Periodic Reporting for period 1 - TissueLymphOmics (Cellular crosstalk driving Tertiary Lymphoid Structure formation across tissues)
Période du rapport: 2024-04-01 au 2026-03-31
The immune system is an intricate network of cellular and molecular interactions playing out through our bodies. Immune cells serve as sentinels against pathogens and the immune response represents a coordinated action to eliminate these threats. Nonetheless, immune cells may act against other tissues, a defining feature of autoimmunity. Autoimmune conditions can appear in various tissues, having a devastating impact in the quality of life of patients, and limited therapeutic options. These are complex diseases, involving a plethora of immune cell types and states interacting with tissue-specific stromal and epithelial cells. Understanding them requires technologies capable of unraveling cells and their phenotypes with high resolution. Single-cell RNA-sequencing (scRNA-seq) is a technology whose accuracy, scale, and use has exponentially grown in the last decade. scRNA-seq extracts gene expression information from individual cells, revealing cell types and states, gene regulation and intercellular communication in the context of a tissue, in health and disease. Single-cell sequencing technologies have also grown in complexity and scale. This has allowed researchers to profile more cells and more data modalities from individual cells, such as open chromatin regions or surface proteins. Furthermore, gene expression distribution in tissues can also be spatially assessed using spatial transcriptomics approaches.
This project aims to understand the regulation of immune cell infiltration and subsequent formation of Tertiary Lymphoid Structures in various tissues affected by autoimmune diseases, by combining bioinformatics and machine learning approaches with methods to probe the immune system in vivo and in vitro to validate its key interactions. By identifying and functionally characterising the ligands and receptors modulating TLS formation and development across tissues using scRNA-seq, Spatial Transcriptomics (ST), and multiomics, the project can provide a detailed picture of the cell types in tissues affected by autoimmune disease, and unravel their cell-cell interactions. These will be further characterised in the context of TLS morphology and development, and the immune function and underlying regulation of the key signalling molecules will be further unravelled with in vitro studies, ultimately setting a path from discovery to functional validation. This is further expected to serve as a springboard to clinical research by investigating the key targets revealed in the project.
This project aims to understand the regulation of immune cell infiltration and subsequent formation of Tertiary Lymphoid Structures in various tissues affected by autoimmune diseases, by combining bioinformatics and machine learning approaches with methods to probe the immune system in vivo and in vitro to validate its key interactions. By identifying and functionally characterising the ligands and receptors modulating TLS formation and development across tissues using scRNA-seq, Spatial Transcriptomics (ST), and multiomics, the project can provide a detailed picture of the cell types in tissues affected by autoimmune disease, and unravel their cell-cell interactions. These will be further characterised in the context of TLS morphology and development, and the immune function and underlying regulation of the key signalling molecules will be further unravelled with in vitro studies, ultimately setting a path from discovery to functional validation. This is further expected to serve as a springboard to clinical research by investigating the key targets revealed in the project.
The key approaches for this project were the use of scRNA-seq and spatial transcriptomics data to unravel cell-cell communication events across autoimmune diseases, and relate them to the formation of Tertiary Lymphoid Structures.
In a first stage, this was achieved by collecting published scRNA-seq datasets of autoimmune diseases affecting different organs. To this, cell-cell communication inference methods were applied, creating the first comparative reference of cell communication events across tissues and autoimmune diseases.
To then contextualise the inferred interactions, two approaches were undertaken. First, tissue samples from salivary glands of Sjögren's Disease (an autoimmune disease) patients were obtained to generate spatial transcriptomics data using Visium, Visium HD, and Xenium. Together, these are the largest generated dataset of autoimmune patients, with several samples achieving single-cell resolution. Second, a mouse model for Sjögren's Disease was implemented, allowing for the study of the outcomes of this disease on demand. While the model takes between 10 to 30 weeks to develop the disease, it is amenable to perturbations. Tests of overexpression and KO of pathways identified in this project are already planned or underway.
Lastly, to mimick the function of Tertiary Lymphoid Structures in vitro, human tonsil organoids were implemented. These can form functioning Germinal Centres in vitro, as are also found in Tertiary Lymphoid Structures. This project optimised the conditions for their culturing, which primes them for use in subsequent validations of the function of the identified signalling molecules. Furthermore, the planned generation of multiome data will allow for a direct gene expression and regulation comparison to the immune dysregulation observed in autoimmune diseases.
In a first stage, this was achieved by collecting published scRNA-seq datasets of autoimmune diseases affecting different organs. To this, cell-cell communication inference methods were applied, creating the first comparative reference of cell communication events across tissues and autoimmune diseases.
To then contextualise the inferred interactions, two approaches were undertaken. First, tissue samples from salivary glands of Sjögren's Disease (an autoimmune disease) patients were obtained to generate spatial transcriptomics data using Visium, Visium HD, and Xenium. Together, these are the largest generated dataset of autoimmune patients, with several samples achieving single-cell resolution. Second, a mouse model for Sjögren's Disease was implemented, allowing for the study of the outcomes of this disease on demand. While the model takes between 10 to 30 weeks to develop the disease, it is amenable to perturbations. Tests of overexpression and KO of pathways identified in this project are already planned or underway.
Lastly, to mimick the function of Tertiary Lymphoid Structures in vitro, human tonsil organoids were implemented. These can form functioning Germinal Centres in vitro, as are also found in Tertiary Lymphoid Structures. This project optimised the conditions for their culturing, which primes them for use in subsequent validations of the function of the identified signalling molecules. Furthermore, the planned generation of multiome data will allow for a direct gene expression and regulation comparison to the immune dysregulation observed in autoimmune diseases.
Cell-cell communication analysis revealed that most identified interactions are shared between at least two organs. This provides us with a list of cross-disease interactions, that can be explored as potential targets in novel therapies. The in vivo and in vitro systems implemented in this project will serve as platforms for validation of the importance of these interactions.
This project has also generated a wealth of spatial transcriptomics data. This first in-depth deep dive into Sjögren's Disease histology and gene expression have so far revealed the co-localization of key cell types to immune cell recruitment with the infiltrates that form in salivary glands in this autoimmune disease.
The skills gained when implementing the validation models will also be key for future projects. These are systems with vastly unexplored capabilities, both in their ability to characterise autoimmunity, and generaly test new hypothesis about adaptive immune functions.
This project has also generated a wealth of spatial transcriptomics data. This first in-depth deep dive into Sjögren's Disease histology and gene expression have so far revealed the co-localization of key cell types to immune cell recruitment with the infiltrates that form in salivary glands in this autoimmune disease.
The skills gained when implementing the validation models will also be key for future projects. These are systems with vastly unexplored capabilities, both in their ability to characterise autoimmunity, and generaly test new hypothesis about adaptive immune functions.