Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Targeted removal of macrophage-secreted interleukin-1 receptor antagonist protects against lethal Candida albicans sepsis.

Invasive fungal infections are associated with high mortality rates, and the lack of efficient treatment options emphasizes an urgency to identify underlying disease mechanisms. We report that disseminated Candida albicans infection is facilitated by interleukin-1 receptor antagonist (IL-1Ra) secreted from macrophages in two temporally and spatially distinct waves. Splenic CD169+ macrophages release IL-1Ra into the bloodstream, impeding early neutrophil recruitment. IL-1Ra secreted by monocyte-derived tissue macrophages further impairs pathogen containment. Therapeutic IL-1Ra neutralization restored the functional competence of neutrophils, corrected maladapted hyper-inflammation, and eradicated the otherwise lethal infection. Conversely, augmentation of macrophage-secreted IL-1Ra by type I interferon severely aggravated disease mortality. Our study uncovers how a fundamental immunoregulatory mechanism mediates the high disease susceptibility to invasive candidiasis. Furthermore, interferon-stimulated IL-1Ra secretion may exacerbate fungal dissemination in human patients with secondary candidemia. Macrophage-secreted IL-1Ra should be considered as an additional biomarker and potential therapeutic target in severe systemic candidiasis.

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages.

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

Cross-presenting Langerhans cells are required for the early reactivation of resident CD8+ memory T cells in the epidermis.

Tissue-resident memory CD8+ T cells (TRM) reside at sites of previous infection, providing protection against reinfection with the same pathogen. In the skin, TRM patrol the epidermis, where keratinocytes are the entry site for many viral infections. Epidermal TRM react rapidly to cognate antigen encounter with the secretion of cytokines and differentiation into cytotoxic effector cells, constituting a first line of defense against skin reinfection. Despite the important protective role of skin TRM, it has remained unclear, whether their reactivation requires a professional antigen-presenting cell (APC). We show here, using a model system that allows antigen targeting selectively to keratinocytes in a defined area of the skin, that limited antigen expression by keratinocytes results in rapid, antigen-specific reactivation of skin TRM. Our data identify epidermal Langerhans cells that cross-present keratinocyte-derived antigens, as the professional APC indispensable for the early reactivation of TRM in the epidermal layer of the skin.

Classical DC2 subsets and monocyte-derived DC: Delineating the developmental and functional relationship.

To specifically tailor immune responses to a given pathogenic threat, dendritic cells (DC) are highly heterogeneous and comprise many specialized subtypes, including conventional DC (cDC) and monocyte-derived DC (MoDC), each with distinct developmental and functional characteristics. However, the functional relationship between cDC and MoDC is not fully understood, as the overlapping phenotypes of certain type 2 cDC (cDC2) subsets and MoDC do not allow satisfactory distinction of these cells in the tissue, particularly during inflammation. However, precise cDC2 and MoDC classification is required for studies addressing how these diverse cell types control immune responses and is therefore currently one of the major interests in the field of cDC research. This review will revise murine cDC2 and MoDC biology in the steady state and under inflammatory conditions and discusses the commonalities and differences between ESAMlo cDC2, inflammatory cDC2, and MoDC and their relative contribution to the initiation, propagation, and regulation of immune responses.

Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

Antigen targeting to dendritic cells: Still a place in future immunotherapy?

The hallmark of DCs is their potent and outstanding capacity to activate naive resting T cells. As such, DCs are the sentinels of the immune system and instrumental for the induction of immune responses. This is one of the reasons, why DCs became the focus of immunotherapeutical strategies to fight infections, cancer, and autoimmunity. Besides the exploration of adoptive DC-therapy for which DCs are generated from monocytes or purified in large numbers from the blood, alternative approaches were developed such as antigen targeting of DCs. The idea behind this strategy is that DCs resident in patients' lymphoid organs or peripheral tissues can be directly loaded with antigens in situ. The proof of principle came from mouse models; subsequent translational studies confirmed the potential of this therapy. The first clinical trials demonstrated feasibility and the induction of T-cell immunity in patients. This review will cover: (i) the historical aspects of antigen targeting, (ii) briefly summarize the biology of DCs and the immunological functions upon which this concept rests, (iii) give an overview on attempts to target DC receptors with antibodies or (glycosylated) ligands, and finally, (iv) discuss the translation of antigen targeting into clinical therapy.

Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non-lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single-cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single-cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells.

Mature dendritic cells (DCs) are established as unrivaled antigen-presenting cells (APCs) in the initiation of immune responses, whereas steady-state DCs induce peripheral T cell tolerance. Using various genetic approaches, we depleted CD11c(+) DCs in mice and induced autoimmune CNS inflammation. Unexpectedly, mice lacking DCs developed aggravated disease compared to control mice. Furthermore, when we engineered DCs to present a CNS-associated autoantigen in an induced manner, we found robust tolerance that prevented disease, which coincided with an upregulation of the PD-1 receptor on antigen-specific T cells. Additionally, we showed that PD-1 was necessary for DC-mediated induction of regulatory T cells. Our results show that a reduction of DCs interferes with tolerance, resulting in a stronger inflammatory response, and that other APC populations could compensate for the loss of immunogenic APC function in DC-depleted mice.

Transcutaneous immunization with CD40 ligation boosts cytotoxic T lymphocyte mediated antitumor immunity independent of CD4 helper cells in mice.

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8+ and CD4+ T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4+ T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI-Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI.

p38 MAPK controls prothrombin expression by regulated RNA 3' end processing.

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3' end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3' end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3'UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3' end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 3' end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.

Innate Effector-Memory T-Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution.

RATIONALE: Immune cells play an important role during the generation and resolution of thrombosis. T cells are powerful regulators of immune and nonimmune cell function, however, their role in sterile inflammation in venous thrombosis has not been systematically examined. OBJECTIVE: This study investigated the recruitment, activation, and inflammatory activity of T cells in deep vein thrombosis and its consequences for venous thrombus resolution. METHODS AND RESULTS: CD4+ and CD8+ T cells infiltrate the thrombus and vein wall rapidly on deep vein thrombosis induction and remain in the tissue throughout the thrombus resolution. In the vein wall, recruited T cells largely consist of effector-memory T (TEM) cells. Using T-cell receptor transgenic reporter mice, we demonstrate that deep vein thrombosis-recruited TEM receive an immediate antigen-independent activation and produce IFN-γ (interferon) in situ. Mapping inflammatory conditions in the thrombotic vein, we identify a set of deep vein thrombosis upregulated cytokines and chemokines that synergize to induce antigen-independent IFN-γ production in CD4+ and CD8+ TEM cells. Reducing the number of TEM cells through a depletion recovery procedure, we show that intravenous TEM activation determines neutrophil and monocyte recruitment and delays thrombus neovascularization and resolution. Examining T-cell recruitment in human venous stasis, we show that superficial varicose veins preferentially contain activated memory T cells. CONCLUSIONS: TEM orchestrate the inflammatory response in venous thrombosis affecting thrombus resolution.

IL-36R signalling activates intestinal epithelial cells and fibroblasts and promotes mucosal healing in vivo.

OBJECTIVE: Interleukin (IL)-36R signalling plays a proinflammatory role in different organs including the skin, but the expression of IL-36R ligands and their molecular function in intestinal inflammation are largely unknown. DESIGN: We studied the characteristics of IL-36R ligand expression in IBDs and experimental colitis. The functional role of IL-36R signalling in the intestine was addressed in experimental colitis and wound healing models in vivo by using mice with defective IL-36R signalling (IL-36R-/-) or Myd88, neutralising anti-IL-36R antibodies, recombinant IL-36R ligands and RNA-seq genome expression analysis. RESULTS: Expression of IL-36α and IL-36γ was significantly elevated in active human IBD and experimental colitis. While IL-36γ was predominantly detected in nuclei of the intestinal epithelium, IL-36α was mainly found in the cytoplasm of CD14+ inflammatory macrophages. Functional studies showed that defective IL-36R signalling causes high susceptibility to acute dextran sodium sulfate colitis and impairs wound healing. Mechanistically, IL-36R ligands released upon mucosal damage activated IL-36R+ colonic fibroblasts via Myd88 thereby inducing expression of chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6. Moreover, they induced proliferation of intestinal epithelial cells (IECs) and expression of the antimicrobial protein lipocalin 2. Finally, treatment of experimental intestinal wounds with IL-36R ligands significantly accelerated mucosal healing in vivo. CONCLUSIONS: IL-36R signalling is activated upon intestinal damage, stimulates IECs and fibroblasts and drives mucosal healing. Modulation of the IL-36R pathway emerges as a potential therapeutic strategy for induction of mucosal healing in IBD.

Regulatory T cell-derived adenosine induces dendritic cell migration through the Epac-Rap1 pathway.

Dendritic cells (DC) are one target for immune suppression by regulatory T cells (Treg), because their interaction results in reduced T cell stimulatory capacity and secretion of inhibitory cytokines in DC. We show that DC in the presence of Treg are more mobile as compared with cocultures with conventional CD4(+) T cells and form DC-Treg aggregates within 2 h of culture. The migration of DC was specifically directed toward Treg, as Treg, but not CD4(+) T cells, attracted DC in Boyden chambers. Treg deficient for the ectonucleotidase CD39 were unable to attract DC. Likewise, addition of antagonists for A2A adenosine receptors abolished the formation of DC-Treg clusters, indicating a role for adenosine in guiding DC-Treg interactions. Analysis of the signal transduction events in DC after contact to Treg revealed increased levels of cAMP, followed by activation of Epac1 and the GTPase Rap1. Subsequently activated Rap1 localized to the subcortical actin cytoskeleton in DC, providing a means by which directed locomotion of DC toward Treg is facilitated. In aggregate, these data show that Treg degrade ATP to adenosine via CD39, attracting DC by activating Epac1-Rap1-dependent pathways. As a consequence, DC-Treg clusters are formed and DC are rendered less stimulatory. This adenosine-mediated attraction of DC may therefore act as one mechanism by which Treg regulate the induction of immune responses by DC.

Regulation of the tolerogenic function of steady-state DCs.

Dendritic cells (DCs) are master regulators of T-cell responses. After sensing pathogen-derived molecular patterns (PAMPs), or signals of inflammation and cellular stress, DCs differentiate into potent activators of naïve CD4(+) and CD8(+) T cells through a process that is termed DC maturation. By contrast, DCs induce and maintain peripheral T-cell tolerance in the steady state, that is in the absence of overt infection or inflammation. However, the immunological steady state is not devoid of DC-activating stimuli, such as commensal microorganisms, subclinical infections, or basal levels of proinflammatory mediators. In the presence of these activating stimuli, DC maturation must be calibrated to ensure self-tolerance yet allow for adequate T-cell responses to infections. Here, we review the factors that are known to control DC maturation in the steady state and discuss their effect on the tolerogenic function of steady-state DCs.

A CD40/CD40L feedback loop drives the breakdown of CD8(+) T-cell tolerance following depletion of suppressive CD4(+) T cells.

Dendritic cells (DCs) are the key APCs not only for the priming of naïve T cells, but also for the induction and maintenance of peripheral T-cell tolerance. We have recently shown that cognate interactions between Foxp3(+) Tregs and steady-state DCs are crucial to maintain the tolerogenic potential of DCs. Using DIETER mice, which allow the induction of antigen presentation selectively on DCs without altering their maturation status, we show here that breakdown of CD8(+) T-cell tolerance, which ensues after depletion of suppressive CD4(+) T cells, is driven by a positive feedback loop in which autoreactive CD8(+) T cells activate DCs via CD40. These data identify ligation of CD40 on DCs as a stimulus that promotes autoreactive T-cell priming when regulatory T-cell suppression fails and suggest that feedback from autoreactive T cells to DCs may contribute to the well-documented involvement of CD40 in many autoimmune diseases.

Release of dendritic cells from cognate CD4+ T-cell recognition results in impaired peripheral tolerance and fatal cytotoxic T-cell mediated autoimmunity.

Resting dendritic cells (DCs) induce tolerance of peripheral T cells that have escaped thymic negative selection and thus contribute significantly to protection against autoimmunity. We recently showed that CD4(+)Foxp3(+) regulatory T cells (Tregs) are important for maintaining the steady-state phenotype of DCs and their tolerizing capacity in vivo. We now provide evidence that DC activation in the absence of Tregs is a direct consequence of missing DC-Treg interactions rather than being secondary to generalized autoimmunity in Treg-less mice. We show that DCs that lack MHC class II and thus cannot make cognate interactions with CD4(+) T cells are completely unable to induce peripheral CD8(+) T-cell tolerance. Consequently, mice in which interactions between DC and CD4(+) T cells are not possible develop spontaneous and fatal cytotoxic T lymphocyte-mediated autoimmunity.

Radiotherapy promotes tumor-specific effector CD8+ T cells via dendritic cell activation.

Radiotherapy is an important treatment for cancer. The main mode of action is thought to be the irreversible damage to tumor cell DNA, but there is evidence that irradiation mobilizes tumor-specific immunity, and recent studies showed that the efficacy of high-dose radiotherapy depends on the presence of CD8(+) T cells. We show in this study that the efficacy of radiotherapy given as a single, high dose (10 Gy) crucially depends on dendritic cells and CD8(+) T cells, whereas CD4(+) T cells or macrophages are dispensable. We show that local high-dose irradiation results in activation of tumor-associated dendritic cells that in turn support tumor-specific effector CD8(+) T cells, thus identifying the mechanism that underlies radiotherapy-induced mobilization of tumor-specific immunity. We propose that in the absence of irradiation, the activation status of dendritic cells rather than the amount of tumor-derived Ag is the bottleneck, which precludes efficient anti-tumor immunity.

FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells.

Peripheral T-cell tolerance is thought to significantly contribute to the prevention of autoimmunity, and it has been shown that antigen-presenting steady-state dendritic cells efficiently induce peripheral tolerance. We previously showed that dendritic-cell-induced tolerance is a T-cell-intrinsic process that depends on coinhibitory molecules such as programmed death-1. Here we specifically analyze the involvement of FoxP3(+) regulatory T cells, which are known to be important for maintenance of self-tolerance. We show that antigen presentation by steady-state dendritic cells failed to induce peripheral tolerance in the absence of FoxP3(+) regulatory T cells but induced protective CD8(+) T-cell-mediated immunity instead. Regulatory T-cell-depleted mice had massively increased numbers of dendritic cells in lymph nodes. Dendritic cells isolated from mice without regulatory T cells had up-regulated costimulatory molecules and showed stronger T-cell stimulatory capacity ex vivo, suggesting that regulatory T cells contribute to peripheral tolerance by keeping the dendritic cells in an immature state. Using blocking antibodies, we demonstrate that CTLA-4 but not IL-10 is necessary for control of dendritic cells by regulatory T cells.