Deciphering the effects of genetic variability in regulatory T cells - links with autoimmunity

The role of the immune system is to defend an individual from infectious agents and clear the body from harmful own elements, such as e.g. cancerous cells. This is orchestrated by a number of specialised immune cells that recognise and eliminate potentially dangerous components. At the same time, the immune system undergoes careful negative control that restricts the immune response to the necessary minimum. This is carried out by regulatory T cells (Tregs). However, under certain conditions, the immune cells can react against individual self tissues as if they were harmful factors. If such an immune response is not restrained, it can lead to the development of chronic inflammation and pathogenic conditions known as autoimmune diseases.

Autoimmune diseases can be debilitating, difficult to diagnose and frequent - they affect one in ten individuals in Western countries. The exact origin of autoimmune diseases is unknown, although they are thought to result from a combination of environmental and genetic factors. To date, hundreds of studies have linked thousands of genetic variants with increased risk of developing immune diseases. However, establishing the functional effects of these genetic variants remains challenging.

The role of genetic variants associated to autoimmune diseases has been linked with modulating function of Tregs. In this project, I propose to use large scale genomic and immunologic assays to characterise the activity of Tregs from healthy individuals and correlate it with genetic variants. Genomic assays will provide the measurement of gene activity, while the immune assays will provide information on cell activity. I will use data from cells in resting and stimulated states - the later is used to mimic physiologically occurring immune response.

My goal in this project will be to develop new computational tools and methods that will pioneer an integrative framework to combine these data in a meaningful way. Ultimately, I want to identify what are the exact causal molecular mechanisms through which genetic variants predispose to autoimmune diseases.

The findings of this project will have benefits on both scientific and social levels. In a direct way, our results will help to understand the mechanisms that cause the failures in the immune system that lead to the onset and progression of autoimmunity. This information potentially can be of high impact for the patients as it will identify new therapeutic targets.

Aims: The majority of the causal variants underlying associations to complex traits remain unknown. This proposal aims to define the causal variants at loci associated with autoimmune diseases, by studying the effects of of associated variants on CD4+ regulatory T cells (Tregs).

Objectives: Specifically, I will 1) construct a catalogue of chromatin and gene activity in Tregs from 100 individuals, 2) functionally prioritise putative causal variants based on their effects on gene regulatory features, as well as 3) gene and cytokine expression levels and 4) assess if the observed effects are specific cellular states, resting or stimulated.

Methodology: Dr Trynka's group is generating functional genomic data from Tregs from 100 healthy blood donors. The assays include: 1) genome wide genotyping, 2) characterising active promoters by ChIP-seq for H3K4me3 histone modification , 3) mapping open chromatin regions using ATAC-seq, and 4) quantifying gene expression with RNA-seq. Assays 2-4 include both resting and stimulated Tregs. Using these datasets I will identify genetic variants that co-localise with H3K4me3 and ATAC features and test for correlation between alleles and the number of mapped sequence reads. Variants with allele-specific effects on histone modification levels or chromatin accessibility will comprise a pool of prioritised causal variants. I will further correlate these alleles with gene expression levels and assess the genetic effects on cytokine levels. Finally, if a subset of nominated causal alleles point to disrupted transcription factor binding (TF), I will perform functional validation using TF-specific ChIP-seq.

Scientific and medical opportunities: We will gain insights into the causal molecular mechanisms underlying the immune disease loci that may lead to identification of novel drug targets and disease biomarkers. Finally, the framework proposed here can be easily adapted to other cell types and complex traits.

Although individually ADs may be perceived as rare diseases, collectively autoimmune disorders affect 5-10% of the population in Western countries. These disorders are often chronic and disabling, causing great patient suffering. Furthermore, autoimmune disorders often co-occur and aggregate in families, increasing the familial burden of the affected individuals. Patient care and treatment is expensive and typically life-long, posing high costs for the public health system. The translation of our findings to patient care is a long term but essential aim of the present project. Often diagnosis of ADs is difficult and can delay treatment, which normally consists of a combination of biologic agents and conventional disease-modifying drugs, such as methotrexate. These treatments affect pleiotropic molecules and pathways and often result in drug-induced intolerances and discontinuation of the treatment becomes necessary. The proposed study will deliver a better understanding of biology of regulatory T cells (Tregs) and the identification of either the target genes or the transcription factors whose binding is altered by the genetic variants associated to autoimmune disease. This may lead to the selection of novel relevant molecules or pathways that could be used as potential therapeutic targets. Importantly, rather than acting generically they could directly impact pathways specific to Tregs. As a consequence, these results may provide both improved specificity of treatment and increased patient tolerance. Therefore, great benefits for patient treatment and prognosis may arise from these initiatives, saving human and economic costs for the society.

Because of the cross-disciplinary nature of our study, bridging genomics with immunology, I expect that our results will impact a diverse scientific community and provide a valuable resource to researchers in the fields of immunology, cellular biology, and those working on autoimmune diseases with strong links to Tregs not covered in our analysis. For instance, publically available genomic data from human Tregs is scarce and our project will fill this gap by providing a comprehensive catalogue of gene regulatory regions and key pathways active in stimulated and resting Tregs. Similarly, the analytical approaches that I will develop here will be generally applicable to a wider range of cell types, conditions, and diseases, amplifying the scope and relevance of our investigation to other fields.