Periodic Reporting for period 1 - ThymEForT (Dissecting the role of thymic epithelial cells in T lymphocytes maturation during human foetal development)
Reporting period: 2021-09-01 to 2023-08-31
The thymus plays an essential role in the establishment of adaptive immunity and central tolerance as it provides a nurturing environment for the differentiation of T cells, a process orchestrated by their interaction with multiple cell types. Despite advances in the knowledge of human thymus biology, how it functionally develops remains elusive. This project aimed at dissecting molecular mechanisms responsible for functional maturation of T cells by recapitulating human thymic development within a novel in vitro 3D system. Epithelial and T cell compartments were isolated and characterised at relevant developmental stages to underpin when thymic stroma acquires competence to sustain the production of functional T cells. We identified an epithelial stem cell population that sustain thymic function. Thymic epithelial stem cells self-renew in vitro and differentiate into specialised epithelial cell types that, when cocultured with T-cell progenitors, give rise to mature thymocytes within a unique 3D thymic natural matrix. After characterising the thymic epithelial compartment at single-cell level, we uncovered a new medullary progenitor population expressing specific transcription factors. Thymic epithelial stem/progenitor cells were genetically modified in vitro to knock-out these novel genes to explore whether they impact on T cell maturation. Finally, this project has revealed that human thymic epithelial stem cells can be co-cultured with thymocytes progenitors within a novel, whole human 3D system. This allows to tackle fundamental immunological questions regarding human thymus development including maturation of T cells and regulation of central tolerance. In addition, this will lay the foundation to develop new treatments for thymic diseases and for congenital immune deficiencies based upon correction of the underlying molecular defects.
Aim1. Phenotypic characterisation of thymic stroma ex vivo and in vitro to define functional cortex/medulla specification and functional T cells
We performed comprehensive analyses of mature postnatal thymic tissue and collected human thymic foetal samples to study changes in epithelial cells during thymic maturation. We identified novel subtypes of cortical and medullary epithelial cells, characterized their transcriptional features, and isolated them in vitro. We explored the appearance of Hassall’s Bodies structures during foetal thymic maturation and observed a significantly higher medullary content in foetal thymi compared to postnatal counterparts. Furthermore, we isolated and expanded thymic epithelial stem cells across different ages, as well as RNA-seq and epigenetic profiling to understand transcriptional changes during development. Single-cell RNA-seq was performed on postnatal thymocytes and integrated with human foetal thymocytes datasets to understand the trajectory of progenitors undergoing T cell maturation. The study concludes by defining the properties of thymic epithelial cells in supporting T cell function and identifying epithelial stem cells in the human thymus.
Aim2. Recapitulate human foetal T cell development within a 3D natural matrix in vitro by modulating the introduction of stromal components
We next investigated the interaction between thymic epithelial cells and thymocytes across different human developmental stages. Initially, we established working conditions using postnatal cells. We utilized a rat thymic natural matrix scaffold injected with thymic stem cells and other stromal components, which reorganized along the three-dimensional extracellular matrix structure. Thymocyte precursors were injected into the scaffolds to evaluate their functionality in supporting T cell development. The resulting cells mirrored the ratio found in the native human thymus. The study confirmed the differentiation of thymic epithelial stem cells and their re-expression of functional molecules. This ex vivo system is now utilized to analyse T cell maturation upon injection of thymic epithelial cells from different developmental ages and anatomical compartments. T cell output is assessed to understand various aspects of human T cell development, including the establishment of T cell subsets and the role of human-specific genes expressed by stromal cells in driving the immune maturation process.
Aim3. Genetic ablation of stromal factors during foetal development and evaluation of its impact on T cell
maturation.
After extensive analysis of thymic epithelial compartment, we found a transcription factor to be highly expressed in several medullary cell subtypes, suggesting its importance in thymic function. Knocking out this transcription regulator in human thymic epithelial cells has led to a decrease in medullary markers, prompting further investigation into its molecular mechanisms in thymic function and across organ development.
Exploitation and dissemination:
-Thymic epithelial stem cell properties across development have now been published and the bioinformatic datasets of the study are publicly available: doi: 10.1016/j.devcel.2023.08.017.
-Aim1-2 results have been disseminated in presentations for teaching activities at University College London (UCL) and in two conferences: International Society for Stem Cell Research (ISSCR) in 2023 and London Stem Cell Symposium in 2021 and workshop in 2024
-Human foetal epithelial cells data generated under this project are currently under revision in the following manuscript:
“Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments”. Authors: Savvas Savvidis, Roberta Ragazzini, Valeria Conde de Rafael, J. Ciaran Hutchinson, Lorenzo Massimi, Fabio A. Vittoria, Sara Campinoti, Olumide K. Ogunbiyi, Alessia Atzeni, Neil J. Sebire, Paolo De Coppi, Alberto Mittone, Alberto Bravin, Paola Bonfanti and Alessandro Olivo.
-Spatial transcriptomics data about thymic stem cell niche during development will be made publicly available upon publication.
We performed comprehensive analyses of mature postnatal thymic tissue and collected human thymic foetal samples to study changes in epithelial cells during thymic maturation. We identified novel subtypes of cortical and medullary epithelial cells, characterized their transcriptional features, and isolated them in vitro. We explored the appearance of Hassall’s Bodies structures during foetal thymic maturation and observed a significantly higher medullary content in foetal thymi compared to postnatal counterparts. Furthermore, we isolated and expanded thymic epithelial stem cells across different ages, as well as RNA-seq and epigenetic profiling to understand transcriptional changes during development. Single-cell RNA-seq was performed on postnatal thymocytes and integrated with human foetal thymocytes datasets to understand the trajectory of progenitors undergoing T cell maturation. The study concludes by defining the properties of thymic epithelial cells in supporting T cell function and identifying epithelial stem cells in the human thymus.
Aim2. Recapitulate human foetal T cell development within a 3D natural matrix in vitro by modulating the introduction of stromal components
We next investigated the interaction between thymic epithelial cells and thymocytes across different human developmental stages. Initially, we established working conditions using postnatal cells. We utilized a rat thymic natural matrix scaffold injected with thymic stem cells and other stromal components, which reorganized along the three-dimensional extracellular matrix structure. Thymocyte precursors were injected into the scaffolds to evaluate their functionality in supporting T cell development. The resulting cells mirrored the ratio found in the native human thymus. The study confirmed the differentiation of thymic epithelial stem cells and their re-expression of functional molecules. This ex vivo system is now utilized to analyse T cell maturation upon injection of thymic epithelial cells from different developmental ages and anatomical compartments. T cell output is assessed to understand various aspects of human T cell development, including the establishment of T cell subsets and the role of human-specific genes expressed by stromal cells in driving the immune maturation process.
Aim3. Genetic ablation of stromal factors during foetal development and evaluation of its impact on T cell
maturation.
After extensive analysis of thymic epithelial compartment, we found a transcription factor to be highly expressed in several medullary cell subtypes, suggesting its importance in thymic function. Knocking out this transcription regulator in human thymic epithelial cells has led to a decrease in medullary markers, prompting further investigation into its molecular mechanisms in thymic function and across organ development.
Exploitation and dissemination:
-Thymic epithelial stem cell properties across development have now been published and the bioinformatic datasets of the study are publicly available: doi: 10.1016/j.devcel.2023.08.017.
-Aim1-2 results have been disseminated in presentations for teaching activities at University College London (UCL) and in two conferences: International Society for Stem Cell Research (ISSCR) in 2023 and London Stem Cell Symposium in 2021 and workshop in 2024
-Human foetal epithelial cells data generated under this project are currently under revision in the following manuscript:
“Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments”. Authors: Savvas Savvidis, Roberta Ragazzini, Valeria Conde de Rafael, J. Ciaran Hutchinson, Lorenzo Massimi, Fabio A. Vittoria, Sara Campinoti, Olumide K. Ogunbiyi, Alessia Atzeni, Neil J. Sebire, Paolo De Coppi, Alberto Mittone, Alberto Bravin, Paola Bonfanti and Alessandro Olivo.
-Spatial transcriptomics data about thymic stem cell niche during development will be made publicly available upon publication.
The project advanced understanding of epithelial stem cell biology in the thymus, showing their multilineage potency and contribution to various cell types. This insight is crucial for comprehending medullary microenvironments, thymic tumours, and autoimmune diseases. Discoveries also highlight the potential for cell replacement therapies, akin to those using other stem cells. Investigating how thymic stem cells adapt as the organ shrinks could lead to strategies for enhancing immune responses, including vaccinations and cancer immunity. The study's findings offer promise for future clinical applications, particularly in athymic patients, with potential for further development using biocompatible matrices and patient-derived stem cells.