SummaryThe mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant BrafV600E mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in BrafV600E mouse melanoma cells, as well as in NrasG12D melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.

Following immunogenic challenge, infiltrating and dividing lymphocytes significantly increase lymph node (LN) cellularity leading to organ expansion1,2. Here we report that the physical elasticity of LNs is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells (DCs). We show that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-kinase. 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 immunised mice augments LN expansion. In contrast, the latter is significantly constrained in mice selectively lacking CLEC-2 expression in DCs. Thus, the same DCs that initiate immunity by presenting antigens to T lymphocytes3 also initiate remodeling of LNs by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid LN expansion driven by lymphocyte influx and proliferation that is the critical hallmark of adaptive immunity.

SUMMARY Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögren’s syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.

Mononuclear phagocytes are classified as macrophages or dendritic cells (DCs) based on cell morphology, phenotype, or select functional properties. However, these attributes are not absolute and often overlap, leading to difficulties in cell-type identification. To circumvent this issue, we describe a mouse model to define DCs based on their ontogenetic descendence from a committed precursor. We show that precursors of mouse conventional DCs, but not other leukocytes, are marked by expression of DNGR-1. Genetic tracing of DNGR-1 expression history specifically marks cells traditionally ascribed to the DC lineage, and this restriction is maintained after inflammation. Notably, in some tissues, cells previously thought to be monocytes/macrophages are in fact descendants from DC precursors. These studies provide an in vivo model for fate mapping of DCs, distinguishing them from other leukocyte lineages, and thus help to unravel the functional complexity of the mononuclear phagocyte system.

Over the past decade, a series of discoveries relating to fibroblastic reticular cells (FRCs) — immunologically specialized myofibroblasts found in lymphoid tissue — has promoted these cells from benign bystanders to major players in the immune response. In this Review, we focus on recent advances regarding the immunobiology of lymph node-derived FRCs, presenting an updated view of crucial checkpoints during their development and their dynamic control of lymph node expansion and contraction during infection. We highlight the robust effects of FRCs on systemic B cell and T cell responses, and we present an emerging view of FRCs as drivers of pathology following acute and chronic viral infections. Lastly, we review emerging therapeutic advances that harness the immunoregulatory properties of FRCs.

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.

SummaryTo 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.

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.