Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development.

ABSTRACT: Antiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface-expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)-dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL-induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain-dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

Tumor-infiltrating CCR2+ inflammatory monocytes counteract specific immunotherapy.

Tumor development and progression is shaped by the tumor microenvironment (TME), a heterogeneous assembly of infiltrating and resident host cells, their secreted mediators and intercellular matrix. In this context, tumors are infiltrated by various immune cells with either pro-tumoral or anti-tumoral functions. Recently, we published our non-invasive immunization platform DIVA suitable as a therapeutic vaccination method, further optimized by repeated application (DIVA2). In our present work, we revealed the therapeutic effect of DIVA2 in an MC38 tumor model and specifically focused on the mechanisms induced in the TME after immunization. DIVA2 resulted in transient tumor control followed by an immune evasion phase within three weeks after the initial tumor inoculation. High-dimensional flow cytometry analysis and single-cell mRNA-sequencing of tumor-infiltrating leukocytes revealed cytotoxic CD8+ T cells as key players in the immune control phase. In the immune evasion phase, inflammatory CCR2+ PDL-1+ monocytes with immunosuppressive properties were recruited into the tumor leading to suppression of DIVA2-induced tumor-reactive T cells. Depletion of CCR2+ cells with specific antibodies resulted in prolonged survival revealing CCR2+ monocytes as important for tumor immune escape in the TME. In summary, the present work provides a platform for generating a strong antigen-specific primary and memory T cell immune response using the optimized transcutaneous immunization method DIVA2. This enables protection against tumors by therapeutic immune control of solid tumors and highlights the immunosuppressive influence of tumor infiltrating CCR2+ monocytes that need to be inactivated in addition for successful cancer immunotherapy.

Optimized dithranol-imiquimod-based transcutaneous immunization enables tumor rejection.

Introduction: Transcutaneous immunization (TCI) is a non-invasive vaccination method promoting strong cellular immune responses, crucial for the immunological rejection of cancer. Previously, we reported on the combined application of the TLR7 agonist imiquimod (IMQ) together with the anti-psoriatic drug dithranol as novel TCI platform DIVA (dithranol/IMQ based vaccination). In extension of this work, we further optimized DIVA in terms of drug dose, application pattern and established a new IMQ formulation. Methods: C57BL/6 mice were treated on the ear skin with dithranol and IMQ-containing ointments together with ovalbumin-derived peptides. T cell responses were determined by flow cytometry and IFN-ɤ ELISpot assay, local skin inflammation was characterized by ear swelling. Results: Applying the adjuvants on separate skin sites, a reduced number of specific CD8+ T cells with effector function was detectable, indicating that the local concurrence of adjuvants and peptide antigens is required for optimal vaccination. Likewise, changing the order of dithranol and IMQ resulted in an increased skin inflammatory reaction, but lower frequencies of antigen-specific CD8+ T cells indicating that dithranol is essential for superior T cell priming upon DIVA. Dispersing nanocrystalline IMQ in a spreadable formulation (IMI-Sol+) facilitated storage and application rendering comparable immune responses. DIVA applied one or two weeks after the first immunization resulted in a massive increase in antigen-specific T cells and up to a ten-fold increased memory response. Finally, in a prophylactic tumor setting, double but no single DIVA treatment enabled complete control of tumor growth, resulting in full tumor protection. Discussion: Taken together, the described optimized transcutaneous vaccination method leads to the generation of a strong cellular immune response enabling the effective control of tumor growth and has the potential for clinical development as a novel non-invasive vaccination method for peptide-based cancer vaccines in humans.

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.

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.

Dithranol as novel co-adjuvant for non-invasive dermal vaccination.

Transcutaneous immunization (TCI) utilizing the TLR7 agonist imiquimod (IMQ-TCI) induces T cell-driven protective immunity upon application onto intact skin. In our present work, we combine the anti-psoriatic agent dithranol with IMQ-TCI to boost vaccination efficacy (Dithranol/IMQ-based transcutaneous vaccination (DIVA)). Using ovalbumin-derived peptides as model antigens in mice, DIVA induced superior cytolytic CD8+ T cells and CD4+ T cells with a TH1 cytokine profile in the priming as well as in the memory phase. Regarding the underlying mechanisms, dithranol induced an oxidant-dependent, monocyte-attracting inflammatory milieu in the skin boosting TLR7-dependent activation of dendritic cells and macrophages leading to superior T cell priming and protective immunity in vaccinia virus infection. In conclusion, we introduce the non-invasive vaccination method DIVA to induce strong primary and memory T cell responses upon a single local treatment. This work provides relevant insights in cutaneous vaccination approaches, paving the way for clinical development in humans.

CD27 expression on Treg cells limits immune responses against tumors.

Regulatory T cells (Tregs) suppress immune responses and thus contribute to immune homeostasis. On the downside, Tregs also limit immune responses against tumors promoting the progression of cancer. Among the many mechanisms implied in Treg-mediated suppression, the inhibition of dendritic cells (DCs) has been shown to be central in peripheral tolerance induction as well as in cancers. We have shown previously that the maintenance of peripheral T cell tolerance critically depends on cognate interactions between Tregs and DCs and that the CTL priming by unsuppressed steady state DCs is mediated via CD70. Here, we have investigated whether the CD70/CD27 axis is also involved in Treg-mediated suppression of anti-tumor immunity. Using a mixed bone marrow chimeric mouse model in which we can deplete regulatory T cells in a temporally controlled fashion, we show that Treg-expressed CD27 prevents the breakdown of peripheral tolerance and limits anti-tumor immunity. Furthermore, ablation of Treg expressed CD27 acts synergistically with PD-1 checkpoint inhibition to improve CTL mediated immunity against a solid tumor. Our data thus identify Treg-expressed CD27 as a potential target in cancer immunotherapy. KEY MESSAGES : Treg expressed CD27 maintains steady state DC tolerogenic Treg expressed CD27 limits anti-tumor immunity Ablation of Treg expressed CD27 synergizes with PD-1 blockade to improve CTL mediated tumor control.

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.

Dermal CD207-Negative Migratory Dendritic Cells Are Fully Competent to Prime Protective, Skin Homing Cytotoxic T-Lymphocyte Responses.

Dendritic cells (DCs) are important inducers and regulators of T-cell responses. They are able to activate and modulate the differentiation of CD4+ and CD8+ T cells. In the skin, there are at least five phenotypically distinct DC subpopulations that can be distinguished by differential expression of the cell surface markers CD207, CD103, and CD11b. Previous studies have suggested that dermal CD11b-CD207+ conventional type 1 DCs are indispensable for the priming of a skin homing cytotoxic T-lymphocyte response. However, conventional type 1 DCs are also the only skin DC subset capable of cross-presenting exogenous antigens on major histocompatibility complex class I. Thus, it remained unclear whether for antigens that do not require cross-presentation, such as viruses that infect DCs, other DC subtypes in the skin can contribute to cytotoxic T-lymphocyte priming. To address this question, we used a transgenic mouse model that allows inducible expression and presentation of a model antigen on selected subsets of dermal DCs. We show that for antigens presented via the conventional major histocompatibility complex class I presentation pathway, CD207- dermal DCs are fully competent to prime a skin homing cytotoxic T-lymphocyte response that is capable of protection against a local virus challenge and gives rise to skin resident memory CD8+ T cells.

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.

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.

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.

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.

Cholesterol-induced conformational changes in the oxytocin receptor.

Recent studies suggest that cholesterol binding is widespread among GPCRs (G-protein-coupled receptors). In the present study, we analysed putative cholesterol-induced changes in the OTR [OT (oxytocin) receptor], a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant OTRs that are able to bind two small-sized fluorescence-labelled ligands simultaneously. An OTR antagonist was chosen as one of the ligands. To create a second ligand-binding site, a small-sized α-BTB (bungarotoxin binding) site was inserted at the N-terminus or within the third extracellular loop of the OTR. All receptor constructs were functionally active and bound both ligands with high affinity in the nanomolar range. Measurements of the quenching behaviour, fluorescence anisotropy and energy transfer of both receptor-bound ligands were performed to monitor receptor states at various cholesterol concentrations. The quenching studies suggested no major changes in the molecular environment of the fluorophores in response to cholesterol. The fluorescence anisotropy data indicated that cholesterol affects the dynamics or orientation of the antagonist. The energy transfer efficiency between both ligands clearly increased with increasing cholesterol. Overall, cholesterol induced both a changed orientation and a decreased distance of the receptor-bound ligands, suggesting a more compact receptor state in association with cholesterol.

Women with polycystic ovary syndrome (PCOS) often undergo protracted treatment with metformin and are disinclined to stop: indications for a change in licensing arrangements?

Women with polycystic ovary syndrome (PCOS) are increasingly being treated with metformin as an insulin sensitizing agent to reduce symptoms of hyperandrogenism and promote fertility. Indications such as hirsutism and cycle regulation require long term treatment. The drug is also being used through pregnancy. It is not licensed for any indication specific to PCOS, which means that much of this prescribing is taking place in an environment short of reliable information concerning safety. We describe the failure of recruitment to a study undertaken to explore the effects of metformin treatment discontinuation in women with PCOS, to provide both clinical and aetiological information. The study failed because the patients did not wish to stop treatment, and it illustrates the problems facing doctors working in this area. To achieve a safer prescribing environment, we recommend that action be taken by the manufacturer of metformin to work with regulatory agencies on a European base to extend prescribing indications for metformin to women with PCOS.