Summary Anergic B cells are characterized by impaired signaling and activation following aggregation of their antigen receptors (BCR). The molecular basis of this impairment is not understood. In studies reported here Src homology-2 (SH2)-containing inositol 5-phosphatase SHIP-1 and its adaptor Dok-1 were found to be constitutively phosphorylated in anergic B cells, and activation of this inhibitory circuit was dependent on Src-family kinase activity and consequent to biased BCR immunoreceptor tyrosine-based activation motif (ITAM) monophosphorylation. B cell-targeted deletion of SHIP-1 caused severe lupus-like disease. Moreover, absence of SHIP-1 in B cells led to loss of anergy as indicated by restoration of BCR signaling, loss of anergic surface phenotype and production of autoantibodies. Thus chronic BCR signals maintain anergy in part via ITAM monophosphorylation-directed activation of an inhibitory signaling circuit involving SHIP-1 and Dok-1.

SummaryBackgroundIntradermal MVA85A, a candidate vaccine against tuberculosis, induces high amounts of Ag85A-specific CD4 T cells in adults who have already received the BCG vaccine, but aerosol delivery of this vaccine might offer immunological and logistical advantages. We did a phase 1 double-blind trial to compare the safety and immunogenicity of aerosol-administered and intradermally administered MVA85AMethodsIn this phase 1, double-blind, proof-of-concept trial, 24 eligible BCG-vaccinated healthy UK adults were randomly allocated (1:1) by sequentially numbered, sealed, opaque envelopes into two groups: aerosol MVA85A and intradermal saline placebo or intradermal MVA85A and aerosol saline placebo. Participants, the bronchoscopist, and immunologists were masked to treatment assignment. The primary outcome was safety, assessed by the frequency and severity of vaccine-related local and systemic adverse events. The secondary outcome was immunogenicity assessed with laboratory markers of cell-mediated immunity in blood and bronchoalveolar lavage samples. Safety and immunogenicity were assessed for 24 weeks after vaccination. Immunogenicity to both insert Ag85A and vector modified vaccinia virus Ankara (MVA) was assessed by ex-vivo interferon-γ ELISpot and serum ELISAs. Since all participants were randomised and vaccinated according to protocol, our analyses were per protocol. This trial is registered with ClinicalTrials.gov, number NCT01497769.FindingsBoth administration routes were well tolerated and immunogenic. Respiratory adverse events were rare and mild. Intradermal MVA85A was associated with expected mild local injection-site reactions. Systemic adverse events did not differ significantly between the two groups. Three participants in each group had no vaccine-related systemic adverse events; fatigue (11/24 [46%]) and headache (10/24 [42%]) were the most frequently reported symptoms. Ag85A-specific systemic responses were similar across groups. Ag85A-specific CD4 T cells were detected in bronchoalveolar lavage cells from both groups and responses were higher in the aerosol group than in the intradermal group. MVA-specific cellular responses were detected in both groups, whereas serum antibodies to MVA were only detectable after intradermal administration of the vaccine.InterpretationFurther clinical trials assessing the aerosol route of vaccine delivery are merited for tuberculosis and other respiratory pathogens.FundingThe Wellcome Trust and Oxford Radcliffe Hospitals Biomedical Research Centre.

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Although dogma predicts that under normal circumstances, potentially offensive autoreactive cells are silenced by mechanisms of immune tolerance, islet antigen–reactive B lymphocytes are known to play a crucial role in the development of autoimmunity in type 1 diabetes (T1D). Thus, participation of these cells in T1D may reflect escape from silencing mechanisms. Consistent with this concept, we found that in healthy subjects, high-affinity insulin-binding B cells occur exclusively in the anergic naive IgD+, IgM− B-cell (BND) compartment. Antigen receptors expressed by these cells are polyreactive and have N-region additions, Vh usage, and charged complementarity-determining region 3 consistent with autoreactivity. Consistent with a potential early role in autoimmunity, these high-affinity insulin-binding B cells are absent from the anergic compartment of some first-degree relatives and all prediabetic and new-onset (<1 year) T1D patients tested, but return to normal levels in individuals diabetic for >1 year. Interestingly, these changes were correlated by transient loss of the entire BND compartment. These findings suggest that environmental events such as infection or injury may, by disrupting B-cell anergy, dispose individuals toward autoimmunity, the precise nature of which is specified by genetic risk factors, such as HLA alleles.

Type 1 diabetes mellitus (T1DM) is an autoimmune disorder that affects an estimated 30 million people worldwide. It is characterized by the destruction of pancreatic β cells by the immune system, which leads to lifelong dependency on exogenous insulin and imposes an enormous burden on patients and health-care resources. T1DM is also associated with an increased risk of comorbidities, such as cardiovascular disease, retinopathy, and diabetic kidney disease (DKD), further contributing to the burden of this disease. Although T cells are largely considered to be responsible for β-cell destruction in T1DM, increasing evidence points towards a role for B cells in disease pathogenesis. B cell-depletion, for example, delays disease progression in patients with newly diagnosed T1DM. Loss of tolerance of islet antigen-reactive B cells occurs early in disease and numbers of pancreatic CD20 B cells correlate with β-cell loss. Although the importance of B cells in T1DM is increasingly apparent, exactly how these cells contribute to disease and its comorbidities, such as DKD, is not well understood. Here we discuss the role of B cells in the pathogenesis of T1DM and how these cells are activated during disease development. Finally, we speculate on how B cells might contribute to the development of DKD.

The purpose of this study was to determine whether β-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/β-catenin pathway gene expression suggested a role for β-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by β-catenin. This issue was overcome by analysis of β-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for β-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for β-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of β-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent β-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells.

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37°C, but not 4°C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37°C that facilitate virus entry.Human coronaviruses HCoV-229E in serogroup I and HCoV-OC43 in serogroup II are, after rhinoviruses, the second most important cause of the common cold (7, 13). The first step in HCoV-229E infection is binding of the trimeric 200-kDa viral spike glycoprotein (S) to human aminopeptidase N (hAPN, CD13) on human cells (26). The three-dimensional structures of the spike glycoproteins of the plus-strand RNAcontaining coronaviruses are not yet known. However, the structure of S is predicted to be generally similar to that of type 1 viral fusion proteins of large negative-strand RNA viruses (12,22,25). The approximately 547-amino-acid (aa)-long Nterminal S1 domain of HCoV-229E spike protein has receptorbinding activity (2), and the membrane-anchored S2 domain contains several heptad repeats (3). APNs of the normal host species are also receptors for porcine, feline, and canine coronaviruses in group I (5, 6, 15, 23). The metalloprotease APN is a 150-kDa, class II glycoprotein that is expressed as a dimer on apical membranes of polarized epithelial cells, at synaptic junctions, and on antigen-presenting cells (11,17,19,21).Soluble receptors for many viruses have been used to identify critical receptor-binding sites on the viral attachment proteins, explore the specificity of virus-receptor interactions, and characterize conformational changes in the viral attachment protein that are induced by receptor binding (10,14,18,20,28). Temperature-dependent triggering of such conformational changes in a virus attachment protein by receptors on host cell membranes leads to penetration of the viral genome into the cytoplasm of the host cell. Binding of receptor to the N-terminal domain of a type 1 viral fusion protein at 37°C can trigger a conformational change in the membrane-anchored domain, leading to fusion of the viral envelope with host cell membranes (9,10,14,27). This paper reports the expression in insect cells and characterization of a soluble recombinant hAPN protein (shAPN) and interactions of shAPN with HCoV-229E virions, the S1 domain of the viral spike glycoprotein, and truncated S1 proteins. Characterization of shAPN.A soluble form of hAPN lacking the stalk, transmembrane, and intracellular domains (anchor/ stalk-minus APN), was previously expressed in mammalian cells and shown to be enzymatically active (24). For these studies, we expressed in insect cells the 95-to 98-kDa soluble, anchor/stalk-minus protein (shAPN). The open reading frame included the BiP cleavable signal sequence (MKLCILLAVVA FVGLSGL), a linker (RS), and aa 66 to 967 of hAPN with a C-terminal V5 epitope and His tag. We purified the protein on Ni-nitrilotriacetic acid (NTA) resin...

Despite the almost universal clinical observation that inflammation of the gut is frequently associated with inflammation of the joints and vice versa, the nature of this relationship remains elusive. In the present review, we provide evidence for how the interaction of dietary lectins with enterocytes and lymphocytes may facilitate the translocation of both dietary and gut-derived pathogenic antigens to peripheral tissues, which in turn causes persistent peripheral antigenic stimulation. In genetically susceptible individuals, this antigenic stimulation may ultimately result in the expression of overt rheumatoid arthritis (RA) via molecular mimicry, a process whereby foreign peptides, similar in structure to endogenous peptides, may cause antibodies or T-lymphocytes to cross-react with both foreign and endogenous peptides and thereby break immunological tolerance. By eliminating dietary elements, particularly lectins, which adversely influence both enterocyte and lymphocyte structure and function, it is proposed that the peripheral antigenic stimulus (both pathogenic and dietary) will be reduced and thereby result in a diminution of disease symptoms in certain patients with RA.