The Regulators of Self-antigen Expression in the Thymus: Guardians of Immunological Tolerance – SelfExpress

CONTEXT: The thymus plays a pivotal role in the establishment of immune central tolerance by producing a diverse repertoire of non-autoreactive T lymphocytes. In particular, the expression of a large diversity of tissue-restricted self-antigens (TRAs) by mature medullary thymic epithelial cells (mTECs) is critical to the elimination of autoreactive thymocytes and selection of regulatory T cells (Tregs) able to suppress autoimmunity. Although a number of TRAs is controlled by AIRE and FEZF2 transcription factors (TFs), half of TRAs lack known regulators to date. This lack of knowledge leaves considerable gaps in our understanding of how the “self” is expressed in the thymus and how immune central tolerance is established.

OBJECTIVES: Overall, the SelfExpress project aims at identifying the “missing regulators” that compose the TRA regulome and characterizing their respective mode of action in mTECs. The specific aims will determine:
1. the potential TFs 1) that are upregulated by the thymic crosstalk that we recently found to promote TRA expression or 2) having recognition sequences in TRA genes that lie within regions of open chromatin, as profiled by scATAC-seq. TFs showing a robust expression in the mature mTEC subset will be considered for further investigation.
2. the ability of these candidate TFs to induce TRA expression using a CRISPR interference (CRISPRi) perturbation screening in primary human mTECs in a 3D organotypic co-culture setup. In addition to assess the breadth of individual TF effects, this screening will evaluate their inter-dependency, thereby delineating TF regulatory modules. TFs that are independent to AIRE’s action will be formally identified by a secondary lower scale CRISPRi screening in WT and Aire-KO mouse mTECs.
3. whether the two most active TFs as well as Tbx21 that we previously identified as a very promising candidate TF, directly target TRA genes by characterizing their DNA binding profile by ChIP-seq experiments in primary human mTECs. We will further refine their mode of action by exploring the chromatin landscape at their DNA binding sites, focusing on active H3K4me3 and repressive H3K27me3 marks but also on the H3K27Ac enhancer mark that could pinpoint an indirect regulation.
4. the impact of the two most active TFs and of Tbx21 in the establishment of self-tolerance by generating conditional knockout mice lacking these TFs specifically in TECs using FoxN1Cre x TF LoxP mice. These unique mouse models will allow to assess in vivo the repertoire of TRA genes induced by the tested TFs and its functional impact on the elimination of highly autoreactive thymocytes and the development of Tregs. Importantly, the effect of these TFs on self-tolerance induction will be evaluated by analyzing signs of autoimmunity in peripheral tissues.

CONSORTIUM: This project is a close collaboration between two French research teams, expert in the biology of mTECs and central tolerance. It brings together a fully complementary expertise on transcriptomics, epigenetics and bioinformatics (Team #1) and immunology, autoimmunity and in vivo models (Team #2).

SCIENTIFIC AND BIOMEDICAL IMPACTS: This project will undoubtedly lead to the identification of “missing factors” controlling the expression of TRAs. It is thus expected to provide new insights into the understanding of how the immune system acquires tolerance to self-antigens and thereby prevents the organism from life-threatening autoimmune diseases. Beyond this fundamental understanding of central tolerance, the SelfExpress project will help in designing novel therapeutic strategies, based on the use of mature mTECs expressing the full TRA regulome, to treat patients with autoimmune diseases or a transplanted organ.