Monophosphorylation of CD79a and CD79b ITAM motifs initiates a SHIP-1 phosphatase-mediated inhibitory signaling cascade required for B cell anergy.

Anergic B cells are characterized by impaired signaling and activation after 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.

Inhibition of Interleukin-23-Mediated Inflammation with a Novel Small Molecule Inverse Agonist of RORγt.

Blockade of interleukin (IL)-23 or IL-17 with biologics is clinically validated as a treatment of psoriasis. However, the clinical impact of targeting other nodes within the IL-23/IL-17 pathway, especially with small molecules, is less defined. We report on a novel small molecule inverse agonist of retinoid acid-related orphan receptor (ROR) γt and its efficacy in preclinical models of psoriasis and arthritis. 1-(2,4-Dichloro-3-((1,4-dimethyl-6-(trifluoromethyl)-1H-indol-2-yl)methyl)benzoyl)piperidine-4-carboxylic acid (A-9758) was optimized from material identified from a high-throughput screening campaign. A-9758 is selective for RORγt and exhibits robust potency against IL-17A release both in vitro and in vivo. In vivo, we also show that IL-23 is sufficient to drive the accumulation of RORγt+ cells, and inhibition of RORγt significantly attenuates IL-23-driven psoriasiform dermatitis. Therapeutic treatment with A-9758 (i.e., delivered during active disease) was also effective in blocking skin and joint inflammation. Finally, A-9758 exhibited efficacy in an ex vivo human whole blood assay, suggesting small molecule inverse agonists of RORγt could be efficacious in human IL-17-related diseases. SIGNIFICANCE STATEMENT: Using a novel small molecule inverse agonist, and preclinical assays, we show that RORγt is a viable target for the inhibition of RORγt/Th17-driven diseases such as psoriasis. Preclinical models of psoriasis show that inhibition of RORγt blocks both the accumulation and effector function of IL-17-producing T cells.

Discovery of novel quinoline sulphonamide derivatives as potent, selective and orally active RORγ inverse agonists.

A high-throughput screen against Inventiva's compound library using a Gal4/RORγ-LBD luciferase reporter gene assay led to the discovery of a new series of quinoline sulphonamides as RORγ inhibitors, eventually giving rise to a lead compound having an interesting in vivo profile after oral administration. This lead was evaluated in a target engagement model in mouse, where it reduced IL-17 cytokine production after immune challenge. It also proved to be active in a multiple sclerosis model (EAE) where it reduced the disease score. The synthesis, structure activity relationship (SAR) and biological activity of these derivatives is described herein.

B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection.

Manipulation of host cellular pathways is a strategy employed by gammaherpesviruses, including mouse gammaherpesvirus 68 (MHV68), in order to negotiate a chronic infection. Ataxia-telangiectasia mutated (ATM) plays a unique yet incompletely understood role in gammaherpesvirus infection, as it has both proviral and antiviral effects. Chronic gammaherpesvirus infection is poorly controlled in a host with global ATM insufficiency, whether the host is a mouse or a human. In contrast, ATM facilitates replication, reactivation, and latency establishment of several gammaherpesviruses in vitro, suggesting that ATM is proviral in the context of infected cell cultures. The proviral role of ATM is also evident in vivo, as myeloid-specific ATM expression facilitates MHV68 reactivation during the establishment of viral latency. In order to better understand the complex relationship between host ATM and gammaherpesvirus infection, we depleted ATM specifically in B cells, a cell type critical for chronic gammaherpesvirus infection. B cell-specific ATM deficiency attenuated the establishment of viral latency due to compromised differentiation of ATM-deficient B cells. Further, we found that during long-term infection, peritoneal B-1b, but not related B-1a, B cells display the highest frequency of gammaherpesvirus infection. While ATM expression did not affect gammaherpesvirus tropism for B-1 B cells, B cell-specific ATM expression was necessary to support viral reactivation from peritoneal cells during long-term infection. Thus, our study reveals a role of ATM as a host factor that promotes chronic gammaherpesvirus infection of B cells.IMPORTANCE Gammaherpesviruses infect a majority of the human population and are associated with cancer, including B cell lymphomas. ATM is a unique host kinase that has both proviral and antiviral roles in the context of gammaherpesvirus infection. Further, there is insufficient understanding of the interplay of these roles in vivo during chronic infection. In this study, we show that ATM expression by splenic B cells is required for efficient establishment of gammaherpesvirus latency. We also show that ATM expression by peritoneal B cells is required to facilitate viral reactivation during long-term infection. Thus, our study defines a proviral role of B cell-specific ATM expression during chronic gammaherpesvirus infection.

B-cell anergy: from transgenic models to naturally occurring anergic B cells?

Anergy, a condition in which cells persist in the periphery but are unresponsive to antigen, is responsible for silencing many self-reactive B cells. Loss of anergy is known to contribute to the development of autoimmune diseases, including systemic lupus erythematosus and type 1 diabetes. Multiple transgenic mouse models have enabled the dissection of mechanisms that underlie anergy, and recently, anergic B cells have been identified in the periphery of wild-type mice. Heterogeneity of mechanistic concepts developed using model systems has complicated our understanding of anergy and its biological features. In this Review, we compare and contrast the salient features of anergic B cells with a view to developing unifying mechanistic hypotheses that explain their lifestyles.

The Use of Salmonella Typhim Vaccine to Diagnose Antibody Deficiency.

PURPOSE: The specific antibody response to the unconjugated 23-valent pneumococcal polysaccharide vaccine is one of the most common tests used to assess for possible humoral immunodeficiency. The results can be difficult to interpret because most people have been immunized with one or more of the pneumococcal vaccines and there is controversy regarding what constitutes a normal response. To circumvent this problem, we developed an ELISA to measure IgG-specific antibodies to the Salmonella Vi Typhim (S. Typhim) vaccine, a pure polysaccharide vaccine, which is a neoantigen for the vast majority of people in the USA. METHODS: We compared the pre- and post-vaccination serum titers to the Vi Typhim vaccine in healthy controls (n = 22), patients previously diagnosed with a primary immunodeficiency (n = 30), and patients referred for possible humoral immune deficiency (n = 29). We also determined if the S. Typhim vaccine could be used to assess specific antibody responses in people on antibody replacement therapy. RESULTS: Following immunization with the S. Typhim vaccine, we found that a 2-fold increase in titers is 100% sensitive and specific in detecting known humoral immune deficiencies as determined by ROC curve analysis. This cut-off value was successfully applied to possible immune deficiency patients (n = 29), resulting in the diagnosis of seven subjects with humoral immunodeficiency. The use of immunoglobulin replacement therapy did not affect the median response ratios compared to subjects not receiving gammaglobulin. CONCLUSION: This study suggests that measurement of the specific antibody response to the S. Typhim vaccine may have advantages over pneumococcal vaccination in the evaluation of the humoral immune response.

Tumor Suppressor Interferon-Regulatory Factor 1 Counteracts the Germinal Center Reaction Driven by a Cancer-Associated Gammaherpesvirus.

UNLABELLED: Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis. IMPORTANCE: Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible, as the host risk factors that predispose individuals toward viral lymphomagenesis are poorly understood. The current study identifies interferon-regulatory factor 1 (IRF-1) to be one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies.

Cutting edge: in the absence of regulatory T cells, a unique Th cell population expands and leads to a loss of B cell anergy.

The absence of regulatory T cells (Tregs) results in significant immune dysregulation that includes autoimmunity. The mechanism(s) by which Tregs suppress autoimmunity remains unclear. We have shown that B cell anergy, a major mechanism of B cell tolerance, is broken in the absence of Tregs. In this study, we identify a unique subpopulation of CD4(+) Th cells that are highly supportive of Ab production and promote loss of B cell anergy. Notably, this novel T cell subset was shown to express the germinal center Ag GL7 and message for the B cell survival factor BAFF, yet failed to express markers of the follicular Th cell lineage. We propose that the absence of Tregs results in the expansion of a unique nonfollicular Th subset of helper CD4(+) T cells that plays a pathogenic role in autoantibody production.

Autoreactive preplasma cells break tolerance in the absence of regulation by dendritic cells and macrophages.

The ability to induce Ab responses to pathogens while maintaining the quiescence of autoreactive cells is an important aspect of immune tolerance. During activation of TLR4, dendritic cells (DCs) and macrophages (MFs) repress autoantibody production through their secretion of IL-6 and soluble CD40L (sCD40L). These soluble mediators selectively repress B cells chronically exposed to Ag, but not naive cells, suggesting a means to maintain tolerance during TLR4 stimulation, yet allow immunity. In this study, we identify TNF-α as a third repressive factor, which together with IL-6 and CD40L account for nearly all the repression conferred by DCs and MFs. Similar to IL-6 and sCD40L, TNF-α did not alter B cell proliferation or survival. Instead, it reduced the number of Ab-secreting cells. To address whether the soluble mediators secreted by DCs and MFs functioned in vivo, we generated mice lacking IL-6, CD40L, and TNF-α. Compared to wild-type mice, these mice showed prolonged anti-nuclear Ab responses following TLR4 stimulation. Furthermore, adoptive transfer of autoreactive B cells into chimeric IL-6(-/-) × CD40L(-/-) × TNF-α(-/-) mice showed that preplasma cells secreted autoantibodies independent of germinal center formation or extrafollicular foci. These data indicate that in the absence of genetic predisposition to autoimmunity, loss of endogenous IL-6, CD40L, and TNF-α promotes autoantibody secretion during TLR4 stimulation.

Modulation of B-cell tolerance by murine gammaherpesvirus 68 infection: requirement for Orf73 viral gene expression and follicular helper T cells.

Viruses such as Epstein-Barr virus (EBV) have been linked to mechanisms that support autoantibody production in diseases such as systemic lupus erythematosus. However, the mechanisms by which viruses contribute to autoantibody production remain poorly defined. This stems in part, from the high level of seropositivity for EBV (> 95%) and the exquisite species specificity of EBV. In this study we overcame these problems by using murine gammaherpesvirus 68 (MHV68), a virus genetically and biologically related to EBV. We first showed that MHV68 drives autoantibody production by promoting a loss of B-cell anergy. We next showed that MHV68 infection resulted in the expansion of follicular helper T (Tfh) cells in vivo, and that these Tfh cells supported autoantibody production and a loss of B-cell anergy. Finally, we showed that the expansion of Tfh cells and autoantibody production was dependent on the establishment of viral latency and expression of a functional viral gene called Orf73. Collectively, our studies highlighted an unexpected role for viral latency in the development of autoantibodies following MHV68 infection and suggest that virus-induced expansion of Tfh cells probably plays a key role in the loss of B-cell anergy.

Expansion of follicular helper T cells in the absence of Treg cells: implications for loss of B-cell anergy.

The maintenance of B-cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B-cell extrinsic mechanisms that regulate B-cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B-cell anergy. We now show that in Treg-cell-deficient mice, helper T cells are necessary and sufficient for loss of B-cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4(+) cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC-T(FH) ) cells. These GC-T(FH) cells, but not those from Treg-cell-sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T(FH) cells, especially GC-T(FH) cells, which support autoantibody production.

Ataxia telangiectasia mutated kinase controls chronic gammaherpesvirus infection.

Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.

Requirements for growth and IL-10 expression of highly purified human T regulatory cells.

Human regulatory T cells (T(R)) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human T(R) cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9.3) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting T(R) cell proliferation, and under these conditions the proliferative capacity of T(R) cells was comparable to conventional CD4 lymphocytes. Blocking TGF-ß activity abrogated IL-10 expression from these cells, while addition of TGF-ß resulted in IL-10 production. These data demonstrate that highly purified populations of T(R) cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of T(R) cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-ß is critical to enable human T(R) cells to express IL-10.

Altered B cell development and anergy in the absence of Foxp3.

The importance of regulatory T cells in immune tolerance is illustrated by the human immune dysregulatory disorder IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), caused by a lack of regulatory T cells due to decreased or absent expression of Foxp3. Although the majority of work on regulatory T cells has focused on their ability to suppress T cell responses, the development of significant autoantibody titers in patients with IPEX suggests that regulatory T cells also contribute to the suppression of autoreactive B cells. Using a murine model, deficient in the expression of Foxp3, we show that B cell development is significantly altered in the absence of regulatory T cells. Furthermore, we identify a loss of B cell anergy as a likely mechanism to explain the production of autoantibodies that occurs in the absence of regulatory T cells. Our results suggest that regulatory T cells, by either direct or indirect mechanisms, modulate B cell development and anergy.

Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis.

Immune cells are fundamental regulators of extracellular matrix (ECM) production by fibroblasts and have important roles in determining extent of fibrosis in response to inflammation. Although much is known about fibroblast signaling in fibrosis, the molecular signals between immune cells and fibroblasts that drive its persistence are poorly understood. We therefore analyzed skin and lung samples of patients with diffuse cutaneous systemic sclerosis, an autoimmune disease that causes debilitating fibrosis of the skin and internal organs. Here, we define a critical role of epiregulin-EGFR signaling between dendritic cells and fibroblasts to maintain elevated ECM production and accumulation in fibrotic tissue. We found that epiregulin expression marks an inducible state of DC3 dendritic cells triggered by type I interferon and that DC3-derived epiregulin activates EGFR on fibroblasts, driving a positive feedback loop through NOTCH signaling. In mouse models of skin and lung fibrosis, epiregulin was essential for persistence of fibrosis in both tissues, which could be abrogated by epiregulin genetic deficiency or a neutralizing antibody. Therapeutic administration of epiregulin antibody reversed fibrosis in patient skin and lung explants, identifying it as a previously unexplored biologic drug target. Our findings reveal epiregulin as a crucial immune signal that maintains skin and lung fibrosis in multiple diseases and represents a promising antifibrotic target.

ACT1 Is Required for Murine IL-23-Induced Psoriasiform Inflammation Potentially Independent of E3 Ligase Activity.

Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.

CD23-mediated cell signaling in human B cells differs from signaling in cells of the monocytic lineage.

CD23 is the low affinity receptor for IgE and in B cells CD23 has been proposed to play a role in the regulation of IgE synthesis. CD23 is expressed also on other cell types including monocytes/macrophages, eosinophils, follicular dendritic cells and intestinal epithelial cells none of which is capable of expressing IgE. The diverse nature of the expressing cells suggests that either the CD23-mediated signal transduction pathway may be different among the cell types or biological outcomes differ in different cells in response to the same signaling pathway. To address this issue, the CD23 signaling pathway was analyzed and compared in primary tonsillar B cells and in the monocytic cell lines U937 and THP-1. Activation of the tyrosine kinase Fyn and the serine/threonine kinase Akt were only observed in B cells. These results suggest that the CD23-mediated signal transduction pathways in human B cells and human monocytes are different.

Silencing of autoreactive B cells by anergy: a fresh perspective.

B-cell antigen receptor (BCR) signals are crucial for initiation of humoral immune responses and must be actively modulated and/or terminated in preparation for receipt of subsequent cues for progression. BCR signaling is also actively inhibited in autoreactive cells in which unresponsiveness is maintained by anergy. This serves to prevent cell activation and autoimmunity. Importantly, the feedback mechanisms that modulate and/or terminate signaling during normal antigen-induced B-cell activation appear to also be involved in maintaining B-cell anergy. In fact, it is suggested that anergy reflects nothing more than the normal inability of cells to respond to antigen following preceding stimulation of normal inhibitory feedback mechanisms. Thus, the time-honored two-signal hypothesis is almost certainly correct, with second signals being required to release the cell from inhibitory BCR-specific and trans-active feedback regulation.

IL-23 induces regulatory T cell plasticity with implications for inflammatory skin diseases.

Foxp3+ regulatory T cells (Tregs) represent a major fraction of skin resident T cells. Although normally protective, Tregs have been shown to produce pro-inflammatory cytokines in human diseases, including psoriasis. A significant hurdle in the Treg field has been the identification, or development, of model systems to study this Treg plasticity. To overcome this gap, we analyzed skin resident Tregs in a mouse model of IL-23 mediated psoriasiform dermatitis. Our results demonstrate that IL-23 drove the accumulation of Tregs; including a subpopulation that co-expressed RORγt and produced IL-17A. Genesis of this population was attenuated by a RORγt inverse agonist compound and clinically relevant therapeutics. In vitro, IL-23 drove the generation of CD4+Foxp3+RORγt+IL-17A+ cells from Treg cells. Collectively, our data shows that IL-23 drives Treg plasticity by inducing a population of CD4+Foxp3+RORγt+IL-17A+ cells that could play a role in the disease pathogenesis. Through this work, we define an in vitro system and a pre-clinical in vivo mouse model that can be used to further study Treg homeostasis and plasticity in the context of psoriasis.

Monocytes/Macrophages play a pathogenic role in IL-23 mediated psoriasis-like skin inflammation.

Psoriasis is an immune-mediated inflammatory skin disease that affects millions worldwide. Studying immune cells involved in psoriasis pathogenesis is essential to identify effective and safe therapeutics for the disease. Using human psoriasis skin, activated macrophages were observed in both lesional and non-lesional skin, but were elevated in lesional skin. Activation of the IL-23/IL-17 pathway is integral to the development of psoriasis. To further characterize the monocyte/macrophage (Mon/Mac) population when the IL-23 pathway is activated, a murine model of intradermal injection of IL-23 was used. Flow cytometry revealed that Mon/Mac cells were the dominant immune population, particularly late in the model, highlighted by strong presence of Ly6ChiMHC IIhi cells. The Mon/Mac cells were also shown to have high expression for TNFα but not IL-17A. Prophylactic dosing of a CSF-1R inhibitor to deplete Mon/Mac cells significantly reduced several inflammatory mediators from the skin tissue suggesting a pathogenic role for Mon/Mac. Treatment dosing of the inhibitor produced a less robust effect. Mon/Mac cells were also differentiated by levels of Ki67 and TNFα expression. These data point to an important contribution of Mon/Mac cells in IL-23 related skin inflammation and suggest that these cells are a significant player in the underlying pathophysiology of psoriasis.