Stephen B. Gauld

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.

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.

Differential Roles for Extracellularly Regulated Kinase-Mitogen-Activated Protein Kinase in B Cell Antigen Receptor-Induced Apoptosis and CD40-Mediated Rescue of WEHI-231 Immature B Cells

One of the major unresolved questions in B cell biology is how the B cell Ag receptor (BCR) differentially signals to transduce anergy, apoptosis, proliferation, or differentiation during B cell maturation. We now report that extracellularly regulated kinase-mitogen-activated protein kinase (Erk-MAP kinase) can play dual roles in the regulation of the cell fate of the immature B cell lymphoma, WEHI-231, depending on the kinetics and context of Erk-MAP kinase activation. First, we show that the BCR couples to an early (≤2 h) Erk-MAP kinase signal which activates a phospholipase A2 pathway that we have previously shown to mediate collapse of mitochondrial membrane potential, resulting in depletion of cellular ATP and cathepsin B execution of apoptosis. Rescue of BCR-driven apoptosis by CD40 signaling desensitizes such early extracellularly regulated kinase (Erk) signaling and hence uncouples the BCR from the apoptotic mitochondrial phospholipase A2 pathway. A second role for Erk-MAP kinase in promoting the growth and proliferation of WEHI-231 immature B cells is evidenced by data showing that proliferating and CD40-stimulated WEHI-231 B cells exhibit a sustained cycling pattern (8–48 h) of Erk activation that correlates with cell growth and proliferation. This growth-promoting role for Erk signaling is supported by three key pieces of evidence: 1) signaling via the BCR, under conditions that induce growth arrest, completely abrogates sustained Erk activation; 2) CD40-mediated rescue from growth arrest correlates with restoration of cycling Erk activation; and 3) sustained inhibition of Erk prevents CD40-mediated rescue of BCR-driven growth arrest of WEHI-231 immature B cells. Erk-MAP kinase can therefore induce diverse biological responses in WEHI-231 cells depending on the context and kinetics of activation.

Cutting Edge: Acute and Chronic Exposure of Immature B Cells to Antigen Leads to Impaired Homing and SHIP1-Dependent Reduction in Stromal Cell-Derived Factor-1 Responsiveness

An encounter of B cells with cognate self Ags in the periphery can lead to anergy, a condition characterized by altered anatomical localization, shortened life span, and refractility to Ag stimulation. We recently reported that an immature B cell encounter with cognate self-Ag in the bone marrow can also lead to anergy. In this study we show that anergic as well as acutely Ag-stimulated immature B cells are defective in stromal cell-derived factor-1 (SDF-1)-induced calcium mobilization and migration and do not localize to bone marrow following adoptive transfer. This hyporesponsiveness does not involve CXCR4 modulation. However, BCR signal-mediated hyporesponsiveness to SDF-1 is associated with phosphorylation of the 5-inositol phosphatase SHIP1 and requires SHIP1 expression. Therefore, an encounter with cognate Ag may, by preventing SDF-1-induced phosphatidylinositol 3,4,5-triphosphate accumulation, trigger premature emigration of immature B cells from bone marrow.

Conserved gammaherpesvirus kinase and histone variant H2AX facilitate gammaherpesvirus latency in vivo

Many herpesvirus-encoded protein kinases facilitate viral lytic replication. Importantly, the role of viral kinases in herpesvirus latency is less clear. Mouse gammaherpesvirus-68 (MHV68)-encoded protein kinase orf36 facilitates lytic replication in part through activation of the host DNA damage response (DDR). Here we show that MHV68 latency was attenuated in the absence of orf36 expression. Unexpectedly, our study uncovered enzymatic activity-independent role of orf36 in the establishment of MHV68 latency following intraperitoneal route of infection. H2AX, an important DDR protein, facilitates MHV68 lytic replication and may be directly phosphorylated by orf36 during lytic infection. In this study, H2AX deficiency, whether systemic or limited to infected cells, attenuated the establishment of MHV68 latency in vivo. Thus, our work reveals viral kinase-dependent regulation of gammaherpesvirus latency and illuminates a novel link between H2AX, a component of a tumor suppressor DDR network, and in vivo latency of a cancer-associated gammaherpesvirus.

Ig Allotypic Inclusion Does Not Prevent B Cell Development or Response

B cells expressing two different Igκ L chains (allotype included) have been occasionally observed. To determine frequency and function of these cells, we have analyzed gene-targeted mice that carry a human and a mouse Igk C region genes. Using different methodologies, we found that cells expressing two distinct κ-chains were 1.4–3% of all B cells and that they were present in the follicular, marginal zone, and B1 mature B cell subsets. When stimulated in vitro with anti-IgM, dual κ surface-positive cells underwent activation that manifested with cell proliferation and/or up-regulation of activation markers and similar to single κ-expressing B cells. Yet, when activated by divalent reagents that bound only one of the two κ-chains, dual κ B cells responded suboptimally in vitro, most likely because of reduced Ag receptor cross-linking. Nonetheless, dual κ B cells participated in a SRBC-specific immune response in vivo. Finally, we found that Ig allotype-included B cells that coexpress autoreactive and nonautoreactive Ag receptors were also capable of in vitro responses following BCR aggregation. In summary, our studies demonstrate that Igκ allotype-included B cells are present in the mouse mature B cell population and are responsive to BCR stimulation both in vitro and in vivo. Moreover, because in vitro activation in response to anti-IgM was also observed in cells coexpressing autoreactive and nonautoreactive Abs, our studies suggest a potential role of allotype-included B cells in both physiological and pathological immune responses.

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.

Ataxia telangiectasia mutated kinase controls chronic gammaherpesvirus infection.

ABSTRACT 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.

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‐TFH) cells. These GC‐TFH 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 TFH cells, especially GC‐TFH cells, which support autoantibody production.

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.