The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called El). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the

To obtain a molecular definition of regulatory T (Treg) cell identity, we performed proteomics and transcriptomics on various populations of human regulatory and conventional CD4 T (Tconv) cells. A protein expression signature was identified that defines all Treg cells, and another signature that defines effector Treg cells. These signatures could not be extrapolated from transcriptome data. Unique cell-biological and metabolic features in Treg cells were defined, as well as specific adaptations in cytokine, TCR, and costimulatory receptor signaling pathways. One such adaptation-selective STAT4 deficiency-prevented destabilization of Treg cell identity and function by inflammatory cytokines, while these signals could still induce critical transcription factors and homing receptors via other pathways. Furthermore, our study revealed surface markers that identify FOXP3CD4 T cells with distinct functional properties. Our findings suggest that adaptation in signaling pathways protect Treg cell identity and present a resource for further research into Treg cell biology.

BackgroundThe genetic cause of primary immunodeficiency disease (PID) carries prognostic information.ObjectiveWe conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource–Rare Diseases cohort.MethodsIn the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes 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 means of 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 cosegregation analyses.ResultsBoth sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated 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 an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases.ConclusionWe show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.

Key Points Activated neutrophils can suppress T-cell proliferation in a CD11b-dependent multistep process involving ROS production and degranulation. MDSC activity results in nonapoptotic T-cell damage.

Disclosure of potential conflict of interest: S. Gray has received travel support from Medtronic. E. Holbrook has consultant arrangements with 480 Biomedical and has provided expert testimony for Margol & Margol PA. B. S. Bleier has received grants from MEEI Curing Kids Fund and Cook Medical; has consultant arrangements with Olympus, Canon, and Storz; has provided expert testimony on ENT-related cases; has a patent for P-gp inhibition for chronic rhinosinusitis and receives royalties from this patent; and has stock/stock options in Interscope. The rest of the authors declare that they have no relevant conflicts of interest.

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Highlights d Dissection of transcriptome-proteome networks underlying neutrophil differentiation d Distinct patterns of RNA-protein kinetics correlate with biological processes d Discordant dynamics allows for functional annotation of granule proteins d Anabolic collapse paradoxically coincides with gain in neutrophil function

Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33 scFv , and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 3 Ala/ Ala (fVIII YFP 2092/93) and 2 single mutants Lys2092 3 Ala and Phe2093 3 Ala. KM33 scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII YFP 2092/93 had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33 scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33 scFv inhibited binding to platelets and fVIII YFP 2092/93 had reduced binding to A23187-stimulated platelets. fVIII YFP 2092 exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII YFP 2093 showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII YFP 2092/93 had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain. (Blood. 2009;114:3938-3946) Introduction Factor VIII functions as a cofactor in the membrane-bound intrinsic factor Xase complex. Together with the enzyme factor IXa, activated factor VIII binds to phosphatidyl-L-serine (Ptd-L-Ser)-containing membranes 1,2 to form an enzyme complex that cleaves the zymogen factor X to factor Xa. 3,4 Factor Xa is thereafter responsible for catalyzing prothrombin cleavage to thrombin. 5 The importance of the factor Xase complex is illustrated by the disease hemophilia, in which a deficiency of factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Despite the central importance of membrane binding, this aspect of factor VIII function remains poorly understood. Factor VIII is synthesized as a single polypeptide chain containing 2351 amino acids (molecular weight, 280 kDa) and shows a domain structure of A1-a1-A2-a2-B-a3-A3-C1-C2, where a1, a2, and a3 are spacer regions that separate the domains from each other. 6 Factor VIII is homologous to factor V in amino acid sequence and domain structure. 7 The A domains are homologous with ceruloplasmin, the C domains with discoidin I, and with lactadherin, 8,9 and the B domain is unique to each protein. 10 The A domains mediate the dominant interactions with factor IXa and factor X in the factor Xase complex, whereas binding to Ptd-L-Ser-containing membranes is mediated predominantly by the C2 domain. 11-15 The structure-function relationships of factor V resemble those of factor VIII in that the A domains mediate the dominant interactions with the enzyme and substrate and the C2 domain mediates the dominant membrane-binding inter...