Shannon J. Turley

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.

TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells

Therapeutic antibodies that block the programmed death-1 (PD-1)–programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor β (TGFβ) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFβ-blocking and anti-PD-L1 antibodies reduced TGFβ signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFβ shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

Stromal infrastructure of the lymph node and coordination of immunity

The initiation of adaptive immune responses depends upon the careful maneuvering of lymphocytes and antigen into and within strategically placed lymph nodes (LNs). Non-hematopoietic stromal cells form the cellular infrastructure that directs this process. Once regarded as merely structural features of lymphoid tissues, these cells are now appreciated as essential regulators of immune cell trafficking, fluid flow, and LN homeostasis. Recent advances in the identification and in vivo targeting of specific stromal populations have resulted in striking new insights to the function of stromal cells and reveal a level of complexity previously unrealized. We discuss here recent discoveries that highlight the pivotal role that stromal cells play in orchestrating immune cell homeostasis and adaptive immunity.

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.

Fibroblastic Reticular Cells: Organization and Regulation of the T Lymphocyte Life Cycle

The connective tissue of any organ in the body is generally referred to as stroma. This complex network is commonly composed of leukocytes, extracellular matrix components, mesenchymal cells, and a collection of nerves, blood, and lymphoid vessels. Once viewed primarily as a structural entity, stromal cells of mesenchymal origin are now being intensely examined for their ability to directly regulate various components of immune cell function. There is particular interest in the ability of stromal cells to influence the homeostasis, activation, and proliferation of T lymphocytes. One example of this regulation occurs in the lymph node, where fibroblastic reticular cells support the maintenance of naive T cells, induce Ag-specific tolerance, and restrict the expansion of newly activated T cells. In an effort to highlight the varied immunoregulatory properties of fibroblastic reticular cells, we reviewed the most recent advances in this field and provide some insights into potential future directions.

Hepatic immune regulation by stromal cells

A metabolic organ, the liver also has a central role in tolerance induction. Stromal cells lining the hepatic sinusoids, such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), are the first liver cells to encounter gut-derived and systemic antigens, thereby shaping local and systemic tolerance. Recent studies have demonstrated that stromal cells can modulate immune responses by antigen-dependent and independent mechanisms. Stromal cells interfere with the function of other antigen-presenting cells (APCs) and induce non-responsive T cells as well as regulatory T cells and myeloid-derived suppressor cells (MDSCs). The immunosuppressive microenvironment thus created provides a means to protect the liver from tissue damage. Such tolerized surroundings, however, can be exploited by certain pathogens, promoting persistent liver infections.

A short field guide to fibroblast function in immunity

Fibroblasts in secondary lymphoid organs, or fibroblastic reticular cells (FRC), are gate-keepers of immune responses. Here, we frame how these cells regulate immune responses via a three-part scheme in which FRC can setup, support or suppress immune responses. We also review how fibroblasts from non-lymphoid tissues influence immunity and highlight how they resemble and differ from FRC. Overall, we aim to focus attention on the emerging roles of lymphoid tissue and non-lymphoid tissue fibroblasts in control of innate and adaptive immunity.

Macrophage Death following Influenza Vaccination Initiates the Inflammatory Response that Promotes Dendritic Cell Function in the Draining Lymph Node

The mechanism by which inflammation influences the adaptive response to vaccines is not fully understood. Here, we examine the role of lymph node macrophages (LNMs) in the induction of the cytokine storm triggered by inactivated influenza virus vaccine. Following vaccination, LNMs undergo inflammasome-independent necrosis-like death that is reliant on MyD88 and Toll-like receptor 7 (TLR7) expression and releases pre-stored interleukin-1α (IL-1α). Furthermore, activated medullary macrophages produce interferon-β (IFN-β) that induces the autocrine secretion of IL-1α. We also found that macrophage depletion promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of CD11b+ LNDCs to capture virus and express co-stimulatory molecules. Inhibition of the IL-1α-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1α increased the humoral response. Stimulation of the IL-1α inflammatory pathway might therefore represent a strategy to enhance antigen presentation by LNDCs and improve the humoral response against influenza vaccines.

Testosterone is an endogenous regulator of BAFF and splenic B cell number

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.Testosterone deficiency is associated with autoimmunity and increased B cell numbers, but the underlying mechanism is unclear. Here the authors show that testosterone may modulate the production of B cell survival factor BAFF by fibroblastic reticular cells via regulation of splenic neurotransmitter levels.

Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity

MYD88 signaling in fibroblastic reticular cells drives the initiation of immune responses in fat-associated lymphoid clusters. Setting the stage for attack In classical secondary lymphoid organs (SLOs) such as lymph nodes, tonsils, and Peyer’s patches, it is well established that fibroblastic reticular cells (FRCs) play an integral role in the generation of immune responses. Nonclassical SLOs, including fat-associated lymphoid clusters (FALCs), also play important roles in systemic immunity. However, the role of FRCs in FALCs has not been previously examined. Here, using Ccl19-driven cre to delete MYD88 in FALC-associated FRCs, Perez-Shibayama et al. report that FRCs in FALCs play both organizational and immunomodulatory roles. These studies add to the growing recognition of the importance of stromal cells in shaping immune organs and immune responses. Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in nonclassical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). Compared with classical secondary lymphoid organs such as lymph nodes and Peyer’s patches, FALCs develop along distinct differentiation trajectories and display a reduced structural complexity. Although it is well established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of classical secondary lymphoid organs, the role of FRCs in FALC-dependent peritoneal immunity remains unclear. Using FRC-specific gene targeting, we found that FRCs play an essential role in FALC-driven immune responses. Specifically, we report that initiation of peritoneal immunity was governed through FRC activation in a myeloid differentiation primary response 88 (MYD88)–dependent manner. FRC-specific ablation of MYD88 blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell–dependent B-cell activation and IgG class switching. Moreover, containment of Salmonella infection was compromised in mice lacking MYD88 expression in FRCs, indicating that FRCs in FALCs function as an initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.