Exploring lymphatic endothelium – innate immune system crosstalk in lymph nodes and its impact on adaptive immunity – Lymphinnate

Lymph nodes (LN) are prototypical lymphoid organs where adaptive immune responses against local infections are initiated. It has become well established that peripheral dendritic cells (DC) arriving from afferent lymphatic vessels in the subcapsular sinus (SCS) migrate via the interfollicular areas (IFA) into the paracortical T cell zone. There, DC present processed protein antigen in the form of peptide-major histocompatibility complex (pMHC) to naive T cells for the initiation of cellular responses. This process has been exhaustively studied. Yet, IFA are strongly enriched for cells of the innate immune system such as gamma-delta T cells, innate-like CD8+ T cells, natural killer T cells (NKT), innate lymphoid cells (ILC), natural killer (NK) cells, macrophages and neutrophils. The physiological function of this multicellular innate system has been assigned to limit pathogen spread by fostering antimicrobial activity of macrophages and stromal cell-mediated repair processes. To date, however, it is unknown how the specific IFA niche for innate immune cells is created and maintained by the stromal microenvironment. Further, it remains unclear whether and how a microbial challenge impacts on innate cells of the IFA-resident multicellular immune system and their cross-talk with cells of the adaptive immune system.
The team of C. Mueller, partner in this project, has recently demonstrated that LN stromal mesenchymal cells located just beneath the SCS express the TNF family member TNFSF11 (also known as RANKL) to activate the overlaying TNFRSF11a+ (RANK+) lymphatic endothelial cells (LEC). This creates a specific niche for the recruitment and retention of sinusoidal macrophage (SM) populations. Deletion of mesenchymal RANKL or lymphatic RANK precipitates the loss of SM, with a concomitant impairment of humoral immune responses. In addition, LEC lose their activated phenotype, which leads to altered expression of numerous immunoregulatory genes. These include chemokine scavenging receptors and the chemoattractants CXCL4 and CCL20, which are known to attract and/or retain cells of the innate immune system. An attractive hypothesis is therefore that the RANKL-RANK signaling axis linking LN mesenchymal cells and LEC orchestrates the establishment and maintenance of the multicellular innate immune system of the IFA.
Here, we will combine transgenic mouse lines with disrupted RANK and CSF-1 signaling in combination with reporter mouse lines for innate immune cells to critically examine the role of LEC in establishing the innate immune system in IFA. To this end, we will employ multiplex immunofluorescence in combination with whole-organ and intravital imaging, flow cytometry, as well as single cell and spatial transcriptomics of the IFA niche in the steady state and after microbial challenge. We will use this knowledge to examine how activated LEC and the innate immune system of the IFA regulate adaptive immunity during a microbial challenge. From these data, we anticipate to unravel the complex cross-talk balancing innate and adaptive immune responses in reactive LN, which may eventually lead to specifically-tailored vaccination strategies.