway of Treg cells in humans. The differentiation of Tregs begins early during thymic development and both the cells dual-specificity and AIRE probably affect the final commitment of Treg cells.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.

Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.

Finnish Dosimetric Practice for Epithermal Neutron Beam Dosimetry in Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a form of chemically targeted radiotherapy that utilises the high neutron capture cross-section of boron-10 isotope to achieve a preferential dose increase in the tumour. The BNCT dosimetry poses a special challenge as the radiation dose absorbed by the irradiated tissues consists of several dose different components. Dosimetry is important as the effect of the radiation on the tissue is correlated with the radiation dose. Consistent and reliable radiation dose delivery and dosimetry are thus basic requirements for radiotherapy. The international recommendations for are not directly applicable to BNCT dosimetry. The existing dosimetry guidance for BNCT provides recommendations but also calls for investigating for complementary methods for comparison and improved accuracy. In this thesis the quality assurance and stability measurements of the neutron beam monitors used in dose delivery are presented. The beam monitors were found not to be affected by the presence of a phantom in the beam and that the effect of the reactor core power distribution was less than 1%. The weekly stability test with activation detectors has been generally reproducible within the recommended tolerance value of 2%. An established toolkit for epithermal neutron beams for determination of the dose components is presented and applied in an international dosimetric intercomparison. The measured quantities (neutron flux, fast neutron and photon dose) by the groups in the intercomparison were generally in agreement within the stated uncertainties. However, the uncertainties were large, ranging from 3-30% (1 standard deviation), emphasising the importance of dosimetric intercomparisons if clinical data is to be compared between different centers. Measurements with the Exradin type 2M ionisation chamber have been repeated in the epithermal neutron beam in the same measurement configuration over the course of 10 years. The presented results exclude severe sensitivity changes to thermal neutrons that have been reported for this type of chamber. Microdosimetry and polymer gel dosimetry as complementary methods for epithermal neutron beam dosimetry are studied. For microdosimetry the comparison of results with ionisation chambers and computer simulation showed that the photon dose measured with microdosimetry was lower than with the two other methods. The disagreement was within the uncertainties. For neutron dose the simulation and microdosimetry results agreed within 10% while the ionisation chamber technique gave 10-30% lower neutron dose rates than the two other methods. The response of the BANG-3 gel was found to be linear for both photon and epithermal neutron beam irradiation. The dose distribution normalised to dose maximum measured by MAGIC polymer gel was found to agree well with the simulated result near the dose maximum while the spatial difference between measured and simulated 30% isodose line was more than 1 cm. In both the BANG-3 and MAGIC gel studies, the interpretation of the results was complicated by the presence of high-LET radiation.

Proton magnetic resonance spectroscopy of a boron neutron capture therapy 10B-carrier, L-p-boronophenylalanine-fructose complex

Boron neutron capture therapy (BNCT) is a radiotherapy that has mainly been used to treat malignant brain tumours, melanomas, and head and neck cancer. In BNCT, the patient receives an intravenous infusion of a 10B-carrier, which accumulates in the tumour area. The tumour is irradiated with epithermal or thermal neutrons, which result in a boron neutron capture reaction that generates heavy particles to damage tumour cells. In Finland, boronophenylalanine fructose (BPA-F) is used as the 10B-carrier. Currently, the drifting of boron from blood to tumour as well as the spatial and temporal accumulation of boron in the brain, are not precisely known. Proton magnetic resonance spectroscopy (1H MRS) could be used for selective BPA-F detection and quantification as aromatic protons of BPA resonate in the spectrum region, which is clear of brain metabolite signals. This study, which included both phantom and in vivo studies, examined the validity of 1H MRS as a tool for BPA detection. In the phantom study, BPA quantification was studied at 1.5 and 3.0 T with single voxel 1H MRS, and at 1.5 T with magnetic resonance imaging (MRSI). The detection limit of BPA was determined in phantom conditions at 1.5 T and 3.0 T using single voxel 1H MRS, and at 1.5 T using MRSI. In phantom conditions, BPA quantification accuracy of ± 5% and ± 15% were achieved with single voxel MRS using external or internal (internal water signal) concentration references, respectively. For MRSI, a quantification accuracy of <5% was obtained using an internal concentration reference (creatine). The detection limits of BPA in phantom conditions for the PRESS sequence were 0.7 (3.0 T) and 1.4 mM (1.5 T) mM with 20 × 20 × 20 mm3 single voxel MRS, and 1.0 mM with acquisition-weighted MRSI (nominal voxel volume 10(RL) × 10(AP) × 7.5(SI) mm3), respectively. In the in vivo study, an MRSI or single voxel MRS or both was performed for ten patients (patients 1-10) on the day of BNCT. Three patients had glioblastoma multiforme (GBM), and five patients had a recurrent or progressing GBM or anaplastic astrocytoma gradus III, and two patients had head and neck cancer. For nine patients (patients 1-9), MRS/MRSI was performed 70-140 min after the second irradiation field, and for one patient (patient 10), the MRSI study began 11 min before the end of the BPA-F infusion and ended 6 min after the end of the infusion. In comparison, single voxel MRS was performed before BNCT, for two patients (patients 3 and 9), and for one patient (patient 9), MRSI was performed one month after treatment. For one patient (patient 10), MRSI was performed four days before infusion. Signals from the tumour spectrum aromatic region were detected on the day of BNCT in three patients, indicating that in favourable cases, it is possible to detect BPA in vivo in the patient’s brain after BNCT treatment or at the end of BPA-F infusion. However, because the shape and position of the detected signals did not exactly match the BPA spectrum detected in the in vitro conditions, assignment of BPA is difficult. The opportunity to perform MRS immediately after the end of BPA-F infusion for more patients is necessary to evaluate the suitability of 1H MRS for BPA detection or quantification for treatment planning purposes. However, it could be possible to use MRSI as criteria in selecting patients for BNCT.