Periodic Reporting for period 5 - TEC_Pro (Molecular control of self-renewal and lineage specification in thymic epithelial cell progenitors in vivo.) Reporting period: 2021-07-01 to 2022-10-31 Summary of the context and overall objectives of the project The development of vaccines for the treatment of infectious diseases, cancer and autoimmunity depends on our knowledge of T-cell differentiation. This project focused on studying the thymus, the organ that generates immunologically competent and self-tolerant T cells. Within the thymus, thymic epithelial cells (TECs) provide key inductive microenvironments for the development and selection of T cells. Defects in TEC differentiation cause syndromes that range from immunodeficiency to autoimmunity, which makes the study of TECs key to understand immunity and tolerance induction. TECs are divided into two functionally distinct cortical (cTECs) and medullary (mTECs) subsets, which derive from bipotent TEC progenitors (TEPs). Our goals were to identify TEC progenitors, define new molecular components involved in their self-renewal and lineage potential, and elucidate the genetic programs that regulate cTEC/mTEC fate decision. Using advanced research tools that combine reporter mice, organotypic cultures, high-throughput screen and genome-wide transcriptomic, we defined novel principles that underlie the self-renewal lineage differentiation and function of TECs in vivo. Our findings have the potential to contribute with knowledge that can help tackling one of the great challenges of modern immunology – modulate thymic function through the induction of TEPs - and therefore, represents a major advance in Health Sciences. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Cortical (cTEC) and medullary (mTEC) TECs form specialized niches for T cell differentiation and arise from TEC progenitors (TEP). Yet, the niches occupied by TEPs in the post-natal thymus remain poorly defined. In the search for epithelial stemness, we set clonogenic assays that were reported to select stem cells from other epithelial tissues. We have identified a new subset of bipotent TEP that reside within the thymic cortex and whose abundance is dynamically controlled by continual interactions with thymocytes (Meireles et al, EJI 2017). These findings potentially explain how changes in TEP bioavailability impact on the maintenance of TEC niches, and ultimately thymic output. Moreover, mTECs play a chief role in tolerance induction and prevention of autoimmunity. Still, it remains elusive whether their differentiation is linearly related or independently specified (as reviewed in Rodrigues et al Trends Immunology 2017, Alves et al EJI 2016, Pinheiro et al Frontiers in Immunology 2021, Ribeiro et al Immunology Letters 2019). Despite considerable advances, the identification of specialized mTEC populations depends on exclusive lineage-tracing mouse models and complex single-cell RNA sequencing analysis. We revealed an efficient phenotype-based method to resolve Early-, Late- and Post-Aire stages of mTEC differentiation. These findings not only close gaps in our comprehension of the mTEC differentiation program, but also offer a useful road map for the study of Post-Aire mTEC in T-cell development, tolerance induction and autoimmunity (Ferreirinha et al EJI 2021). Our study was selected by the publishing journal (EJI) as the research highlight. Previously, we showed that TECs are the main source of thymic IL-7 (Alves et al PNAS). IL-7 is a central cytokine for lymphopoiesis in the bone marrow and thymus. The role of sex steroid ablation (SSA) in thymic regeneration is a well-documented process. Nevertheless, to which extent this regenerative intervention acts in the bone marrow and/or thymus remained elusive. We showed that thymic IL-7 is dispensable to SSA-driven thymopoiesis, being the later dependent on the engagement of hematopoietic progenitors in the bone marrow (Rodrigues et al JI 2018). Despite the chief contribution of TECs, recent studies emphasize the regulatory role of mesenchymal cells in thymic function. Employing advanced flow cytometry, genome-wide transcriptomic and fate mapping analyses, we have identified novel fibroblast progenitors within the developing thymus, whose bioavailability is controlled by thymic crosstalk (Ferreirinha et al Development 2022). Our study was selected by the journal “Development” under the section “Research Highlights”.To seek for new determinants that regulate TEC lineage specification in vivo, we obtained the genome-wide transcriptomic analysis (RNA seq) of embryonic TEC (eTEC), cTEC, mTEC and ClonoTEC (related with aim1). Global PCA analysis positioned ClonoTEC, eTEC and cTEC more closely related to each other, with mTEC defining a more distant population. These findings reinforce the notion that TEP transverse first through the cortical lineage prior to their commitment to mTECs (Alves et al EJI 2014). We defined lineage-specific profiles for these TEC subsets, which represent an integrative framework for selection of genes for further functional analysis. Using a combinatory in silico analyses, we selected target genes (transcription factors/regulators) that presented the greatest changes and an unreported role in TEC/Thymus differentiation. We imported several new floxed mouse lines for genes that were specifically enriched in certain TEC subsets. To examine their intrinsic role in TEC development and thymopoiesis, we generated novel cKO mice by crossing floxed mice to TEC-specific Foxn1:Cre. We have a series of on-going studies (4 manuscripts still in preparation) that aimed at defining the new role of TFs/regulators in TEC differentiation. Although these studies will be concluded beyond the time frame, the support of the ERC starting grant was central to implementing these research lines, and the future publications will acknowledge the support of the ERC. Moreover, we identified a novel immunoregulatory role for the transcription p53 in TECs to orchestrate their role in T-cell development and tolerance induction (Rodrigues et al Blood 2017). Our study received highlights from Blood (companion commentary article and selection for the snapshot of “This Week in Blood”) and Science (selection for the “Editor’s Choice” section). More recently, we reported a new role for LAMP2 in T cell differentiation and function. Using a genetic model that inactivates LAMP-2 in TECs, we showed that LAMP-2 specifically regulates the positive selection of CD4 T cells (Rodrigues et al Autophagy 2022). Lastly, the recognitions and commentaries on our studies by publishing journals also attest on the innovative nature of our findings. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our research provided fundamental knowledge that can be incorporated in future therapeutics to regenerate thymus function and immunity in aged individuals, patients undergoing bone marrow transplantation, under anti-cancer regimens or suffering from autoimmune disease. We defined new molecular determinants that control the role of thymic stromal microenvironment in T-cell development and induction of self-tolerance. We have used several in vivo models of great relevance to immune pathologies in humans. Thus, our research scope has important translational implications in clinical contexts where normal T cell responses are disturbed, including congenital (e.g. DiGeorge Syndrome) and acquired (e.g. HIV infection and Bone Marrow Transplantation in Leukemia) immunodeficiencies, age-related thymic involution and autoimmunity. erc-copy-2.jpg
