Distinct ontogenetic lineages dictate cDC2 heterogeneity.

Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet+ cDC2As and T-bet- cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H+ pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H- CD8α+ pre-cDC2As in tissues, which differentiated into T-bet+ cDC2As. In contrast, a Siglec-H- fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet- cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment.

Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity.

Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses.

The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.

Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8+ T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.

NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control.

Conventional type 1 dendritic cells (cDC1) are critical for antitumor immunity, and their abundance within tumors is associated with immune-mediated rejection and the success of immunotherapy. Here, we show that cDC1 accumulation in mouse tumors often depends on natural killer (NK) cells that produce the cDC1 chemoattractants CCL5 and XCL1. Similarly, in human cancers, intratumoral CCL5, XCL1, and XCL2 transcripts closely correlate with gene signatures of both NK cells and cDC1 and are associated with increased overall patient survival. Notably, tumor production of prostaglandin E2 (PGE2) leads to evasion of the NK cell-cDC1 axis in part by impairing NK cell viability and chemokine production, as well as by causing downregulation of chemokine receptor expression in cDC1. Our findings reveal a cellular and molecular checkpoint for intratumoral cDC1 recruitment that is targeted by tumor-derived PGE2 for immune evasion and that could be exploited for cancer therapy.

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.

Antagonistic Inflammatory Phenotypes Dictate Tumor Fate and Response to Immune Checkpoint Blockade.

Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.

Genetic tracing via DNGR-1 expression history defines dendritic cells as a hematopoietic lineage.

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.

Sex Differences in the Impact of Electronic Nicotine Vapor on Corticotropin-Releasing Factor Receptor 1 Neurons in the Mouse Ventral Tegmental Area.

Nicotine engages dopamine neurons in the ventral tegmental area (VTA) to encode reward and drive the development of nicotine addiction, however how nicotine alters a stress associated VTA population remains unclear. Here, we used male and female CRF1-GFP mice and nicotine vapor exposure to examine the effects of nicotine in VTA corticotropin-releasing factor receptor 1 (CRF1) neurons. We use immunohistochemistry and electrophysiology to examine neuronal activity, excitability, and inhibitory signaling. We found that VTA CRF1 neurons are mainly dopaminergic and project to the nucleus accumbens (NAc; VTA-NAcCRF1 neurons). VTA-NAcCRF1 neurons show greater phasic inhibition in naive females and greater focal nicotine-induced increases in firing in naive males. Following acute nicotine vapor exposure, phasic inhibition was not altered, but focal nicotine-induced tonic inhibition was enhanced in females and diminished in males. Acute nicotine vapor exposure did not affect firing in VTA-NAcCRF1 neurons, but females showed lower baseline firing and higher focal nicotine-induced firing. Activity (cFos) was increased in the CRF1 dopaminergic VTA population in both sexes, but with greater increases in females. Following chronic nicotine vapor exposure, both sexes displayed reduced basal phasic inhibition and the sex difference in tonic inhibition following acute vapor exposure was no longer observed. Additionally, activity of the CRF1 dopaminergic VTA population was no longer elevated in either sex. These findings reveal sex-dependent and exposure-dependent changes in mesolimbic VTA-NAc CRF1 neuronal activity, inhibitory signaling, and nicotine sensitivity following nicotine vapor exposure. These changes potentially contribute to nicotine-dependent behaviors and the intersection between stress, anxiety, and addiction.SIGNIFICANCE STATEMENT Nicotine is known to engage reward systems in the brain historically centering the neurotransmitter dopamine however, how nicotine impacts other neurons in the reward pathway is less clear. The current study investigates the impact of acute and chronic electronic nicotine vapor exposure in a genetically-defined cell population containing the stress receptor corticotropin-releasing factor 1 (CRF1) that is located in the reward circuitry. This study employs functional measures of neuronal activity and identifies important sex differences in nicotine's effects across time and exposure.

Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells.

RNA interference (RNAi) elicited by long double-stranded (ds) or base-paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence-specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN-responsive cells with type I IFN Notably, transfection with long dsRNA specifically vaccinates IFN-deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.

Correction: Syk Signaling in Dendritic Cells Orchestrates Innate Resistance to Systemic Fungal Infection.

[This corrects the article DOI: 10.1371/journal.ppat.1004276.].

An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma.

Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation.

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.

Simultaneous observation of auroral substorm onset in Polar satellite global images and ground-based all-sky images.

Substorm onset has originally been defined as a longitudinally extended sudden auroral brightening (Akasofu initial brightening: AIB) followed a few minutes later by an auroral poleward expansion in ground-based all-sky images (ASIs). In contrast, such clearly marked two-stage development has not been evident in satellite-based global images (GIs). Instead, substorm onsets have been identified as localized sudden brightenings that expand immediately poleward. To resolve these differences, optical substorm onset signatures in GIs and ASIs are compared in this study for a substorm that occurred on December 7, 1999. For this substorm, the Polar satellite ultraviolet global imager was operated with a fixed-filter (170 nm) mode, enabling a higher time resolution (37 s) than usual to resolve the possible two-stage development. These data were compared with 20-s resolution green-line (557.7 nm) ASIs at Muonio in Finland. The ASIs revealed the AIB at 2124:50 UT and the subsequent poleward expansion at 2127:50 UT, whereas the GIs revealed only an onset brightening that started at 2127:49 UT. Thus, the onset in the GIs was delayed relative to the AIB and in fact agreed with the poleward expansion in the ASIs. The fact that the AIB was not evident in the GIs may be attributed to the limited spatial resolution of GIs for thin auroral arc brightenings. The implications of these results for the definition of substorm onset are discussed herein.

Oncogenic Transformation of Dendritic Cells and Their Precursors Leads to Rapid Cancer Development in Mice.

Dendritic cells (DCs) are powerful APCs that can induce Ag-specific adaptive immune responses and are increasingly recognized as important players in innate immunity to both infection and malignancy. Interestingly, although there are multiple described hematological malignancies, DC cancers are rarely observed in humans. Whether this is linked to the immunogenic potential of DCs, which might render them uniquely susceptible to immune control upon neoplastic transformation, has not been fully investigated. To address the issue, we generated a genetically engineered mouse model in which expression of Cre recombinase driven by the C-type lectin domain family 9, member a (Clec9a) locus causes expression of the Kirsten rat sarcoma viral oncogene homolog (Kras)(G12D) oncogenic driver and deletion of the tumor suppressor p53 within developing and differentiated DCs. We show that these Clec9a(Kras-G12D) mice rapidly succumb from disease and display massive accumulation of transformed DCs in multiple organs. In bone marrow chimeras, the development of DC cancer could be induced by a small number of transformed cells and was not prevented by the presence of untransformed DCs. Notably, activation of transformed DCs did not happen spontaneously but could be induced upon stimulation. Although Clec9a(Kras-G12D) mice showed altered thymic T cell development, peripheral T cells were largely unaffected during DC cancer development. Interestingly, transformed DCs were rejected upon adoptive transfer into wild-type but not lymphocyte-deficient mice, indicating that immunological control of DC cancer is in principle possible but does not occur during spontaneous generation in Clec9a(Kras-G12D) mice. Our findings suggest that neoplastic transformation of DCs does not by default induce anti-cancer immunity and can develop unhindered by immunological barriers.

Altered lymph node composition in diphtheria toxin receptor-based mouse models to ablate dendritic cells.

Dendritic cells (DCs) are key regulators of innate and adaptive immunity. Our understanding of immune function has benefited greatly from mouse models allowing for selective ablation of DCs. Many such models rely on transgenic diphtheria toxin receptor (DTR) expression driven by DC-restricted promoters. This renders DCs sensitive to DT but is otherwise thought to have no effect on immune physiology. In this study, we report that, unexpectedly, mice in which DTR is expressed on conventional DCs display marked lymph node (LN) hypocellularity and reduced frequency of DCs in the same organs but not in spleen or nonlymphoid tissues. Intriguingly, in mixed bone marrow chimeras the phenotype conferred by DTR-expressing DCs is dominant over control bone marrow-derived cells, leading to small LNs and an overall paucity of DCs independently of the genetic ability to express DTR. The finding of alterations in LN composition and size independently of DT challenge suggests that caution must be exercised when interpreting results of experiments obtained with mouse models to inducibly deplete DCs. It further indicates that DTR, a member of the epidermal growth factor family, is biologically active in mice. Its use in cell ablation experiments needs to be considered in light of this activity.

Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses.

IFN-α/ß allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2-like DExH-box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN-α/ß by retinoic acid inducible gene 1-like receptors (RLRs) that detect the presence of RNA viruses in a cell-intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN-α/ß induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60-deficient mice revealed no impairment in IFN-α/ß production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60-deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus-1. These results put in question the reported role of DDX60 as a broad-acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses.

Syk signaling in dendritic cells orchestrates innate resistance to systemic fungal infection.

Host protection from fungal infection is thought to ensue in part from the activity of Syk-coupled C-type lectin receptors and MyD88-coupled toll-like receptors in myeloid cells, including neutrophils, macrophages and dendritic cells (DCs). Given the multitude of cell types and receptors involved, elimination of a single pathway for fungal recognition in a cell type such as DCs, primarily known for their ability to prime T cell responses, would be expected to have little effect on innate resistance to fungal infection. Here we report that this is surprisingly not the case and that selective loss of Syk but not MyD88 in DCs abrogates innate resistance to acute systemic Candida albicans infection in mice. We show that Syk expression by DCs is necessary for IL-23p19 production in response to C. albicans, which is essential to transiently induce GM-CSF secretion by NK cells that are recruited to the site of fungal replication. NK cell-derived-GM-CSF in turn sustains the anti-microbial activity of neutrophils, the main fungicidal effectors. Thus, the activity of a single kinase in a single myeloid cell type orchestrates a complex series of molecular and cellular events that underlies innate resistance to fungal sepsis.

Identification of a dendritic cell receptor that couples sensing of necrosis to immunity.

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair. In addition, antigens present in necrotic cells can sometimes provoke a specific immune response and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection. In the mouse, the CD8alpha+ subset of dendritic cells phagocytoses dead cell remnants and cross-primes CD8+ T cells against cell-associated antigens. Here we show that CD8alpha+ dendritic cells use CLEC9A (also known as DNGR-1), a recently-characterized C-type lectin, to recognize a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair the uptake of necrotic cell material by CD8+ dendritic cells, but specifically reduces cross-presentation of dead-cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue in its intracellular tail that allows the recruitment and activation of the tyrosine kinase SYK, which is also essential for cross-presentation of dead-cell-associated antigens. Thus, CLEC9A functions as a SYK-coupled C-type lectin receptor to mediate sensing of necrosis by the principal dendritic-cell subset involved in regulating cross-priming to cell-associated antigens.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.