Kantonsspital St.Gallen

Deciphering the molecular landscape of the stromal cells in human lymph nodes at the single-cell level

Burkhard Ludewig

abstract Secondary lymphoid organs are strategically positioned at routes of pathogen invasion and thereby increase the likelihood of lymphocytes to encounter their cognate antigens at a particular location and during a certain time window. Lymph nodes are found at convergence points of afferent lymph vessels and surveil extracellular fluids from separate areas of peripheral tissue. The structural integrity of all lymphoid organs is determined by fibroblastic stromal cells that build, for example, the capsule of lymph nodes or the spleen. In addition, specialized immune-interacting fibroblasts, generally termed fibroblastic reticular cells (FRC), form the scaffold structures that underpin the distinct microenvironments required for efficient immune cell interactions. Importantly, while the knowledge on FRC in murine lymph nodes is constantly growing, the identity and molecular landscape of FRC in human lymph node has remained unknown.
The main hypothesis underlying the proposed MD thesis research project is that human lymph nodes harbor distinct subsets of FRC. To address this question, it is planned (i) to determine the heterogeneity of fibroblast subsets in human lymph nodes and to (ii) to compare the molecular landscape of lymph node FRC with fibroblastic stromal cell properties that are currently elaborated within the frame of a PhD project in the host laboratory. The candidate will be trained in current molecular biological technologies including single-cell RNA-seq analysis and cell biological techniques including flow cytometry and confocal laser scanning microscopy.
The main goal of the first Aim is to identify fibroblast subsets in human lymph nodes microenvironment on the basis of established marker combinations and to pinpoint the localization of fibroblasts in the complex microenvironment of lymph nodes. To this end, a panel of at least 10 fibroblast markers will be established for comprehensive flow cytometric analyses. The flow cytometric analysis will be complemented with high-resolution confocal microscopic imaging to achieve a 3D reconstruction of the fibroblastic landscape in the lymph node. We anticipate that the work on Aim 1 will provide a comprehensive molecular topological analysis of the fibroblast subsets present in different lymph nodes microenvironment. The overarching goal of Aim 2 is to reconstruct the fibroblastic ecosystem in human lymph nodes using high-dimensional single-cell analyses that facilitate the molecular definition of distinct fibroblast subsets. It is planned to perform single-cell RNA-seq analysis using cell suspensions obtained from at least five human lymph nodes obtained from gall bladder and nephrectomy surgery at the Clinics of Visceral Surgery and Urology, respectively. We anticipate that our approach will leverage the advantages of single-cell RNA-seq data to identify the transcriptome and signaling pathways in immune-interacting fibroblast subsets within human lymph nodes. Findings from the single-cell analyses in Aim 2 will be validated using the expertise generated in the work on Aim 1 to facilitate the corroboration of molecular marker combinations at the protein level.
The candidate, Mr. Yves Stanossek, shows a high commitment to improve his scientific profile and has expressed his aspiration to follow an academic career trajectory. The subsequent mentoring will be taken over by Prof. Stöckli who has offered Mr. Stanossek a position as Specialist Registrar in the Ear, Nose and Throat Clinic.
type of project fundamental research
status ongoing - recruiting phase
start of project 2019
end of project 2019
project manager Prof. Burkhard Ludewig