The regulatory role of genetic risk factors for autoimmune diseases on thymic antigen-presenting cells and self-tolerance

Autoimmune diseases affect about 5% of the population and are caused by an immunological attack against the body´s own tissues. Several hundred genetic variants are known to predispose to autoimmune diseases, but their biological effects are largely unknown. A hallmark of the genetic risk variants is that they are mostly located in regulatory regions of immune genes. With this proposal, we are expanding the understanding of the role of autoimmune risk variants on thymic antigen-presenting cells and their influence on self-tolerance by first generating a comprehensive overview of all individual cell populations existing in human thymus. We have established laboratory protocols and data analyses pipelines for single cell data. This has required rigorous optimization to ensure that the full repertoire of cell types of interest is covered. These are recent and rapidly evolving technologies, which now offer the opportunity to study several data modalities at single cell resolution. We have generated and analyzed single cell data with information on RNA and protein expression levels (CITE-seq), immune receptors (scAIRR-seq), chromatin accessibility (scATAC-seq), and binding sites for the three selected transcription factors AIRE, FEZF2, and DEAF1 (scCut&Tag). In addition, we have generated and analyzed RNA sequencing data with spatial information (spatial transcriptomics). A combined use of these data modalities has enabled us to study novel aspects that may not be possible to elucidate based on one modality alone. The results have provided novel insights into the role of distinct cell type subsets during immune cell development, how nearby cell populations residing in distinct thymic microenvironments communicate, and how cell types vary in immune receptor profiles and chromatin landscapes. We have focused in particular on the development of unconventional groups of T-cells which are involved in immune regulation and self-tolerance, and how different antigen presenting cell types affect the development of such unconventional T cells. These findings are of interest for understanding how genetic variants that affect immune cell development in thymus entail susceptibility to autoimmunity, and highlight mechanisms that may be of relevance for the development of future diagnostics and therapies. Further, the generated data represent a resource for future single-cell studies, including studies aiming to understand how the identified cell populations differ between healthy individuals and individuals with autoimmune disease. As a part of the work, the doctoral research fellow on the project has had an virtual research stay (due to COVID-19) with Sarah Teichmanns group, which is world leading in single cell biology. A paper treating the findings have been published in Frontiers in Immunology, a second paper has been submitted, and a third paper is being prepared for submission. These three papers are included in the submitted thesis by the doctoral research fellow. Further, a fourth paper is in preparation, in collaboration with Anthony Matelier/Centre of molecular medicine Norway (NCMM) and a fifth paper will be written regarding HLA allelic expression. In addition to these publications, the project has been presented at national and international scientific conferences, and also been communicated to a broader audience though popular science blog post and social media.

Funnene fra prosjektet har økt kunnskapen vår om immuncelleutvikling i tymus, spesielt utviklingen fra ukonvensjonelle typer av T-celler som regulatoriske T celler og CD8 T-celler, og hvordan undergrupper av antigen-presenterende og stromale celler påvirker utviklingen. Ved bruk av moderne, multi-modale enkeltcelleteknologier har vi kunnet studere heterogenitet som ikke ville ha kommet frem i studier på bulk-nivå. Denne forståelsen av heterogenitet i tymus er nødvendig for effektiv utvikling av nye terapier mot autoimmun sykdom. Ukonvensjonelle, immun-modulerende T-celler har potensiale i behandling av pasienter med autoimmun sykdom, eksemplifisert av forskning på celleterapier basert på overføring av regulatoriske T celler, eller terapier som målrettet prøver å fremme utviklingen og aktiviteten av slike celler, som behandling med lav-dose IL-2. Våre funn indikerer at det finnes ulike utviklingsveier for ukonvensjonelle, immun-modulerende celler i tymus. Cellene som bøyer av mot de ulike utviklingsveiene varierer i modenhet, respons på immunreseptorsignalering, lokalisering i vevet, og i kommunikasjon med nærliggende antigenpresenterende og stromale celler. Denne heterogeniteten har betydning for målet om å fremme utvikling av stabile, immun-modulerende cellelinjer som trygt kan brukes i behandlingsformål. Prosjektet har også demonstrert hvordan en kombinert bruk av ulike informasjonslag, et felt som er i rask utvikling, kan muliggjøre funn som ikke kan oppdages gjennom én type data alene. Den oppnådde kompetansen innen dette feltet har blitt spredt til forskere både i samme og i andre forskningsgrupper gjennom presentasjoner, faglige diskusjoner og publikasjoner, og vært til hjelp for andre kommende og pågående forskningsprosjekter. Data frembrakt i prosjektet er gjort tilgjengelig i databaser og vil også være en ressurs for fremtidige studier. Prosjektet har fremhevet mekanismer som vil være av interesse for utvikling av fremtidige forskningsprosjekter, med mål om å bedre diagnostikk og behandling av autoimmune sykdommer. Mer effektive og persontilpassede behandlinger for pasienter med autoimmune sykdommer er avgjørende for å redusere den høye individuelle og samfunnsmessige kostnaden av slike tilstander.

Autoimmune diseases affect about 5% of the population and are caused by an immunological attack against the body´s own tissues. The etiology is complex and largely unknown. Human leukocyte antigen (HLA) alleles are major genetic determinants explaining up to half of the heritability, but several hundred other immune loci confer susceptibility. The autoimmune risk variants are mainly located outside coding regions, implying a regulatory role and thereby acting as quantitative trait loci (QTL). We have previously systematically revealed autoimmune risk variants influencing expression levels of immune genes in whole thymic tissue. With this proposal, we will first generate a comprehensive overview of cell populations existing in human thymus and their transcriptomes. Next, we will reveal the regulatory role of genetic risk factors in human thymic antigen-presenting cell type, being crucial for presenting all tissue proteins in order to establish self-tolerance and avoid autoimmunity. The project will employ state of the art mass and flow cytometry, single cell RNA sequencing and next generation ChIP and RNA sequencing. We will identify autoimmune risk variants influence gene expression and alternative splicing (as sQTLs), including comprehensive assessment of HLA allelic expression levels. In the medullary epithelial cells, we will also identify binding sequences for AIRE and FEZF2 that are disturbed by risk variants (bQTLs). AIRE and FEZF2 are important transcription factors for promiscuous gene expression in thymus and thereby establishment of self-tolerance. Ultimately, the benefits of unravelling and elucidating genetic mechanisms for autoimmune diseases in thymic antigen presenting cells include increased knowledge about pathogenesis and thereby reveal treatment targets.