Transcriptional Profiling of Organ‐Specific Autoimmunity in Human

Our understanding of the pathogenesis of organ‐specific autoinflammation has been restricted by limited access to the target organs. Peripheral blood, however, as a preferred transportation route for immune cells, provides a window to assess the entire immune system throughout the body. Transcriptional profiling with RNA stabilizing blood collection tubes reflects in vivo expression profiles at the time the blood is drawn, allowing detection of the disease activity in different samples or within the same sample over time. The main objective of this Ph.D. study was to apply gene‐expression microarrays in the characterization of peripheral blood transcriptional profiles in patients with autoimmune diseases. To achieve this goal a custom cDNA microarray targeted for gene‐expression profiling of human immune system was designed and produced. Sample collection and preparation was then optimized to allow gene‐expression profiling from whole‐blood samples. To overcome challenges resulting from minute amounts of sample material, RNA amplification was successfully applied to study pregnancy related immunosuppression in patients with multiple sclerosis (MS). Furthermore, similar sample preparation was applied to characterize longitudinal genome‐wide expression profiles in children with type 1 diabetes (T1D) associated autoantibodies and eventually clinical T1D. Blood transcriptome analyses, using both the ImmunoChip cDNA microarray with targeted probe selection and genome‐wide Affymetrix U133 Plus 2.0 oligonucleotide array, enabled monitoring of autoimmune activity. Novel disease related genes and general autoimmune signatures were identified. Notably, down‐regulation of the HLA class Ib molecules in peripheral blood was associated with disease activity in both MS and T1D. Taken together, these studies demonstrate the potential of peripheral blood transcriptional profiling in biomedical research and diagnostics. Imbalances in peripheral blood transcriptional activity may reveal dynamic changes that are relevant for the disease but might be completely missed in conventional cross‐sectional studies.

Transcriptional and Epigenetic Regulation of Human CD4+ T Helper Lineage Specification

Activated T helper (Th) cells have ability to differentiate into functionally distinct Th1, Th2 and Th17 subsets through a series of overlapping networks that include signaling and transcriptional control and the epigenetic mechanisms to direct immune responses. However, inappropriate execution in the differentiation process and abnormal function of these Th cells can lead to the development of several immune mediated diseases. Therefore, the thesis aimed at identifying genes and gene regulatory mechanisms responsible for Th17 differentiation and to study epigenetic changes associated with early stage of Th1/Th2 cell differentiation. Genome wide transcriptional profiling during early stages of human Th17 cell differentiation demonstrated differential regulation of several novel and currently known genes associated with Th17 differentiation. Selected candidate genes were further validated at protein level and their specificity for Th17 as compared to other T helper subsets was analyzed. Moreover, combination of RNA interference-mediated downregulation of gene expression, genome-wide transcriptome profiling and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq), combined with computational data integration lead to the identification of direct and indirect target genes of STAT3, which is a pivotal upstream transcription factor for Th17 cell polarization. Results indicated that STAT3 directly regulates the expression of several genes that are known to play a role in activation, differentiation, proliferation, and survival of Th17 cells. These results provide a basis for constructing a network regulating gene expression during early human Th17 differentiation. Th1 and Th2 lineage specific enhancers were identified from genome-wide maps of histone modifications generated from the cells differentiating towards Th1 and Th2 lineages at 72h. Further analysis of lineage-specific enhancers revealed known and novel transcription factors that potentially control lineage-specific gene expression. Finally, we found an overlap of a subset of enhancers with SNPs associated with autoimmune diseases through GWASs suggesting a potential role for enhancer elements in the disease development. In conclusion, the results obtained have extended our knowledge of Th differentiation and provided new mechanistic insights into dysregulation of Th cell differentiation in human immune mediated diseases.