Immunogenetic Study of Autoimmunity-Associated Variants in Selected TNF Superfamily Genes

Abstract

Major advances in human genetics have paved the way for the development of Genome Wide Association Studies (GWAS) in an effort to understand complex traits - including autoimmune diseases such as Multiple Sclerosis (MS). Using unbiased genome screens of affected individuals and appropriately matched controls, these studies aim to establish an association between genetic variants and a phenotype of interest. However, whilst GWAS studies have identified thousands of loci associated with a host of traits, only a handful of these associations were successfully translated into functional understanding. The vast majority of variants identified by GWAS studies map to non-coding regions and therefore thought to promote disease by regulating gene expression, but it is often unclear which gene a specific variant regulates, in which cell subset and under what physiological state. The overarching aim of my work was to study selected MS-associated variants in an effort to understand how they functionally contribute to disease pathogenesis. My choice of gene candidates to take forward for validation was guided by publicly available functional genomics datasets, which I used to prioritise genes and signalling pathways from GWAS summary data. This pointed to several Single Nucleotide Polymorphisms (SNPs) proximal to or intronic within genes involved in LIGHT (TNFSF14) signalling. To establish the settings in which these SNPs regulate gene expression, I first recruited healthy volunteers that are heterozygous for my SNPs of interest and assayed Allele Specific Expression (ASE) under different activation conditions. This was done in a number of immune cell subsets derived from Peripheral Blood Mononuclear Cells (PBMC) and revealed subset- and state-dependent regulation of gene expression. Notably, the MS-associated variant rs1077667-C was shown to downregulate expression of TNFSF14 in monocytes – and this disappeared as cells were activated or in-vitro differentiated to monocyte-derived macrophages (MDMs). To assess whether this translates to differences in protein expression, I recruited 60 healthy volunteers that are homozygous for either the risk (CC) or the protective (TT) allele on rs1077667. In line with changes detected at the message level, monocytes from individuals homozygous for the risk allele (CC) had significantly lower expression of LIGHT on their surface (as assessed by flow cytometry), lower serum levels of LIGHT, and lower levels of secreted LIGHT in monocyte culture supernatants (both measured with Bead-Based Immunoassay). Next, I followed up on these findings by studying LIGHT signalling in monocytes and MDMs cultured in vitro under different activating/polarising conditions. I showed that, similar to the effect of the protective allele (T), treatment of cultured monocytes with Vitamin D (1α,25-Dihydroxyvitamin D3) results in upregulation of TNFSF14 gene expression. Moreover, I present preliminary data to show that treatment of cultured monocytes and MDMs with recombinant human LIGHT resulted in upregulation of immunoregulatory markers. In addition to blood-derived cells, the brain harbours tissue-resident macrophages that are distinct in ontogeny and function (microglia). Therefore, I extended my work to human microglia I differentiated from induced Pluripotent Stem Cells (iPSCs) derived from PBMC of the same individuals recruited for the ASE cohort. This was done in order to study the extent to which the findings presented are applicable to this unique yolk-sac derived myeloid cell population.