The thymus stromal compartment across developmental stages: Determining single cell transcription, chromatin accessibility and spatial position

The Human Cell Atlas (HCA) seeks to create at single cell resolve a comprehensive reference map integrating the multi-dimensional molecular description of cell states. This research proposal will contribute to this ambitious flagship project by providing a high-resolution description of thymic stromal cells. The composite of these cells create the thymic microenvironment which is unique in its ability to promote the development of naïve T cells with a repertoire purged of vital "Self" specificities and poised to react to injurious "Non-Self" antigens. Thymic epithelial cells (TECs) constitute the major component of the thymic stroma and can be categorized into separate cortical (c) and medullary (m) lineages based on their specific molecular, structural and functional characteristics. cTEC induce the commitment of blood-borne precursor cells to a T cell fate, foster the subsequent maturation and control the positive selection of antigen receptor bearing thymocytes. In contrast, mTEC promote the terminal differentiation of thymocytes which includes (together with other thymic stromal cells) the establishment of immunological tolerance to self-antigens via a deletional mechanism and the generation of natural regulatory T cells. In this way, TEC are indispensible throughout life in creating a repertoire of of self-tolerant T cells. This essential capacity depends on the cells' unique ability to express at population level almost all protein coding genes, a process known as promiscuous gene expression.
Age-related postnatal changes in the human thymus stromal composition and architectural organization have been observed as early as the second year of life and progressively increase over time with a severe but not complete involution of the organ at 40 years of age and beyond. These alterations are typically characterized by a loss of the cortico-medullary demarcation, an expansion of perivascular spaces and the replacement of the cortical and medullary compartments by adipose tissue, hence changes that affect the cellular composition and consequently the function of the thymus stroma.
Given the dynamic, senescence-related changes in the composition and function of the thymus stroma, we propose to analyse comparatively and at single cell resolution foetal, infant and adult thymus stromal cells for their transcriptional activity, chromatin accessibility and spatial position within intact thymus tissue. The sequencing data will be used to reveal the cellular complexity of the thymus stroma, to better describe its cellular components, identify rare and novel cell populations and their states, uncover regulatory relationships between genes for and among each of the separate stromal cell states, and track the trajectories of distinct cell lineages in development. Defining these molecular features opens new avenues for an integrative understanding of single-cell states within the complexity of the thymus microenvironment where distinct functions are spatially compartmentalized (cortex vs. medulla) and continuous cell replacement is realized by on-going differentiation within the TEC lineages, a process that however exhausts itself over time. Taken together, the proposed analyses of this unique and indispensible cell population will contribute important information to the Human Cell Atlas essential to understand human health and disease.

This research proposal will describe the transcriptional profile, chromatin accessibility and spatial position of all non-haematopoietic human thymus stromal cells analysing tissue isolated at 10-14 and 16-20 weeks of gestation, within the first 6 months of postnatal life and from individuals 20 and 40 years of age. Two distinct single-cell RNA-sequencing approaches will be employed: (i) a microfluidic-droplet-based and (ii) a fluorescent activated cell sorting (FACS)-based method to identify cell states and spaces. The microfluidic droplet-based 3'-end counting method (GemCode Single-Cell Instrument, 10x Genomics, combined with the GemCode Single-Cell 3' Gel Bead and Library V2 Kit) will be used to enable the survey of thousands of stromal cells (notably at relatively lower coverage) and additionally allows to determine both number and relative abundance of individual stromal cell types. Full-length transcript analysis based on FACS-index sorted thymus epithelial cells (TEC; EpCAM+MHC+CD45-) placed into 384 well plates will be employed to allow for a high sensitivity and deep coverage characterization. Standard methods will be employed for the generation of cDNA and library preparation and pooled libraries will be analysed by next generation sequencing. Chromatin accessibility at single cell resolution will be analysed for specific TEC subpopulations using the Chromium ATAC-Seq platform. This data will be used to link regulatory elements to their target genes. Finally, single thymus stromal cells will also be analysed for their spatial position within the complex thymus microenvironment using sequential image captures of immunohistological analyses of in total as many as 52 separate epitopes using a standard, 3-color fluorescence microscope platform. The proposed analyses will define the distribution of distinct thymus stromal cells within the organ and identify non-random spatial interactions in its microenvironment.

The goal of the Human Cell Atlas (HCA) is to create at single cell resolve a comprehensive multi-dimensional molecular reference map of cell states. The HCA will thus provide a description of a cell's characteristic features at high DNA, RNA, and proteins resolution which, together with the knowledge of their precise spatial position in tissues, will transform our understanding of the complexity of health and form a an important comparator to pathological conditions. The atlas will therefore reveal the precise cellular nature and composition of organs, identify rare and previously unappreciated cell states, track the trajectories of distinct cell lineages in development and uncover regulatory relationships between genes for and among each of the separate cell states. This information will be extremely useful to identify biomarkers of disease onset and progression as it is now conceivable that molecular changes occurring across thousands, or tens of thousands, of individual cells can serve as accurate indicators of transitions from a healthy to a disease state. Moreover, these shifts in cell numbers and locations across a multidimensional space will hence improve our description and understanding of disease mechanisms and form the necessary basis for future therapeutic interventions that are precisely shaped according to the cells' defined molecular content.

The work proposed here will make essential contributions to the overall ambitions of the HCA. It will specifically focus on a central aspect of the immune system, namely the thymus stroma, a cellular compartment indispensible for the establishment and function of T cells. Understanding the molecular and cellular mechanisms that underpin the body's vital competence to distinguish between immunological "Self" and "Non-Self" is critical to gain insight into diverse pathologies ranging from primary and acquired immunodeficiencies to autoimmunity, defective immune reconstitution after cytoablative therapies, and immunological senescence marked by reduced vaccine and tumour responses, to name a few.

The HCA in general and this contribution in particular will have a far reaching impact on biomedical research that extends well beyond the UK science landscape. In addition to providing a cell atlas of unprecedented depth and breadth, the work proposed will foster extensive trans-disciplinary research programmes that bring together biomedical, molecular and computational scientists from different domains of the Life Sciences to develop new collaborative initiatives related to both health and disease. In parallel, the HCA-related efforts will promote new developments in methods and technologies that are expected to generate novel major research capabilities. The research programmes will also provide unique opportunities to train the next generation of researches skilled in combining cell and computational biology, a capability which already is and will continue to be in high demand. The research programmes will also create new impact-related technologies and develop innovative ideas that will likely advance and support novel business ventures in the Life Sciences and thus contributing to the initiative's expected potential to extend supporting capabilities and thus build Innovation and Knowledge Centres.