Dendritic cells (DCs) are short-lived immune cells that continuously roam our body in search for foreign or self-antigens. Upon acquisition of antigen, they mature and start migrating to the lymph node to present the antigen to naïve T cells. Depending on the context wherein the antigen is acquired, DCs will mature in a homeostatic or immunogenic manner. So far, the field is lacking proper tools to distinguish between the two maturation states. Most maturation markers are shared between the two states and therefore inappropriate to use. Still, defining the proper maturation type is crucial as it determines how the DCs will instruct the T cells towards antigen expressing cells. In this study, we used a lipid nanoparticle (LNP)-based approach to steer DC maturation pathways in vivo. CITE-seq analysis allowed us to design a panel of flow cytometry markers that reliably annotates the two DC maturation states, as validated in an infection and in a tumor model. Furthermore, the data corroborated that uptake of empty LNPs in DCs induces their homeostatic maturation, in contrast to uptake of mRNA-LNPs or TLR ligand-adjuvanted LNPs, leading to distinct effector T cell outputs. This reveals that LNPs themselves are not being decoded as “danger” by cDC1s, and that the cargo is essential to provide adjuvants activity, which is highly relevant for targeted design of LNP-based therapies.

The cytokine tumor necrosis factor (TNF) plays important roles in limiting infection but is also linked to sepsis. The mechanisms underlying these paradoxical roles are unclear. Here, we show that TNF limits the antimicrobial activity of Paneth cells (PCs), causing bacterial translocation from the gut to various organs. This TNF-induced lethality does not occur in mice with a PC-specific deletion in the TNF receptor, P55. In PCs, TNF stimulates the IFN pathway and ablates the steady-state unfolded protein response (UPR), effects not observed in mice lacking P55 or IFNAR1. TNF triggers the transcriptional downregulation of IRE1 key genes Ern1 and Ern2, which are key mediators of the UPR. This UPR deficiency causes a significant reduction in antimicrobial peptide production and PC antimicrobial activity, causing bacterial translocation to organs and subsequent polymicrobial sepsis, organ failure, and death. This study highlights the roles of PCs in bacterial control and therapeutic targets for sepsis.

Dendritic cells (DCs) are crucial gatekeepers of the balance between immunity and tolerance. They exist in two functional states, immature or mature, that refer to an information-sensing versus an information-transmitting state, respectively. Historically, the term DC maturation was used to describe the acquisition of immunostimulatory capacity by DCs following their triggering by pathogens or tissue damage signals. As such, immature DCs were proposed to mediate tolerance, whereas mature DCs were associated with the induction of protective T cell immunity. Later studies have challenged this view and unequivocally demonstrated that two distinct modes of DC maturation exist, homeostatic and immunogenic DC maturation, each with a distinct functional outcome. Therefore, the mere expression of maturation markers cannot be used to predict immunogenicity. How DCs become activated in homeostatic conditions and maintain tolerance remains an area of intense debate. Several recent studies have shed light on the signals driving the homeostatic maturation programme, especially in the conventional type 1 DC (cDC1) compartment. Here, we highlight our growing understanding of homeostatic DC maturation and the relevance of this process for immune tolerance. Dendritic cells (DCs) act as gatekeepers between immunity and tolerance. Initially, it was postulated that mature DCs promote effector T cell responses and immature DCs promote tolerance. Recent studies have shown instead that two distinct modes of DC maturation exist — homeostatic and immunogenic. Here, Bosteels and Janssens discuss our current understanding of homeostatic DC maturation and how this contributes to immune tolerance, with a focus on the cDC1 compartment.

Over the past decade, the flow cytometry field has witnessed significant advancements in the number of fluorochromes that can be detected. This enables researchers to analyze more than 40 markers simultaneously on thousands of cells per second. However, with this increased complexity and multiplicity of markers, the manual dispensing of antibodies for flow cytometry experiments has become laborious, time-consuming, and prone to errors. An automated antibody dispensing system could provide a potential solution by enhancing the efficiency, and by improving data quality by faithfully dispensing the fluorochrome-conjugated antibodies and by enabling the easy addition of extra controls. In this study, a comprehensive comparison of different liquid handlers for dispensing fluorochrome-labeled antibodies was conducted for the preparation of flow cytometry stainings. The evaluation focused on key criteria including dispensing time, dead volume, and reliability of dispensing. After benchmarking, the I.DOT, a non-contact liquid handler, was selected and optimized in more detail. In the end, the I.DOT was able to prepare a 25-marker panel in 20 min, including the full stain, all FMOs and all single stain controls for cells and beads. Having all these controls improved the validation of the panel, visualization, and analysis of the data. Thus, automated antibody dispensing by dispensers such as the I.DOT reduces time and errors, enhances data quality, and can be easily integrated in an automated workflow to prepare samples for flow cytometry.

The continuous engulfment of apoptotic cells initiates a homeostatic maturation program in conventional type I dendritic cells (cDC1s), hallmarked by the activation of the transcription factor LXRb, which mediates cholesterol efflux and dampens interferon stimulated gene expression. cDC1s are characterized by a high basal activation of the unfolded protein response (UPR) sensor IRE1, without concomitant induction of a proper UPR gene signature, a finding that has puzzled the field. Here we show that in absence of IRE1, the homeostatic maturation of cDC1s is blocked, while homeostatic maturation of cDC2s remains unaffected. IRE1 activation is strictly dependent on apoptotic cell engulfment and cholesterol influx, explaining its cDC1 subset specific activity. Stimulation of IRE1 endonuclease activity in cDC1s leads to a Regulated IRE1 Dependent Decay (RIDD) response, targeting miRNAs rather than mRNAs. This causes the degradation of miRNA-92a, which targets the cholesterol efflux transporter Abcg1. Loss of IRE1 leads to defects in cholesterol efflux in mature cDC1s and concomitant cell death, while cDC2s do not show any defects. Blocking miRNA synthesis or enforcing cholesterol efflux by treatment with reconstituted high-density lipoproteins rescues cDC1s from cell death. These data highlight the central role of IRE1 as a sensor of cholesterol influx in the ER, extending IRE1’s function beyond its canonical role in protein folding. Furthermore, they underscore the tight control of cholesterol metabolism during cDC1 maturation, uncovering a second pathway to coordinate cholesterol efflux that acts in parallel to LXRb.

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell–mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

Anti-nuclear antibodies are the hallmark of autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma. However, the molecular mechanisms of B cell tolerance breakdown in these pathological contexts are poorly known. The study of rare familial forms of autoimmune diseases could therefore help to better describe common biological mechanisms leading to B cell tolerance breakdown. By Whole-Exome Sequencing, we identified a new heterozygous mutation (p.R594C) in ERN1 gene, encoding IRE1α (Inositol-Requiring Enzyme 1α), in a multiplex family with several members presenting autoantibody-mediated autoimmunity. Using human cell lines and a knock-in (KI) transgenic mouse model, we showed that this mutation led to a profound defect of IRE1α ribonuclease activity on X-Box Binding Protein 1 (XBP1) splicing. The KI mice developed a broad panel of autoantibodies, however in a subclinical manner. These results suggest that a decrease of spliced form of XBP1 (XBP1s) production could contribute to B cell tolerance breakdown and give new insights into the function of IRE1α which are important to consider for the development of IRE1α targeting strategies.

The modulation of dendritic cell (DC) functions by intracellular parasites remains poorly investigated. Toxoplasma gondii (Tg) infects a wide range of warm-blooded animals including humans. In immune-competent individuals, acute Tg infection is mostly asymptomatic but the parasite chronically persists in the brain and cerebral chronic toxoplasmosis has recently emerged as an underestimated cause of mental disorders and neurodegenerative pathologies. Tg infection induces a robust Th1 immune response that is initiated by type I conventional DC (cDC1). cDC1-mediated T cell activation is critical to restrict parasite growth during acute toxoplasmosis and T cells play a major role in keeping chronic cerebral infection under control. The induction of the Unfolded Protein Response (UPR) in immune cells has recently emerged as a central response, not only to the accumulation of misfolded proteins in the Endoplasmic Reticulum (ER) but also to cellular metabolic variations and infections. In particular, TLR stimulation in macrophages and DC induces the expression of the XBP1s and CHOP transcription factors, which directly activate inflammatory cytokine production. The UPR also modulates MHC class I antigen presentation in cDC1. We found that Tg infection of Bone Marrow Derived DC (BMDC) triggers the IRE1α/XBP1 branch of the UPR and that parasite replication is not required to induce this pathway. Using BMDC deleted for XBP1, we demonstrated that the Tg-induced UPR promotes a unique set of pro-inflammatory cytokines in a MyD88-dependent manner. In addition, our in vitro results suggest that the UPR modulates MHC-I presentation of OVA peptides from OVA-expressing parasites. Finally, using reporter mice, XBP1 activation was confirmed in infected mice and specifically detected in splenic cDC1 during the acute phase of the infection. Mice deleted for IRE1α and XBP1 in CD11c+-DC display a severe susceptibility to infection demonstrating the protective role of DC specific activation of the UPR during Tg infection.

Three endoplasmic reticulum (ER) resident proteins, IRE1, PERK and ATF6 monitor the health status of the ER and initiate the unfolded protein response (UPR) to restore ER homeostasis during stress. IRE1, a conserved endonuclease, cleaves Xbp1 mRNA and generates the key transcription factor XBP1s. My lab studies the role of IRE1 in dendritic cells (DCs), antigen presenting cells that bridge innate and adaptive immune responses and are essential both for the generation of effective immunity and tolerance. We noticed that in vivo one subset of conventional DCs -cDC1s - displays constitutive IRE1 activity, in absence of a canonical UPR response. Despite strong activation of IRE1 and concomitant induction of XBP1 splicing in cDC1s, we never observed any typical XBP1-dependent gene expression and we undertook a RNA sequencing approach to retrieve a DC-specific gene signature for XBP1. To this end, we compared WT, XBP1KO and IRE1/XBP1DKO cDC1s, all isolated from the spleen. This comparison allowed us to dissect genes downregulated due to loss of XBP1 transcriptional activity versus genes downregulated due to the activation of Regulated IRE1 Dependent Decay (RIDD), a process driven by hyperactivation of IRE1 endonuclease activity in the absence of XBP1. Our analysis revealed that many genes in cDC1s appeared regulated in an IRE1 endonuclease-but not XBP1-dependent manner. Furthermore, the data point towards a novel physiological role for IRE1 in DC homeostasis and provide unique cues for IRE1 activation in DCs, which will be the main topic of this talk.

Supplementary Figure from Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages

GM-CSF promotes myelopoiesis and inflammation, and GM-CSF blockade is being evaluated as a treatment for COVID-19-associated hyperinflammation. Alveolar GM-CSF is, however, required for monocytes to differentiate into alveolar macrophages (AMs) that control alveolar homeostasis. By mapping cross-species AM development to clinical lung samples, we discovered that COVID-19 is marked by defective GM-CSF-dependent AM instruction and accumulation of pro-inflammatory macrophages. In a multi-center, open-label RCT in 81 non-ventilated COVID-19 patients with respiratory failure, we found that inhalation of rhu-GM-CSF did not improve mean oxygenation parameters compared with standard treatment. However, more patients on GM-CSF had a clinical response, and GM-CSF inhalation induced higher numbers of virus-specific CD8 effector lymphocytes and class-switched B cells, without exacerbating systemic hyperinflammation. This translational proof-of-concept study provides a rationale for further testing of inhaled GM-CSF as a non-invasive treatment to improve alveolar gas exchange and simultaneously boost antiviral immunity in COVID-19. This study is registered at ClinicalTrials.gov (NCT04326920) and EudraCT (2020-001254-22).

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

ABSTRACTAgonistic αCD40 therapy has shown to inhibit cancer progression, but only in a fraction of patients. Hence, understanding the cancer cell-intrinsic and microenvironmental determinants of αCD40 therapy response is crucial to identify responsive patient populations and design efficient combination treatments. Here, we showed that the therapeutic efficacy of αCD40 in responder melanoma tumours, relied on pre-existing cDC1-primed CD8+ T cells, however cDC1s were dispensable after αCD40 administration. Surprisingly, in response to αCD40 the abundance of activated cDCs, potentially derived from cDC2s increased, thereby further activating antitumour CD8+ T cells. Hence, distinct cDC subsets are required to induce αCD40 responses. By contrast, lung tumours, characterised by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumour growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell-death inducing chemotherapy sensitised non-immunogenic tumours to αCD40 therapy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) instructs monocytes to differentiate into alveolar macrophages (AM) that preserve lung homeostasis. By comparing AM development in mouse and human, we discovered that COVID-19 patients showed marked defects in GM-CSF-dependent AM instruction. The multi-center, open-label, randomized, controlled SARPAC-trial evaluated the efficacy and safety of 5 days of inhalation of rhu-GM-CSF (sargramostim, Leukine®) in 81 non-ventilated patients with COVID-19 and hypoxemic respiratory failure identified by PaO2/FiO2 ratio < 350mmHg. At day 6, more patients in the sargramostim group experienced at least 25% improvement in oxygenation compared with the standard of care group. Higher numbers of circulating class-switched B cells and effector virus-specific CD8 lymphocytes were found in the sargramostim group. Treatment adverse events, including signs of cytokine storm, were not different between active and control group. This proof-of-concept study demonstrates the feasibility and safety of inhaled GM-CSF in restoring alveolar gas exchange, while simultaneously boosting anti-COVID-19 immunity. ClinicalTrials.gov (NCT04326920).

The unfolded protein response (UPR) has emerged as a central regulator of immune cell responses in several pathologic contexts including infections. However, how intracellular residing pathogens modulate the UPR in dendritic cells (DCs) and thereby affect T cell‐mediated immunity remains uncharacterized. Here, we demonstrate that infection of DCs with Toxoplasma gondii ( T. gondii ) triggers a unique UPR signature hallmarked by the MyD88‐dependent activation of the IRE1α pathway and the inhibition of the ATF6 pathway. Induction of XBP1s controls pro‐inflammatory cytokine secretion in infected DCs, while IRE1α promotes MHCI antigen presentation of secreted parasite antigens. In mice, infection leads to a specific activation of the IRE1α pathway, which is restricted to the cDC1 subset. Mice deficient for IRE1α and XBP1 in DCs display a severe susceptibility to T. gondii and succumb during the acute phase of the infection. This early mortality is correlated with increased parasite burden and a defect in splenic T‐cell responses. Thus, we identify the IRE1α/XBP1s branch of the UPR as a key regulator of host defense upon T. gondii infection.

Background Infections with SARS-CoV-2 continue to cause significant morbidity and mortality. Interleukin (IL)-1 and IL-6 blockade have been proposed as therapeutic strategies in COVID-19, but study outcomes have been conflicting. We sought to study whether blockade of the IL-6 or IL-1 pathway shortened the time to clinical improvement in patients with COVID-19, hypoxic respiratory failure, and signs of systemic cytokine release syndrome. Methods We did a prospective, multicentre, open-label, randomised, controlled trial, in hospitalised patients with COVID-19, hypoxia, and signs of a cytokine release syndrome across 16 hospitals in Belgium. Eligible patients had a proven diagnosis of COVID-19 with symptoms between 6 and 16 days, a ratio of the partial pressure of oxygen to the fraction of inspired oxygen (PaO2:FiO(2)) of less than 350 mm Hg on room air or less than 280 mm Hg on supplemental oxygen, and signs of a cytokine release syndrome in their serum (either a single ferritin measurement of more than 2000 mu g/L and immediately requiring high flow oxygen or mechanical ventilation, or a ferritin concentration of more than 1000 mu g/L, which had been increasing over the previous 24 h, or lyrnphopenia below 800/mL with two of the following criteria: an increasing ferritin concentration of more than 700 mu g/L, an increasing lactate dehydrogenase concentration of more than 300 international units per L, an increasing C-reactive protein concentration of more than 70 mg/L, or an increasing D-dimers concentration of more than 1000 ng/mL). The COV-AID trial has a 2 x 2 factorial design to evaluate IL-1 blockade versus no IL-1 blockade and IL-6 blockade versus no IL-6 blockade. Patients were randomly assigned by means of permuted block randomisation with varying block size and stratification by centre. In a first randomisation, patients were assigned to receive subcutaneous anakinra once daily (100 mg) for 28 days or until discharge, or to receive no IL-1 blockade (1:2). In a second randomisation step, patients were allocated to receive a single dose of siltuximab (11 mg/kg) intravenously, or a single dose of tocilizumab (8 mg/kg) intravenously, or to receive no IL-6 blockade (1:1:1). The primary outcome was the time to clinical improvement, defined as time from randomisation to an increase of at least two points on a 6-category ordinal scale or to discharge from hospital alive. The primary and supportive efficacy endpoints were assessed in the intention-to-treat population. Safety was assessed in the safety population. This study is registered online with ClinicalTrials.gov (NCT04330638) and EudraCT (2020-001500-41) and is complete. Findings Between April 4, and Dec 6,2020,342 patients were randomly assigned to IL-1 blockade n=112) or no IL-1 blockade (n=230) and simultaneously randomly assigned to IL-6 blockade (n=227; 114 for tocilizumab and 113 for siltuximab) or no IL-6 blockade (n=115). Most patients were male (265 [77%] of 342), median age was 65 years (IQR 54-73), and median Systematic Organ Failure Assessment (SOFA) score at randomisation was 3 (2-4). All 342 patients were included in the primary intention-to-treat analysis. The estimated median time to clinical improvement was 12 days (95% CI 10-16) in the IL-1 blockade group versus 12 days (10-15) in the no IL-1 blockade group (hazard ratio [HR] 0.94 [95% CI 0.73-1.21]). For the IL-6 blockade group, the estimated median time to clinical improvement was 11 days (95% CI 10-16) versus 12 days (11-16) in the no IL-6 blockade group (HR 1.00[0-78-1-29]). 55 patients died during the study, but no evidence for differences in mortality between treatment groups was found. The incidence of serious adverse events and serious infections was similar across study groups. Interpretation Drugs targeting IL-1 or IL-6 did not shorten the time to clinical improvement in this sample of patients with COVID-19, hypoxic respiratory failure, low SOFA score, and low baseline mortality risk. Copyright (C) 2021 Elsevier Ltd. All rights reserved.

The phenotypic and functional dichotomy between IRF8(+) type 1 and IRF4(+) type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4(+) T helper (Th) cell polarization while simultaneously presenting antigen to CD8(+) T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.

The disease course of COVID-19 in patients with immunodeficiencies is unclear, as well as the optimal therapeutic strategy. We report a case of a 37-year old male with common variable immunodeficiency disorder and a severe SARS-CoV-2 infection. After administration of convalescent plasma, the patient’s condition improved rapidly. Despite clinical recovery, viral RNA remained detectable up to 60 days after onset of symptoms. We propose that convalescent plasma might be considered as a treatment option in patients with CVID and severe COVID-19. In addition, in patients with immunodeficiencies, a different clinical course is possible, with prolonged viral shedding.