Illuminating the network: Fibroblastic reticular cells at the crossroads of lymph node architecture and immunity

Lymph nodes (LNs) are small organs distributed throughout the body, which are part of our immune system. Millions of lymphocytes circulate through LNs daily, surveilling for foreign antigens and initiating adaptive immune responses when needed. This process is highly efficient due to the specialized architecture of LNs. Fibroblastic reticular cells (FRCs), a subtype of stromal cells, form the LN architecture by production and deposition of extracellular matrix (ECM) within a conduit scaffold. Beyond structural roles, FRCs can regulate immunity by producing immunomodulatory factors that guide immune cell localization, maintain LN homeostasis, and promote peripheral tolerance. Multiple FRC subtypes exist, primarily defined in murine LNs by location and function, supported by single-cell RNA sequencing in both mice and humans. This thesis investigates the phenotypic and functional heterogeneity of human LN FRCs and how intrinsic (e.g., maturation, autofluorescence) and extrinsic factors (e.g., in vitro culture, ECM composition) shape them. Spectral flow cytometry is commonly used to characterize cells, by detecting protein expression with fluorescently labeled antibodies. However, human LN FRCs exhibit high autofluorescence with multiple distinct spectra, interfering with accurate signal detection. In Chapter 2, I developed an unbiased analysis workflow to identify all unique autofluorescence spectra within a sample. This approach enables reliable flow cytometric analysis of highly autofluorescent cells, including human LN FRCs. In Chapter 3, I examined whether human FRC subsets observed in fresh LNs persist after in vitro culture. Using the workflow from Chapter 2, I analyzed the phenotypic profiles of FRCs ex vivo and post-culture. Nine distinct FRC subsets were identified in freshly digested healthy human LNs. After culture, four of these persisted, while four new subsets emerged, demonstrating sustained heterogeneity of human FRCs both ex vivo and in vitro. FRCs contribute to peripheral tolerance through self-antigen expression on MHC class-II molecules, a function first identified in murine FRCs and increasingly supported for human FRCs. In Chapter 4 I show that MHC class-II expression correlates with FRC maturation. Moreover, I identified the subsets with the highest expression of MHC class-II: murine BST1⁺ FRCs (independent of the immune-inhibitory ligand CD200) and human BST1⁺CD200⁺ FRCs. These populations are likely important contributors to peripheral tolerance. In addition to immunological roles, FRCs provide mechanical support to LNs through ECM production and deposition. In Chapter 5 I reviewed the interplay between FRCs, ECM, and immune cells within LNs, during homeostasis, normal immune responses, and chronic dysfunction. ECM production, composition, deposition and stiffness vary with immune status, potentially influencing both FRCs and immune cells. In Chapter 6 I explored how ECM composition affects human LN FRCs in vitro. FRCs cultured on collagen type I, the most abundant ECM component in human LNs, transcriptionally resemble FRCs grown on native human LN ECM. In contrast, culturing on laminin subtypes produced distinct outcomes: laminin α4 promoted a more homeostatic transcriptomic profile, while laminin α5 induced a more pro-inflammatory profile. These findings indicate that ECM composition directly shapes FRC gene expression and potentially their immunoregulatory functions. Finally, in Chapter 7 I integrate the findings of the preceding chapters, discuss broader implications and future research directions, and propose a model of dynamic feedback between FRCs and the LN microenvironment. As FRC subsets are influenced by maturation and cellular interactions, I hypothesize that that there is a subset-specific ECM remodeling within the LN that reinforces spatial niches, such as T-cell zones and B-cell follicles. The immune status of the LN (e.g., tolerance versus inflammation) alters ECM composition produced by FRCs, which in turn feeds back into FRC transcriptomes and functions. This reciprocal regulation likely underpins LN adaptability during immune responses and tolerance, and its dysregulation may contribute to disease.