Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils

Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67phox

STUDY BACKGROUND: Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in CYBB, the X-linked gene that encodes gp91(phox), the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation in NCF2, the gene that encodes p67(phox), the activator subunit of the NADPH oxidase. METHODS: Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients' NCF2 gene was expressed as Ala202Val-p67(phox) in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67(phox) from the cytosol of the patients' neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification. RESULTS: The exceptional feature of the A67 CGD patients reported here is that the p.Ala202Val mutation in the activation domain of p67(phox) was clearly hypomorphic: substantial expression of p67(phox) protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20-70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67(phox) translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67(phox) in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67(phox). CONCLUSION: The mutation found in NCF2 is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p.Ala202Val mutation has changed the conformation of the activation domain of p67(phox), resulting in reduced activation of gp91(phox)

Kindlin3-Dependent CD11b/CD18-Integrin Activation Is Required for Potentiation of Neutrophil Cytotoxicity by CD47-SIRPα Checkpoint Disruption.

The CD47–signal regulatory protein-alpha (SIRPα) immune checkpoint constitutes a therapeutic target in cancer, and initial clinical studies using inhibitors of CD47–SIRPα interactions in combination with tumor-targeting antibodies show promising results. Blockade of CD47–SIRPα interaction can promote neutrophil antibody-dependent cellular cytotoxicity (ADCC) toward antibody-opsonized targets. Neutrophils induce killing of antibody-opsonized tumor cells by a process identified as trogoptosis, a necrotic/lytic type of cancer cell death that involves trogocytosis, the antibody-mediated endocytic acquisition of cancer membrane fragments by neutrophils. Both trogocytosis and killing strictly depend on CD11b/CD18-(Mac-1)–mediated neutrophil–cancer cell conjugate formation, but the mechanism by which CD47–SIRPα checkpoint disruption promotes cytotoxicity has remained elusive. Here, by using neutrophils from patients with leukocyte adhesion deficiency type III carrying&nbsp;FERMT3&nbsp;gene mutations, hence lacking the integrin-associated protein kindlin3, we demonstrated that CD47–SIRPα signaling controlled the inside-out activation of the neutrophil CD11b/CD18-integrin and cytotoxic synapse formation in a kindlin3-dependent fashion. Our findings also revealed a role for kindlin3 in trogocytosis and an absolute requirement in the killing process, which involved direct interactions between kindlin3 and CD18 integrin. Collectively, these results identified a dual role for kindlin3 in neutrophil ADCC and provide mechanistic insights into the way neutrophil cytotoxicity is governed by CD47–SIRPα interactions</p

Loss of function NFKB1 variants are the most common monogenic cause of CVID in Europeans.

BACKGROUND: The genetic etiology of primary immunodeficiency disease (PID) carries prognostic information. OBJECTIVE: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR-BioResource - Rare Disease cohort. METHODS: In the predominantly European study population of principally sporadic unrelated PID cases (n=846), a novel Bayesian method identified NFKB1 as one most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n=390) in the cohort. Amino-acid substitutions predicted to be pathogenic were assessed by analysis of structural protein data. Immunophenotyping, immunoblotting and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype co-segregation analyses. RESULTS: Both sporadic and familial cases demonstrated evidence of the non-infective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%) and autoimmune disease (48%), features prior studies correlate with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B lymphocyte differentiation. Detailed assessment of B lymphocyte numbers, phenotype and function identifies the presence of a raised CD21lowB cell population: combined with identification of the disease-causing variant, this distinguishes between healthy individuals, asymptomatic carriers and clinically affected cases. CONCLUSION: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID that results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.This study was supported by The National Institute for Health Research England (grant number RG65966), and by the Center of Immunodeficiencies Amsterdam (CIDA). JET is supported by an MRC Clinician Scientist Fellowship (MR/L006197/1). AJT is supported by both the Wellcome Trust (104807/Z/14/Z) and by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. EO receives personal fees from CSL Behring and MSD

Inherited p40(phox) deficiency differs from classic chronic granulomatous disease

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.status: publishe