Immune checkpoint biology in health & disease: Immune checkpoint biology and autoimmunity in cancer patients.

Immune checkpoints (ICs) play a central role in maintaining immune homoeostasis. The discovery that tumours use this physiological mechanism to avoid elimination by the immune system, opened up avenues for therapeutic targeting of ICs as a novel way of treating cancer. However, this therapy a new array of autoimmune side effects, termed immune-related adverse events (irAEs). In this narrative review, we first recapitulate the physiological function of ICs that are approved targets for cancer immunotherapy (CTLA-4, PD-(L)1 and LAG-3), as the groundwork to critically discuss current knowledge on irAEs. Specifically, we summarize clinical aspects and examine a molecular classification and predisposing factors of irAEs. Finally, we discuss irAE treatment, particularly emphasizing how molecular knowledge is changing the current treatment paradigm.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. METHODS: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. FINDINGS: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. INTERPRETATION: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. FUNDING: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients.

The human chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is involved in several homeostatic processes and pathologies through interaction with its cognate G protein-coupled receptor CXCR4. Recent research has shown that CXCL12 is present in the lungs and circulation of patients with coronavirus disease 2019 (COVID-19). However, the question whether the detected CXCL12 is bioactive was not addressed. Indeed, the activity of CXCL12 is regulated by NH2- and COOH-terminal post-translational proteolysis, which significantly impairs its biological activity. The aim of the present study was to characterize proteolytic processing of CXCL12 in broncho-alveolar lavage (BAL) fluid and blood plasma samples from critically ill COVID-19 patients. Therefore, we optimized immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA) for detection of CXCL12 proteoforms. In patient samples, this approach uncovered that CXCL12 is rapidly processed by site-specific NH2- and COOH-terminal proteolysis and ultimately degraded. This proteolytic inactivation occurred more rapidly in COVID-19 plasma than in COVID-19 BAL fluids, whereas BAL fluid samples from stable lung transplantation patients and the non-affected lung of lung cancer patients (control groups) hardly induced any processing of CXCL12. In COVID-19 BAL fluids with high proteolytic activity, processing occurred exclusively NH2-terminally and was predominantly mediated by neutrophil elastase. In low proteolytic activity BAL fluid and plasma samples, NH2- and COOH-terminal proteolysis by CD26 and carboxypeptidases were observed. Finally, protease inhibitors already approved for clinical use such as sitagliptin and sivelestat prevented CXCL12 processing and may therefore be of pharmacological interest to prolong CXCL12 half-life and biological activity in vivo.

C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps.

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.

Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.

Single-cell transcriptomics identifies pathogenic T-helper 17.1 cells and pro-inflammatory monocytes in immune checkpoint inhibitor-related pneumonitis.

BACKGROUND: Immune checkpoint inhibitor (ICI)-related pneumonitis is the most frequent fatal immune-related adverse event associated with programmed cell death protein-1/programmed death ligand-1 blockade. The pathophysiology however remains largely unknown, owing to limited and contradictory findings in existing literature pointing at either T-helper 1 or T-helper 17-mediated autoimmunity. In this study, we aimed to gain novel insights into the mechanisms of ICI-related pneumonitis, thereby identifying potential therapeutic targets. METHODS: In this prospective observational study, single-cell RNA and T-cell receptor sequencing was performed on bronchoalveolar lavage fluid of 11 patients with ICI-related pneumonitis and 6 demographically-matched patients with cancer without ICI-related pneumonitis. Single-cell transcriptomic immunophenotyping and cell fate mapping coupled to T-cell receptor repertoire analyses were performed. RESULTS: We observed enrichment of both CD4+ and CD8+ T cells in ICI-pneumonitis bronchoalveolar lavage fluid. The CD4+ T-cell compartment showed an increase of pathogenic T-helper 17.1 cells, characterized by high co-expression of TBX21 (encoding T-bet) and RORC (ROR-γ), IFN-G (IFN-γ), IL-17A, CSF2 (GM-CSF), and cytotoxicity genes. Type 1 regulatory T cells and naïve-like CD4+ T cells were also enriched. Within the CD8+ T-cell compartment, mainly effector memory T cells were increased. Correspondingly, myeloid cells in ICI-pneumonitis bronchoalveolar lavage fluid were relatively depleted of anti-inflammatory resident alveolar macrophages while pro-inflammatory 'M1-like' monocytes (expressing TNF, IL-1B, IL-6, IL-23A, and GM-CSF receptor CSF2RA, CSF2RB) were enriched compared with control samples. Importantly, a feedforward loop, in which GM-CSF production by pathogenic T-helper 17.1 cells promotes tissue inflammation and IL-23 production by pro-inflammatory monocytes and vice versa, has been well characterized in multiple autoimmune disorders but has never been identified in ICI-related pneumonitis. CONCLUSIONS: Using single-cell transcriptomics, we identified accumulation of pathogenic T-helper 17.1 cells in ICI-pneumonitis bronchoalveolar lavage fluid-a phenotype explaining previous divergent findings on T-helper 1 versus T-helper 17 involvement in ICI-pneumonitis-,putatively engaging in detrimental crosstalk with pro-inflammatory 'M1-like' monocytes. This finding yields several novel potential therapeutic targets for the treatment of ICI-pneumonitis. Most notably repurposing anti-IL-23 merits further research as a potential efficacious and safe treatment for ICI-pneumonitis.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

Lung Aeration in COVID-19 Pneumonia by Ultrasonography and Computed Tomography.

We conducted a prospective single-center observational study to determine lung ultrasound reliability in assessing global lung aeration in 38 hospitalized patients with non-critical COVID-19. On admission, fixed chest CT scans using visual (CTv) and software-based (CTs) analyses along with lung ultrasound imaging protocols and scoring systems were applied. The primary endpoint was the correlation between global chest CTs score and global lung ultrasound score. The secondary endpoint was the association between radiographic features and clinical disease classification or laboratory indices of inflammation. Bland−Altman analysis between chest CT scores obtained visually (CTv) or using software (CTs) indicated that only 1 of the 38 paired measures was outside the 95% limits of agreement (−4 to +4 score). Global lung ultrasound score was highly and positively correlated with global software-based CTs score (r = 0.74, CI = 0.55−0.86; p < 0.0001). Significantly higher median CTs score (p = 0.01) and lung ultrasound score (p = 0.02) were found in severe compared to moderate COVID-19. Furthermore, we identified significantly lower (p < 0.05) lung ultrasound and CTs scores in those patients with a more severe clinical condition manifested by SpO2 < 92% and C-reactive protein > 58 mg/L. We concluded that lung ultrasound is a reliable bedside clinical tool to assess global lung aeration in hospitalized non-critical care patients with COVID-19 pneumonia.

Clinical practices underlie COVID-19 patient respiratory microbiome composition and its interactions with the host.

Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the respiratory microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 respiratory microbiome studies by analyzing the upper (n = 58) and lower (n = 35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We find that time in the intensive care unit and type of oxygen support, as well as associated treatments such as antibiotic usage, explain the most variation within the upper respiratory tract microbiome, while SARS-CoV-2 viral load has a reduced impact. Specifically, mechanical ventilation is linked to altered community structure and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomics of the lower respiratory tract of COVID-19 patients identifies specific oral bacteria in physical association with proinflammatory immune cells, which show higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients.

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

OBJECTIVES: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation. METHODS: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils. RESULTS: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils. CONCLUSION: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages.

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

Increased IL-10-producing regulatory T cells are characteristic of severe cases of COVID-19.

OBJECTIVES: The pandemic spread of the coronavirus SARS-CoV-2 is due, in part, to the immunological properties of the host-virus interaction. The clinical presentation varies from individual to individual, with asymptomatic carriers, mild-to-moderate-presenting patients and severely affected patients. Variation in immune response to SARS-CoV-2 may underlie this clinical variation. METHODS: Using a high-dimensional systems immunology platform, we have analysed the peripheral blood compartment of 6 healthy individuals, 23 mild-to-moderate and 20 severe COVID-19 patients. RESULTS: We identify distinct immunological signatures in the peripheral blood of the mild-to-moderate and severe COVID-19 patients, including T-cell lymphopenia, more consistent with peripheral hypo- than hyper-immune activation. Unique to the severe COVID-19 cases was a large increase in the proportion of IL-10-secreting regulatory T cells, a lineage known to possess anti-inflammatory properties in the lung. CONCLUSION: As IL-10-secreting regulatory T cells are known to possess anti-inflammatory properties in the lung, their proportional increase could contribute to a more severe COVID-19 phenotype. We openly provide annotated data (https://flowrepository.org/experiments/2713) with clinical correlates as a systems immunology resource for the COVID-19 research community.

Capmatinib for the treatment of non-small cell lung cancer.

Introduction: Activation of the MET pathway through MET amplifications or mutations is present in 3-4% of stage IV non-squamous non-small cell lung cancers (NSCLC). High MET amplifications and exon 14 skipping mutations are associated with poor prognosis: new treatments are needed for these patients. Capmatinib is a highly selective, potent small-molecule MET inhibitor with antitumor activity in NSCLC in vitro and in vivo. Areas covered: This article provides an overview of the capmatinib clinical development program in NSCLC, both as monotherapy in NSCLC with a dysregulated MET pathway, and in combination with epidermal growth factor receptor (EGFR) inhibitor therapy in EGFR-mutant NSCLC with MET-based acquired resistance to previous EGFR inhibition. Expert opinion: In the GEOMETRY Mono-1 study, treatment with capmatinib resulted in high response rates in stage IV NSCLC with MET exon 14 skipping mutations, particularly in first line, supporting testing for this biomarker at the time of diagnosis. Durable responses have been reported and results in MET-amplified NSCLC are eagerly anticipated. In EGFR-mutant NSCLC, notable responses have been observed in combination with an EGFR-tyrosine kinase inhibitor (TKI) in case of acquired resistance to EGFR-TKIs based on high MET amplification.