Aspecific binding of anti-NK1.1 antibodies on myeloid cells in an experimental model for malaria-associated acute respiratory distress syndrome.

BACKGROUND: Conventional natural killer (cNK) cells play an important role in the innate immune response by directly killing infected and malignant cells and by producing pro- and anti-inflammatory cytokines. Studies on their role in malaria and its complications have resulted in conflicting results. METHODS: Using the commonly used anti-NK1.1 depletion antibodies (PK136) in an in-house optimized experimental model for malaria-associated acute respiratory distress syndrome (MA-ARDS), the role of cNK cells was investigated. Moreover, flow cytometry was performed to characterize different NK cell populations. RESULTS: While cNK cells were found to be dispensable in the development of MA-ARDS, the appearance of a NK1.1+ cell population was observed in the lungs upon infection despite depletion with anti-NK1.1. Detailed characterization of the unknown population revealed that this population consisted of a mixture of monocytes and macrophages that bind the anti-NK1.1 antibody in an aspecific way. This aspecific binding may occur via Fcγ receptors, such as FcγR4. In contrast, in vivo depletion using anti-NK1.1 antibodies was proved to be specific for cNK cells. CONCLUSION: cNK cells are dispensable in the development of experimental MA-ARDS. Moreover, careful flow cytometric analysis, with a critical mindset in relation to potential aspecific binding despite the use of commercially available Fc blocking reagents, is critical to avoid misinterpretation of the results.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE: We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS: We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

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.

Role for Granulocyte Colony-Stimulating Factor in Neutrophilic Extramedullary Myelopoiesis in a Murine Model of Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is a systemic inflammatory disease with childhood onset. Systemic JIA is associated with neutrophilia, including immature granulocytes, potentially driven by the growth factor granulocyte-colony stimulating factor (G-CSF). This study was undertaken to investigate the role of G-CSF in the pathology of systemic JIA. METHODS: Injection of Freund's complete adjuvant (CFA) in BALB/c mice induces mild inflammation and neutrophilia in wild-type (WT) mice and a more pronounced disease, reminiscent to that of JIA patients, in interferon-γ-knockout (IFNγ-KO) mice. Extramedullary myelopoiesis was studied in CFA-immunized mice by single-cell RNA sequencing, and the effect of G-CSF receptor (G-CSFR) blockage on neutrophil development and systemic JIA pathology was evaluated. Additionally, plasma G-CSF levels were measured in patients. RESULTS: Both in systemic JIA patients and in a corresponding mouse model, plasma G-CSF levels were increased. In the mouse model, we demonstrated that G-CSF is responsible for the observed neutrophilia and extramedullary myelopoiesis and the induction of immature neutrophils and myeloid-derived suppressor-like cells. Administration of a G-CSFR antagonizing antibody blocked the maturation and differentiation of neutrophils in CFA-immunized mice. In IFNγ-KO mice, treatment was associated with almost complete inhibition of arthritis due to reduced neutrophilia and osteoclast formation. Disease symptoms were ameliorated, but slight increases in interleukin-6 (IL-6), tumor necrosis factor, and IL-17 were detected upon G-CSFR inhibition in the IFNγ-KO mice, and were associated with mild increases in weight loss, tail damage, and immature red blood cells. CONCLUSION: We describe the role of G-CSF in a mouse model of systemic JIA and suggest an important role for G-CSF-induced myelopoiesis and neutrophilia in regulating the development of arthritis.