Lucas Onder

B cell homeostasis and follicle confines are governed by fibroblastic reticular cells

Fibroblastic reticular cells (FRCs) are known to inhabit T cell–rich areas of lymphoid organs, where they function to facilitate interactions between T cells and dendritic cells. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools that target this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrated their indispensable role in antiviral T cell responses. Unexpectedly, loss of FRCs also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and identifies a subset of FRCs that control B cell homeostasis and follicle identity.

The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway

Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) beta-receptor-dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTalpha(1)beta(2)-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A-independent, LT- and B cell-dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

IL-7-producing stromal cells are critical for lymph node remodeling

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Endothelial cell–specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation

Endothelial cell ablation of the lymphotoxin-β receptor results in failure to develop peripheral lymph nodes and normal high endothelial venues, which impairs lymphocyte homing.

Specific fibroblastic niches in secondary lymphoid organs orchestrate distinct Notch-regulated immune responses

Fasnacht et al. now show that fibroblasts in secondary lymphoid organs are responsible for the production of Notch ligands regulating the differentiation of immune cells

Interleukin-7 is produced by afferent lymphatic vessels and supports lymphatic drainage

The cytokine interleukin (IL)-7 exerts essential roles in lymph node (LN) organogenesis and lymphocyte development and homeostasis. Recent studies have identified lymphatic endothelial cells (LECs) as a major source of IL-7 in LNs. Here, we report that LECs not only produce IL-7, but also express the IL-7 receptor chains IL-7Rα and CD132. Stimulation with recombinant IL-7 enhanced LEC in vitro activity and induced lymphangiogenesis in the cornea of wild-type (WT) mice. Whereas in IL-7Rα(-/-) mice, dermal lymphatic vessels (LVs) were abnormally organized and lymphatic drainage was compromised, transgenic overexpression of IL-7 in mice resulted in an expanded dermal LV network with increased drainage function. Moreover, systemic treatment with recombinant IL-7 enhanced lymphatic drainage in the skin of WT mice and of mice devoid of lymphocytes. Experiments in IL-7Rα(-/-) bone marrow chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7Rα expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7Rα(-/-) mice revealed that the pumping activity of collecting vessels was normal but fluid uptake into lymphatic capillaries was defective. Overall, our data point toward an unexpected new role for IL-7 as a potential autocrine mediator of lymphatic drainage.

A novel bacterial artificial chromosome-transgenic podoplanin-cre mouse targets lymphoid organ stromal cells in vivo.

Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes (LNs). Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the Pdpn–Cre mouse revealed transgene expression in subsets of fibroblastic reticular cells and lymphatic endothelial cells in LNs. Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen expression in the genetically tagged stromal cells in Pdpn–Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn–Cre expression is well-suited to characterize the phenotype and to dissect the function of lymphoid organ stromal cells.

Topological Small-World Organization of the Fibroblastic Reticular Cell Network Determines Lymph Node Functionality

Fibroblastic reticular cells (FRCs) form the cellular scaffold of lymph nodes (LNs) and establish distinct microenvironmental niches to provide key molecules that drive innate and adaptive immune responses and control immune regulatory processes. Here, we have used a graph theory-based systems biology approach to determine topological properties and robustness of the LN FRC network in mice. We found that the FRC network exhibits an imprinted small-world topology that is fully regenerated within 4 wk after complete FRC ablation. Moreover, in silico perturbation analysis and in vivo validation revealed that LNs can tolerate a loss of approximately 50% of their FRCs without substantial impairment of immune cell recruitment, intranodal T cell migration, and dendritic cell-mediated activation of antiviral CD8+ T cells. Overall, our study reveals the high topological robustness of the FRC network and the critical role of the network integrity for the activation of adaptive immune responses.

Alternative NF‐κB signaling regulates mTEC differentiation from podoplanin‐expressing presursors in the cortico‐medullary junction

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)‐expressing progenitor located at the cortico‐medullary junction. PDPN+ junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF‐κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage‐committed mTEC progenitors and shows that NF‐κB‐dependent progression of jTECs to mTECs is critical to secure central tolerance.