The retroelement Lx9 puts a brake on the immune response to virus infection.

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.

T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

The impact of the cytoplasmic ubiquitin ligase TNFAIP3 gene variation on transcription factor NF-κB activation in acute kidney injury.

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

Cutting Edge: Recruitment, Retention, and Migration Underpin Functional Phenotypic Heterogeneity of Regulatory T Cells in Tumors.

Tumor-infiltrating regulatory T cells (Tregs) have been extensively studied as therapeutic targets. However, not all infiltrating T cells exert their functions equally, presumably because of their heterogeneity and substantial turnover in tissues. In this study, we hypothesized that intertissue migration underlies the functional heterogeneity of Tregs. To test this, we applied in vivo photolabeling to examine single-cell diversity of immunosuppressive molecules in mouse Tregs migrating to, remaining in, and emigrating from MC38 tumors. Neuropilin-1 (Nrp1) expression was inversely correlated with that of six other molecules associated with Treg function. Unsupervised clustering analyses revealed that clusters containing Tregs that were retained in tumors expressed high levels of the six functional molecules but not of Nrp1. However, these clusters represented only half of the Tregs migrating to the tumor, suggesting evolving heterogeneity of tumor-infiltrating Tregs. Thus, we propose progressive pathways of Treg activation and migration between tumors and draining lymph nodes.

Near-infrared photoimmunotherapy induced tumor cell death enhances tumor dendritic cell migration.

Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein Kikume Green-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326+ DCs to dLNs. This effect was abolished by blocking adenosine triphosphate (ATP), one of the DAMPs molecules, as well as by inhibition of Gαi signaling by pertussis toxin. Thus, ICD induction by NIR-PIT stimulates Ti-DC migration to dLNs via ATP-P2X7 receptor and Gαi protein-coupled receptor signaling pathways and may augment tumor antigen presentation to induce anti-tumor T cells in dLNs.

Imaging the neutrophil: Intravital microscopy provides a dynamic view of neutrophil functions in host immunity.

Neutrophils are the first cellular responders of the immune system. They employ their impressive arsenal of microbicidal molecules to provide rapid and efficient defense against pathogens. However, the role of neutrophils extends far beyond microbial destruction to include tissue repair and remodeling, provision of signals to the adaptive immune system and body homeostasis. Intravital imaging has allowed the visualization of neutrophils in their native environment in both health and disease and provided crucial insights into their mechanisms of action. In the last few years the power of intravital imaging has been considerably extended by the introduction of photoconvertible proteins and intracellular signaling reporter mice. This review will highlight recent advances in our understanding of neutrophil biology based on the use of intravital microscopy to visualize their modus operandi in vivo including migration in and out of inflamed tissues, host-pathogen interactions and cell fate.

The lymph node neutrophil.

Secondary lymphoid organs provide a specialized microenvironment tailored to foster communication between cells of the innate and adaptive immune systems. These interactions allow immune cells to coordinate multilayered defense against pathogens. Until recently dendritic cells and macrophages were thought to comprise the main innate immune cell subsets responsible for delivering signals that drive the adaptive immune response, while the function of neutrophils was largely confined to the innate immune system. However, the discovery of neutrophils in lymph nodes has raised the question of whether neutrophils might play a more extensive role not only in innate immunity per se, but also in coordinating the interactions between innate and adaptive immune responses. In this review we discuss the mechanisms and consequences of neutrophil recruitment to lymph nodes and how this recruitment influences subsequent immune responses both in situ and at distant sites.

In vivo photolabeling of tumor-infiltrating cells reveals highly regulated egress of T-cell subsets from tumors.

Immune therapy is rapidly gaining prominence in the clinic as a major weapon against cancer. Whereas much attention has been focused on the infiltration of tumors by immune cells, the subsequent fate of these infiltrates remains largely unexplored. We therefore established a photoconversion-based model that allowed us to label tumor-infiltrating immune cells and follow their migration. Using this system, we identified a population of tumor-experienced cells that emigrate from primary tumors to draining lymph nodes via afferent lymphatic vessels. Although the majority of tumor-infiltrating cells were myeloid, T cells made up the largest population of tumor-egressing leukocytes. Strikingly, the subset composition of tumor-egressing T cells was greatly skewed compared with those that had infiltrated the tumor and those resident in the draining lymph node. Some T-cell subsets such as CD8+ T cells emigrated more readily; others including CD4-CD8- T cells were preferentially retained, suggesting that specific mechanisms guide immune cell egress from tumors. Furthermore, tumor-egressing T cells were more activated and displayed enhanced effector function in comparison with their lymph node counterparts. Finally, we demonstrated that tumor-infiltrating T cells migrate to distant secondary tumors and draining lymph nodes, highlighting a mechanism whereby tumor-experienced effector T cells may mediate antitumor immunity at metastatic sites. Thus, our results provide insights into migration and function of tumor-infiltrating immune cells and the role of these cells in tumor immunity outside of primary tumor deposits.

Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node.

Memory T cells circulate through lymph nodes where they are poised to respond rapidly upon re-exposure to a pathogen; however, the dynamics of memory T cell, antigen-presenting cell, and pathogen interactions during recall responses are largely unknown. We used a mouse model of infection with the intracellular protozoan parasite, Toxoplasma gondii, in conjunction with two-photon microscopy, to address this question. After challenge, memory T cells migrated more rapidly than naive T cells, relocalized toward the subcapsular sinus (SCS) near invaded macrophages, and engaged in prolonged interactions with infected cells. Parasite invasion of T cells occurred by direct transfer of the parasite from the target cell into the T cell and corresponded to an antigen-specific increase in the rate of T cell invasion. Our results provide insight into cellular interactions during recall responses and suggest a mechanism of pathogen subversion of the immune response.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

Dynamics of neutrophil migration in lymph nodes during infection.

Although the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning-laser microscopy to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We found that neutrophils formed both small, transient and large, persistent swarms via a coordinated migration pattern. We provided evidence that cooperative action of neutrophils and parasite egress from host cells could trigger swarm formation. Neutrophil swarm formation coincided in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses.

Leukocyte Motility Models Assessed through Simulation and Multi-objective Optimization-Based Model Selection.

The advent of two-photon microscopy now reveals unprecedented, detailed spatio-temporal data on cellular motility and interactions in vivo. Understanding cellular motility patterns is key to gaining insight into the development and possible manipulation of the immune response. Computational simulation has become an established technique for understanding immune processes and evaluating hypotheses in the context of experimental data, and there is clear scope to integrate microscopy-informed motility dynamics. However, determining which motility model best reflects in vivo motility is non-trivial: 3D motility is an intricate process requiring several metrics to characterize. This complicates model selection and parameterization, which must be performed against several metrics simultaneously. Here we evaluate Brownian motion, Lévy walk and several correlated random walks (CRWs) against the motility dynamics of neutrophils and lymph node T cells under inflammatory conditions by simultaneously considering cellular translational and turn speeds, and meandering indices. Heterogeneous cells exhibiting a continuum of inherent translational speeds and directionalities comprise both datasets, a feature significantly improving capture of in vivo motility when simulated as a CRW. Furthermore, translational and turn speeds are inversely correlated, and the corresponding CRW simulation again improves capture of our in vivo data, albeit to a lesser extent. In contrast, Brownian motion poorly reflects our data. Lévy walk is competitive in capturing some aspects of neutrophil motility, but T cell directional persistence only, therein highlighting the importance of evaluating models against several motility metrics simultaneously. This we achieve through novel application of multi-objective optimization, wherein each model is independently implemented and then parameterized to identify optimal trade-offs in performance against each metric. The resultant Pareto fronts of optimal solutions are directly contrasted to identify models best capturing in vivo dynamics, a technique that can aid model selection more generally. Our technique robustly determines our cell populations' motility strategies, and paves the way for simulations that incorporate accurate immune cell motility dynamics.

Skin immunity in wound healing and cancer.

The skin is the body's largest organ. It serves as a barrier to pathogen entry and the first site of immune defense. In the event of a skin injury, a cascade of events including inflammation, new tissue formation and tissue remodeling contributes to wound repair. Skin-resident and recruited immune cells work together with non-immune cells to clear invading pathogens and debris, and guide the regeneration of damaged host tissues. Disruption to the wound repair process can lead to chronic inflammation and non-healing wounds. This, in turn, can promote skin tumorigenesis. Tumors appropriate the wound healing response as a way of enhancing their survival and growth. Here we review the role of resident and skin-infiltrating immune cells in wound repair and discuss their functions in regulating both inflammation and development of skin cancers.

Neutrophil Conversion to a Tumor-Killing Phenotype Underpins Effective Microbial Therapy.

The inflammatory microenvironment of solid tumors creates a protumorigenic milieu that resembles chronic inflammation akin to a subverted wound healing response. Here, we investigated the effect of converting the tumor microenvironment from a chronically inflamed state to one of acute microbial inflammation by injecting microbial bioparticles directly into tumors. Intratumoral microbial bioparticle injection led to rapid and dramatic changes in the tumor immune composition, the most striking of which was a substantial increase in the presence of activated neutrophils. In situ photoconversion and intravital microscopy indicated that tumor neutrophils transiently switched from sessile producers of VEGF to highly motile neutrophils that clustered to make neutrophil-rich domains in the tumor. The neutrophil clusters remodeled tumor tissue and repressed tumor growth. Single-cell transcriptional analysis of microbe-stimulated neutrophils showed a profound shift in gene expression towards heightened activation and antimicrobial effector function. Microbe-activated neutrophils also upregulated chemokines known to regulate neutrophil and CD8+ T-cell recruitment. Microbial therapy also boosted CD8+ T-cell function and enhanced the therapeutic benefit of checkpoint inhibitor therapy in tumor-bearing mice and provided protection in a model of tumor recurrence. These data indicate that one of the major effector mechanisms of microbial therapy is the conversion of tumor neutrophils from a wound healing to an acutely activated cytotoxic phenotype, highlighting a rationale for broader deployment of microbial therapy in the treatment of solid cancers. SIGNIFICANCE: Intratumoral injection of microbial bioparticles stimulates neutrophil antitumor functions, suggesting pathways for optimizing efficacy of microbial therapies and paving the way for their broader utilization in the clinic.

Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation.

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. ARTICLE HIGHLIGHTS: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

Dynamic imaging of T cell-parasite interactions in the brains of mice chronically infected with Toxoplasma gondii.

The intracellular parasite Toxoplasma gondii can establish persistent infection in the brain of a mammalian host, a standoff that involves the active participation of host CD8 T cells to control infection. CD8 T cells generally protect against intracellular pathogens by local delivery of effector molecules upon recognition of specific pathogen Ags on invaded host cells. However, the interactions between CD8 T cells, T. gondii, and APCs in the brain have not yet been examined. In this study we have used a mouse infection model in conjunction with two-photon microscopy of living brain tissue and confocal microscopy of fixed brain sections to examine the interactions between CD8 T cells, parasites, and APCs from chronically infected mice. We found that Ag-specific CD8 T cells were recruited to the brains of infected mice and persisted there in the presence of ongoing Ag recognition. Cerebral CD8 T cells made transient contacts with granuloma-like structures containing parasites and with individual CD11b(+) APCs, including some that did not contain parasites. In contrast, T cells ignored intact Ag-bearing cysts and did not contact astrocytes or neurons, including neurons containing parasites or cysts. Our data represent the first direct observation of the dynamics of T cell-parasite interactions within living tissue and provide a new perspective for understanding immune responses to persistent pathogens in the brain.

Neutrophil Interactions with the Lymphatic System.

The lymphatic system is a complex network of lymphatic vessels and lymph nodes designed to balance fluid homeostasis and facilitate host immune defence. Neutrophils are rapidly recruited to sites of inflammation to provide the first line of protection against microbial infections. The traditional view of neutrophils as short-lived cells, whose role is restricted to providing sterilizing immunity at sites of infection, is rapidly evolving to include additional functions at the interface between the innate and adaptive immune systems. Neutrophils travel via the lymphatics from the site of inflammation to transport antigens to lymph nodes. They can also enter lymph nodes from the blood by crossing high endothelial venules. Neutrophil functions in draining lymph nodes include pathogen control and modulation of adaptive immunity. Another facet of neutrophil interactions with the lymphatic system is their ability to promote lymphangiogenesis in draining lymph nodes and inflamed tissues. In this review, we discuss the significance of neutrophil migration to secondary lymphoid organs and within the lymphatic vasculature and highlight emerging evidence of the neutrophils' role in lymphangiogenesis.

Immunogenic tumor cell death promotes dendritic cell migration and inhibits tumor growth via enhanced T cell immunity.

Immunogenic tumor cell death enhances anti-tumor immunity. However, the mechanisms underlying this effect are incompletely understood. We established a system to induce tumor cell death in situ and investigated its effect on dendritic cell (DC) migration and T cell responses using intravital photolabeling in mice expressing KikGR photoconvertible protein. We demonstrate that tumor cell death induces phagocytosis of tumor cells by tumor-infiltrating (Ti)-DCs, and HMGB1-TLR4 and ATP-P2X7 receptor signaling-dependent Ti-DC emigration to draining lymph nodes (dLNs). This led to an increase in anti-tumor CD8+ T cells of memory precursor effector phenotype and secondary tumor growth inhibition in a CD103+ DC-dependent manner. However, combining tumor cell death induction with lipopolysaccharide treatment stimulated Ti-DC maturation and emigration to dLNs but did not improve tumor immunity. Thus, immunogenic tumor cell death enhances tumor immunity by increasing Ti-DC migration to dLNs where they promote anti-tumor T cell responses and tumor growth inhibition.

Thymocyte-dendritic cell interactions near sources of CCR7 ligands in the thymic cortex.

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 expression are closely associated with capillaries throughout the cortex. The overexpression of the chemokine receptor CCR7 in thymocytes results in an increase in DC-thymocyte interactions, while the loss of CCR7 in the background of a positive-selecting TCR reduces the extent of DC-thymocyte interactions. These observations identify a vasculature-associated microenvironment within the thymic cortex that promotes interactions between DCs and thymocytes that are receiving positive selection signals.

A zebrafish functional genomics model to investigate the role of human A20 variants in vivo.

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.