Automated antibody dispensing to improve high-parameter flow cytometry throughput and analysis.

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.

Loss of function of XBP1 splicing activity of IRE1α favors B cell tolerance breakdown.

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.

LXR signaling controls homeostatic dendritic cell maturation.

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.

Loss of GM-CSF-dependent instruction of alveolar macrophages in COVID-19 provides a rationale for inhaled GM-CSF treatment.

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).

Efficacy of CD40 Agonists Is Mediated by Distinct cDC Subsets and Subverted by Suppressive Macrophages.

Agonistic αCD40 therapy has been shown to inhibit cancer progression in only a fraction of patients. Understanding the cancer cell-intrinsic and microenvironmental determinants of αCD40 therapy response is therefore crucial to identify responsive patient populations and to design efficient combinatorial treatments. Here, we show that the therapeutic efficacy of αCD40 in subcutaneous melanoma relies on preexisting, type 1 classical dendritic cell (cDC1)-primed CD8+ T cells. However, after administration of αCD40, cDC1s were dispensable for antitumor efficacy. Instead, the abundance of activated cDCs, potentially derived from cDC2 cells, increased and further activated antitumor CD8+ T cells. Hence, distinct cDC subsets contributed to the induction of αCD40 responses. In contrast, lung carcinomas, characterized by a high abundance of macrophages, were resistant to αCD40 therapy. Combining αCD40 therapy with macrophage depletion led to tumor growth inhibition only in the presence of strong neoantigens. Accordingly, treatment with immunogenic cell death-inducing chemotherapy sensitized lung tumors to αCD40 therapy in subcutaneous and orthotopic settings. These insights into the microenvironmental regulators of response to αCD40 suggest that different tumor types would benefit from different combinations of therapies to optimize the clinical application of CD40 agonists. SIGNIFICANCE: This work highlights the temporal roles of different dendritic cell subsets in promoting CD8+ T-cell-driven responses to CD40 agonist therapy in cancer.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

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 TCRß 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.

Effect of anti-interleukin drugs in patients with COVID-19 and signs of cytokine release syndrome (COV-AID): a factorial, randomised, controlled trial.

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:FiO2) 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 µg/L and immediately requiring high flow oxygen or mechanical ventilation, or a ferritin concentration of more than 1000 µg/L, which had been increasing over the previous 24 h, or lymphopenia below 800/mL with two of the following criteria: an increasing ferritin concentration of more than 700 µg/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 × 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. FUNDING: Belgian Health Care Knowledge Center and VIB Grand Challenges program.

The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection.

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.

Case Report: Convalescent Plasma, a Targeted Therapy for Patients with CVID and Severe COVID-19.

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.

Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.

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.