Experimental and computational analysis of human GM-CSF producing T-helper cells

<p>T-helper cells are crucial elements of the immune system, and they differentiate into several subsets depending on their cytokine environment. Each subset contributes to a certain type of immune response by producing its characteristic cytokines. Classification of T-helper cells into subsets is a useful conceptual framework to investigate their biological functions. However, this classification is a simplification because the subsets are rather a continuum than discrete types. A necessary condition for health is the balanced presence and activity of the different T-helper subsets. Imbalanced activity of the T-helper subsets contributes to several diseases ranging from cancer to autoimmune and inflammatory diseases.</p><p>This thesis focuses on studying T-helper subsets that have been previously described as being associated with and contributing to autoimmune diseases, for example Multiple Sclerosis. T-helper cells producing the cytokines GM-CSF, IFN-g and/or IL-17 have been described to be important in the pathogenesis of this disease. The biological aspects studied in this thesis include the differentiation, cytokine profiles and gene regulatory patterns of these T-helper subsets. From a methodological point of view, this thesis also explores possibilities to combine experimental and computational approaches.</p><p>Paper I is focused on the (in vitro) differentiation of human GM-CSF producing T-helper cells. Herein, various types of stimuli are tested and analyzed in a data driven way. As a main result, the cytokine TGF-b is identified as a context dependent modulatory factor that can induce or repress the differentiation of human GM-CSF producing T-helper cells depending on activation type or sodium chloride concentration. GM-CSF production is highly correlated with IFN-g on the single cell level and with FOXP3 on the population level. Furthermore, human GM-CSF producing T-helper cells comprise several subpopulations, the composition of which is altered by the cytokine environment.</p><p>Paper II explores the role of splice isoforms of FOXP3 (the key transcription factor of immunosuppressive regulatory T cells) in Crohn’s disease and in the differentiation of human inflammatory T-helper cell subsets. This paper identifies a connection between the pro-inflammatory cytokine IL-1b, FOXP3 alternative splicing, and the differentiation of pro-inflammatory IL-17 producing T-helper cells. Furthermore, the paper reveals a significant correlation between a certain FOXP3 splice isoform and IL-17 expression in affected tissue from Crohn’s disease patients. Paper III presents a web application that allows non-expert users to apply pre-processing and advanced statistical methods on single cell cytometry data. The aim of this tool is to make the statistical approach to cytometry data more accessible to wet-lab biologists, and therefore serve this unmet need.</p><p>Paper IV aims to give an insight and understanding into the gene regulation and the connection between chromatin and transcriptional activity in human T-helper cells. Herein, the focus is on studying the characteristics of memory and GM-CSF producing T-helper cells. For this purpose, chromatin (ATAC-seq) and gene expression (RNA-seq) data are utilized in a combined manner and gene regulatory networks, including transcription factors that appear to be important for defining memory and GM-CSF producing T-helper cells, are identified. These transcription factors might be involved in diseases such as Multiple Sclerosis, and they can be potential candidates for future research towards therapeutic goals.</p><p>In summary, the thesis aims to contribute to our understanding of human T cell biology that is relevant for disease by combining experimental cellular immunology, next generation sequencing and computational approaches.</p><h3>List of scientific papers</h3><p>I. Éliás S, Schmidt A, Kannan V, Andersson J, Tegnér J. TGF-β Affects the Differentiation of Human GM-CSF+ CD4+ T Cells in an Activation- and Sodium-Dependent Manner. Frontiers in Immunology. (2016) 7:603. <br><a href="https://doi.org/10.3389/fimmu.2016.00603">https://doi.org/10.3389/fimmu.2016.00603</a><br><br> </p><p>II. Mailer RKW, Joly A-L, Liu S, Éliás S, Tegnér J, Andersson J. IL-1β promotes Th17 differentiation by inducing alternative splicing of FOXP3. Scientific Reports. (2015) 5:14674. <br><a href="https://doi.org/10.1038/srep14674">https://doi.org/10.1038/srep14674</a><br><br> </p><p>III. Papoutsoglou G, Athineou G, Lagani V, Xanthopoulos I, Schmidt A, Éliás S, Tegnér J, Tsamardinos I. SCENERY: a web application for (causal) network reconstruction from cytometry data. Nucleic Acids Research. (2017) 45:W270-W275. <br><a href="https://doi.org/10.1093/nar/gkx448">https://doi.org/10.1093/nar/gkx448</a><br><br> </p><p>IV. Éliás S, Schmidt A, Gomez-Cabrero D, Tegnér J. Gene regulatory network of human naïve and memory T helper cells focused on GM-CSF producing cells. [Manuscript]</p>