Jillian L. Astarita

Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31− LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α7. Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.

Podoplanin-Rich Stromal Networks Induce Dendritic Cell Motility via Activation of the C-type Lectin Receptor CLEC-2

Summary To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Podoplanin: emerging functions in development, the immune system, and cancer

Podoplanin (PDPN) is a well-conserved, mucin-type transmembrane protein expressed in multiple tissues during ontogeny and in adult animals, including the brain, heart, kidney, lungs, osteoblasts, and lymphoid organs. Studies of PDPN-deficient mice have demonstrated that this molecule plays a critical role in development of the heart, lungs, and lymphatic system. PDPN is widely used as a marker for lymphatic endothelial cells and fibroblastic reticular cells of lymphoid organs and for lymphatics in the skin and tumor microenvironment. Much of the mechanistic insight into PDPN biology has been gleaned from studies of tumor cells; tumor cells often upregulate PDPN as they undergo epithelial-mesenchymal transition and this upregulation is correlated with increased motility and metastasis. The physiological role of PDPN that has been most studied is its ability to aggregate and activate CLEC-2-expressing platelets, as PDPN is the only known endogenous ligand for CLEC-2. However, more recent studies have revealed that PDPN also plays crucial roles in the biology of immune cells, including T cells and dendritic cells. This review will provide a comprehensive overview of the diverse roles of PDPN in development, immunology, and cancer.

Dendritic cells control fibroblastic reticular network tension and lymph node expansion

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion—driven by lymphocyte influx and proliferation—that is the critical hallmark of adaptive immunity.

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.

Integration of Th17- and Lymphotoxin-Derived Signals Initiates Meningeal-Resident Stromal Cell Remodeling to Propagate Neuroinflammation

Tertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αβ (LTαβ) on Th17 cells and LTβR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTβR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.

Lymph node fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine sepsis

Infusion of lymph node–derived fibroblastic reticular cells into septic mice increases survival at late time points after disease onset. A Swiss Army Knife for Treating Sepsis Sepsis is a complication of infection that kills ~7 million people a year, with no successful molecular therapy thus far. But cells are more versatile than molecules: They make products and respond to their environments. Now, Fletcher et al. investigate whether these multifunctional tools are better equipped to battle this multifocal disease. They showed that one injection of anti-inflammatory cells derived from the lymph nodes dramatically increased survival in two mouse models of sepsis, even when delivered late in the disease course and under conditions that mimic those in the clinic. These beneficial cells reduced the deadly sepsis-associated “cytokine storm” by dampening this response in a manner that required the induction of nitric oxide synthase 2. Sepsis is an aggressive inflammatory syndrome and a global health burden estimated to kill 7.3 million people annually. Single-target molecular therapies have not addressed the multiple disease pathways triggered by septic injury. Cell therapies might offer a broader set of mechanisms of action that benefit complex, multifocal disease processes. We describe a population of immune-specialized myofibroblasts derived from lymph node tissue, termed fibroblastic reticular cells (FRCs). Because FRCs have an immunoregulatory function in lymph nodes, we hypothesized that ex vivo–expanded FRCs would control inflammation when administered therapeutically. Indeed, a single injection of ex vivo–expanded allogeneic FRCs reduced mortality in mouse models of sepsis when administered at early or late time points after septic onset. Mice treated with FRCs exhibited lower local and systemic concentrations of proinflammatory cytokines and reduced bacteremia. When administered 4 hours after induction of lipopolysaccharide endotoxemia, or cecal ligation and puncture (CLP) sepsis in mice, FRCs reduced deaths by at least 70%. When administered late in disease (16 hours after CLP), FRCs still conveyed a robust survival advantage (44% survival compared to 0% for controls). FRC therapy was dependent on the metabolic activity of nitric oxide synthase 2 (NOS2) as the primary molecular mechanism of drug action in the mice. Together, these data describe a new anti-inflammatory cell type and provide preclinical evidence for therapeutic efficacy in severe sepsis that warrants further translational study.

FAP delineates heterogeneous and functionally divergent stromal cells in immune-excluded breast tumors

Cancer-associated stromal cells restrain responses to immunotherapy and are associated with poor prognosis. Isolation of mesenchymal cells from fresh breast tumor samples revealed subsets with distinct phenotype and immunoregulatory potential. These observations may lead to better designed immunotherapies. Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.