A survey of new PIs in the UK

The challenges facing a new independent group leader, principal investigator (PI) or university lecturer are formidable: secure funding, recruit staff and students, establish a research programme, give lectures, and carry out various administrative duties. Here we report the results of a survey of individuals appointed as new group leaders, PIs or university lecturers in the UK between 2012 and 2018. The concerns expressed include difficulties in recruiting PhD students, maintaining a good work-life balance and securing permanent positions. Gender differences were also found in relation to starting salary and success with research funding. We make recommendations to employers and funders to address some of these concerns, and offer advice to those applying for PI positions

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 epithelialmesenchymal 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, includingT cells and dendritic cells.This review will provide a comprehensive overview of the diverse roles of PDPN in development, immunology, and cancer

Dendritic cells in remodeling of lymph nodes during immune responses

A critical hallmark of adaptive immune responses is the rapid and extensive expansion of lymph nodes. During this process, the complex internal structure of the organs is maintained revealing the existence of mechanisms able to balance lymph node integrity with structural flexibility. This article reviews the extensive architectural remodeling that occurs within lymph nodes during adaptive immune responses and how it is regulated by dendritic cells (DCs). In particular we focus on previously unappreciated functions of DCs in coordinating remodeling of lymph node vasculature, expansion of the fibroblastic reticular network and maintenance of lymphoid stromal phenotypes. Our increased understanding of these processes indicates that DCs need to be viewed not only as key antigen-presenting cells for lymphocytes but also as broad-acting immune sentinels that convey signals to lymphoid organ stroma and thereby facilitate immune response initiation at multiple levels

Cell-type-specific signaling networks in heterocellular organoids

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids

Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9

In adaptive immunity, CLEC-2+ dendritic cells (DCs) contact fibroblastic reticular cells (FRCs) inhibiting podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. The molecular mechanisms controlling lymph node remodelling are incompletely understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion, and which other proteins are required for the FRC response to DCs. We find that podoplanin and its partner proteins CD44 and CD9 are differentially expressed by specific lymph node stromal populations in vivo, and their expression in FRCs is coregulated by CLEC-2 (encoded by CLEC1B). Both CD44 and CD9 suppress podoplanin-dependent contractility. We find that beyond contractility, podoplanin is required for FRC polarity and alignment. Independently of podoplanin, CD44 and CD9 affect FRC–FRC interactions. Furthermore, our data show that remodelling of the FRC cytoskeleton in response to DCs is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2 and podoplanin binding inhibits FRC contractility, and, secondly, FRCs form protrusions and spread, which requires both CD44 and CD9. Together, we show a multi-faceted FRC response to DCs, which requires CD44 and CD9 in addition to podoplanin

Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular, physical, and structural properties and collectively determine tissue mechanics of lymph nodes, an immunologically relevant adult tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive expansion upon immune challenge. We find that in murine models of immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that C-type lectin-like receptor 2 (CLEC-2)–podoplanin signaling regulates the cell surface mechanics of fibroblasts, providing a mechanically sensitive pathway to regulate lymph node remodeling. Perturbation of fibroblast mechanics through genetic deletion of podoplanin attenuates T cell activation. We find that increased tissue tension through the fibroblastic stromal meshwork is required to trigger the initiation of fibroblast proliferation and restore homeostatic cellular ratios and tissue structure through lymph node expansion

Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilize amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by Rho GTPase signalling. Migrastatic drugs targeting actin polymerization and contractility are therefore a promising treatment option for metastatic melanoma. To predict amoeboid invasion and metastatic potential, biomarkers functionally linked to contractility pathways are needed. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts and is overexpressed in many cancers. We show that podoplanin enhances amoeboid invasion in melanoma. Podoplanin expression in murine melanoma drives rounded cell morphology, increasing motility, and invasion in vivo. Podoplanin expression is increased in a subset of dedifferentiated human melanoma, and in vitro is sufficient to upregulate melanoma-associated marker Pou3f2/Brn2. Together, our data define podoplanin as a functional biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

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

Th17 Cells Induce Ectopic Lymphoid Follicles in Central Nervous System Tissue Inflammation

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