Bioinspired 3D microprinted cell scaffolds: Integration of graph theory to recapitulate complex network wiring in lymph nodes.

Physical networks are ubiquitous in nature, but many of them possess a complex organizational structure that is difficult to recapitulate in artificial systems. This is especially the case in biomedical and tissue engineering, where the microstructural details of 3D cell scaffolds are important. Studies of biological networks-such as fibroblastic reticular cell (FRC) networks-have revealed the crucial role of network topology in a range of biological functions. However, cell scaffolds are rarely analyzed, or designed, using graph theory. To understand how networks affect adhered cells, 3D culture platforms capturing the complex topological properties of biologically relevant networks would be needed. In this work, we took inspiration from the small-world organization (high clustering and low path length) of FRC networks to design cell scaffolds. An algorithmic toolset was created to generate the networks and process them to improve their 3D printability. We employed tools from graph theory to show that the networks were small-world (omega factor, ω = -0.10 ± 0.02; small-world propensity, SWP = 0.74 ± 0.01). 3D microprinting was employed to physicalize networks as scaffolds, which supported the survival of FRCs. This work, therefore, represents a bioinspired, graph theory-driven approach to control the networks of microscale cell niches.

Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function.

Upon initial immune challenge, dendritic cells (DCs) migrate to lymph nodes and interact with fibroblastic reticular cells (FRCs) via C-type lectin-like receptor 2 (CLEC-2). CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs.

Delivery of costimulatory blockade to lymph nodes promotes transplant acceptance in mice.

The lymph node (LN) is the primary site of alloimmunity activation and regulation during transplantation. Here, we investigated how fibroblastic reticular cells (FRCs) facilitate the tolerance induced by anti-CD40L in a murine model of heart transplantation. We found that both the absence of LNs and FRC depletion abrogated the effect of anti-CD40L in prolonging murine heart allograft survival. Depletion of FRCs impaired homing of T cells across the high endothelial venules (HEVs) and promoted formation of alloreactive T cells in the LNs in heart-transplanted mice treated with anti-CD40L. Single-cell RNA sequencing of the LNs showed that anti-CD40L promotes a Madcam1+ FRC subset. FRCs also promoted the formation of regulatory T cells (Tregs) in vitro. Nanoparticles (NPs) containing anti-CD40L were selectively delivered to the LNs by coating them with MECA-79, which binds to peripheral node addressin (PNAd) glycoproteins expressed exclusively by HEVs. Treatment with these MECA-79-anti-CD40L-NPs markedly delayed the onset of heart allograft rejection and increased the presence of Tregs. Finally, combined MECA-79-anti-CD40L-NPs and rapamycin treatment resulted in markedly longer allograft survival than soluble anti-CD40L and rapamycin. These data demonstrate that FRCs are critical to facilitating costimulatory blockade. LN-targeted nanodelivery of anti-CD40L could effectively promote heart allograft acceptance.

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.

Control of Dendritic Cell Function Within the Tumour Microenvironment.

The tumour microenvironment (TME) presents a major block to anti-tumour immune responses and to effective cancer immunotherapy. The inflammatory mediators such as cytokines, chemokines, growth factors and prostaglandins generated in the TME alter the phenotype and function of dendritic cells (DCs) that are critical for a successful adaptive immune response against the growing tumour. In this mini review we discuss how tumour cells and the surrounding stroma modulate DC maturation and trafficking to impact T cell function. Fibroblastic stroma and the associated extracellular matrix around tumours can also provide physical restrictions to infiltrating DCs and other leukocytes. We discuss interactions between the inflammatory TME and infiltrating immune cell function, exploring how the inflammatory TME affects generation of T cell-driven anti-tumour immunity. We discuss the open question of the relative importance of antigen-presentation site; locally within the TME versus tumour-draining lymph nodes. Addressing these questions will potentially increase immune surveillance and enhance anti-tumour immunity.

Immune function and dysfunction are determined by lymphoid tissue efficacy.

Lymphoid tissue returns to a steady state once each immune response is resolved, and although this occurs multiple times throughout life, its structural integrity and functionality remain unaffected. Stromal cells orchestrate cellular interactions within lymphoid tissue, and any changes to the microenvironment can have detrimental outcomes and drive disease. A breakdown in lymphoid tissue homeostasis can lead to a loss of tissue structure and function that can cause aberrant immune responses. This Review highlights recent advances in our understanding of lymphoid tissue function and remodelling in adaptive immunity and in disease states. We discuss the functional role of lymphoid tissue in disease progression and explore the changes to lymphoid tissue structure and function driven by infection, chronic inflammatory conditions and cancer. Understanding the role of lymphoid tissues in immune responses to a wide range of pathologies allows us to take a fuller systemic view of disease progression.

Lymphocyte access to lymphoma is impaired by high endothelial venule regression.

Blood endothelial cells display remarkable plasticity depending on the demands of a malignant microenvironment. While studies in solid tumors focus on their role in metabolic adaptations, formation of high endothelial venules (HEVs) in lymph nodes extends their role to the organization of immune cell interactions. As a response to lymphoma growth, blood vessel density increases; however, the fate of HEVs remains elusive. Here, we report that lymphoma causes severe HEV regression in mouse models that phenocopies aggressive human B cell lymphomas. HEV dedifferentiation occurrs as a consequence of a disrupted lymph-carrying conduit system. Mechanosensitive fibroblastic reticular cells then deregulate CCL21 migration paths, followed by deterioration of dendritic cell proximity to HEVs. Loss of this crosstalk deprives HEVs of lymphotoxin-ß-receptor (LTßR) signaling, which is indispensable for their differentiation and lymphocyte transmigration. Collectively, this study reveals a remodeling cascade of the lymph node microenvironment that is detrimental for immune cell trafficking in lymphoma.

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.

Communication, construction, and fluid control: lymphoid organ fibroblastic reticular cell and conduit networks.

Fibroblastic reticular cells (FRCs) are a crucial part of the stromal cell infrastructure of secondary lymphoid organs (SLOs). Lymphoid organ fibroblasts form specialized niches for immune cell interactions and thereby govern lymphocyte activation and differentiation. Moreover, FRCs produce and ensheath a network of extracellular matrix (ECM) microfibers called the conduit system. FRC-generated conduits contribute to fluid and immune cell control by funneling fluids containing antigens and inflammatory mediators through the SLOs. We review recent progress in FRC biology that has advanced our understanding of immune cell functions and interactions. We discuss the intricate relationships between the cellular FRC and the fibrillar conduit networks, which together form the basis for efficient communication between immune cells and the tissues they survey.

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.

A mouse SWATH-mass spectrometry reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts.

SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here, we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from 'bulk tumour' measurements. We apply the XenoSWATH pipeline to characterize an intraductal xenograft model of breast ductal carcinoma in situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

Molecular mechanisms of dendritic cell migration in immunity and cancer.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells that act to bridge innate and adaptive immunity. DCs are critical in mounting effective immune responses to tissue damage, pathogens and cancer. Immature DCs continuously sample tissues and engulf antigens via endocytic pathways such as phagocytosis or macropinocytosis, which result in DC activation. Activated DCs undergo a maturation process by downregulating endocytosis and upregulating surface proteins controlling migration to lymphoid tissues where DC-mediated antigen presentation initiates adaptive immune responses. To traffic to lymphoid tissues, DCs must adapt their motility mechanisms to migrate within a wide variety of tissue types and cross barriers to enter lymphatics. All steps of DC migration involve cell-cell or cell-substrate interactions. This review discusses DC migration mechanisms in immunity and cancer with a focus on the role of cytoskeletal processes and cell surface proteins, including integrins, lectins and tetraspanins. Understanding the adapting molecular mechanisms controlling DC migration in immunity provides the basis for therapeutic interventions to dampen immune activation in autoimmunity, or to improve anti-tumour immune responses.

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 Cells Control Conduit Matrix Deposition during Lymph Node Expansion.

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-ß and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue.

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.

Immunotherapy: breaching the barriers for cancer treatment.

The great ambition to treat cancer through harnessing a patient's own immune responses has started to become reality. Clinical trials have shown impressive results and some patients reaching the end of existing treatment options have achieved full remission. Yet the response rate even within the most promising trials remain at just 30-40% of patients. To date, the focus of immunotherapy research has been to identify tumour antigens, and to enhance activation of effector lymphocytes. Yet this is only the first step to effective immunotherapy for a broader range of patients. Activated cytotoxic T cells can only act on their tumour cell targets if they have free and easy access to all tumour regions. Solid tumours are complex, heterogeneous environments which vary greatly in their physical properties. We must now focus our efforts on understanding how factors such as the composition, density and geometry of tumour extracellular matrix acts to impede or promote immune cell infiltration and activation, and work to design novel pharmacological interventions which restore and enhance leucocyte trafficking within solid tumours. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

The Stromal Niche Guarding the Gatekeepers.

In this issue of Immunity, Mondor et al. (2019) and Camara et al. (2019) show that lymphatic endothelial cells are essential components of the niche that forms and maintains the subcapsular sinusoidal macrophage network in homeostasis and throughout an immune challenge.

C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.

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.

Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity.

The 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 Braf(V600E) 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 Braf(V600E) mouse melanoma cells, as well as in Nras(G12D) 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.