Non-Antibody-Secreting Functions of B Cells and Their Contribution to Autoimmune Disease.

B cells play multiple important roles in the pathophysiology of autoimmune disease. Beyond producing pathogenic autoantibodies, B cells can act as antigen-presenting cells and producers of cytokines, including both proinflammatory and anti-inflammatory cytokines. Here we review our current understanding of the non-antibody-secreting roles that B cells may play during development of autoimmunity, as learned primarily from reductionist preclinical models. Attention is also given to concepts emerging from clinical studies using B cell depletion therapy, which shed light on the roles of these mechanisms in human autoimmune disease.

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

Therapeutic tactics for targeting B lymphocytes in autoimmunity and cancer.

B lymphocytes have become a very popular therapeutic target in a number of autoimmune indications due to their newly appreciated roles, and approachability, in these diseases. Many of the therapies now applied in autoimmunity were initially developed to deplete malignant B cells. These strategies have also been found to benefit patients suffering from such autoimmune diseases as multiple sclerosis, type I diabetes, systemic lupus erythematosus, and rheumatoid arthritis, to name a few. These observations have supported the expansion of research addressing the mechanistic contributions of B cells in these diseases, as well as blossoming of therapeutics that target them. This review seeks to summarize cutting-edge modalities for targeting B cells, including monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, chimeric antigen receptor-T cells, and small molecule inhibitors. Efforts to refine B-cell targeted therapy to eliminate only pathogenic autoreactive cells will be addressed as well as the potential for future B-cell-based cellular therapeutics. Finally, we also address approaches that seek to silence B-cell function without depletion.

Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

A Precision B Cell-Targeted Therapeutic Approach to Autoimmunity Caused by Phosphatidylinositol 3-Kinase Pathway Dysregulation.

The inositol lipid phosphatases PTEN and SHIP-1 play a crucial role in maintaining B cell anergy and are reduced in expression in B cells from systemic lupus erythematosus and type 1 diabetes patients, consequent to aberrant regulation by miRNA-7 and 155. With an eye toward eventual use in precision medicine therapeutic approaches in autoimmunity, we explored the ability of p110δ inhibition to compensate for PI3K pathway dysregulation in mouse models of autoimmunity. Low dosages of the p110δ inhibitor idelalisib, which spare the ability to mount an immune response to exogenous immunogens, are able to block the development of autoimmunity driven by compromised PI3K pathway regulation resultant from acutely induced B cell-targeted haploinsufficiency of PTEN and SHIP-1. These conditions do not block autoimmunity driven by B cell loss of the regulatory tyrosine phosphatase SHP-1. Finally, we show that B cells in NOD mice express reduced PTEN, and low-dosage p110δ inhibitor therapy blocks disease progression in this model of type 1 diabetes. These studies may aid in the development of precision treatments that act by enforcing PI3K pathway regulation in patients carrying specific risk alleles.

The common HAQ STING variant impairs cGAS-dependent antibacterial responses and is associated with susceptibility to Legionnaires' disease in humans.

The cyclic GMP-AMP synthase (cGAS)-STING pathway is central for innate immune sensing of various bacterial, viral and protozoal infections. Recent studies identified the common HAQ and R232H alleles of TMEM173/STING, but the functional consequences of these variants for primary infections are unknown. Here we demonstrate that cGAS- and STING-deficient murine macrophages as well as human cells of individuals carrying HAQ TMEM173/STING were severely impaired in producing type I IFNs and pro-inflammatory cytokines in response to Legionella pneumophila, bacterial DNA or cyclic dinucleotides (CDNs). In contrast, R232H attenuated cytokine production only following stimulation with bacterial CDN, but not in response to L. pneumophila or DNA. In a mouse model of Legionnaires' disease, cGAS- and STING-deficient animals exhibited higher bacterial loads as compared to wild-type mice. Moreover, the haplotype frequency of HAQ TMEM173/STING, but not of R232H TMEM173/STING, was increased in two independent cohorts of human Legionnaires' disease patients as compared to healthy controls. Our study reveals that the cGAS-STING cascade contributes to antibacterial defense against L. pneumophila in mice and men, and provides important insight into how the common HAQ TMEM173/STING variant affects antimicrobial immune responses and susceptibility to infection. TRIAL REGISTRATION: ClinicalTrials.gov DRKS00005274, German Clinical Trials Register.

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.

B Cell-Intrinsic STING Signaling Triggers Cell Activation, Synergizes with B Cell Receptor Signals, and Promotes Antibody Responses.

Generation of protective immune responses requires coordinated stimulation of innate and adaptive immune responses. An important mediator of innate immunity is stimulator of IFN genes (STING, MPYS, MITA), a ubiquitously but differentially expressed adaptor molecule that functions in the relay of signals initiated by sensing of cytosolic DNA and bacterial cyclic dinucleotides (CDNs). Whereas systemic expression of STING is required for CDN-aided mucosal Ab responses, its function in B cells in particular is unclear. In this study, we show that B cells can be directly activated by CDNs in a STING-dependent manner in vitro and in vivo. Direct activation of B cells by CDNs results in upregulation of costimulatory molecules and cytokine production and this can be accompanied by caspase-dependent cell death. CDN-induced cytokine production by B cells and other cell types also contributes to activation and immune responses. Type I IFN is primarily responsible for this indirect stimulation although other cytokines may contribute. BCR and STING signaling pathways act synergistically to promote Ab responses independent of type I IFN. B cell expression of STING is required for optimal in vivo IgG and mucosal IgA Ab responses induced by T cell-dependent Ags and cyclic-di-GMP but plays no discernable role in Ab responses in which alum is used as an adjuvant. Thus, STING functions autonomously in B cells responding to CDNs, and its activation synergizes with Ag receptor signals to promote B cell activation.

Soluble Antigen Arrays for Selective Desensitization of Insulin-Reactive B Cells.

Autoimmune diseases are believed to be highly dependent on loss of immune tolerance to self-antigens. Currently, no treatments have been successful clinically in inducing autoantigen-specific tolerance, including efforts to utilize antigen-specific immunotherapy (ASIT) to selectively correct the aberrant autoimmunity. Soluble antigen arrays (SAgAs) represent a novel autoantigen delivery system composed of a linear polymer, hyaluronic acid (HA), displaying multiple copies of conjugated autoantigen. We have previously reported that soluble antigen arrays displaying proteolipid peptide (SAgAPLP) induced tolerance to this specific multiple sclerosis (MS) autoantigen. Utilizing SAgA technology, we have developed a new ASIT as a possible type 1 diabetes (T1D) therapeutic by conjugating human insulin to HA, known as soluble antigen array insulin (SAgAIns). Three types were synthesized, low valency lvSAgAIns (2 insulins per HA), medium valency mvSAgAIns (4 insulins per HA), and, high valency hvSAgAIns (9 insulins per HA), to determine if valency differentially modulates the ex vivo activity of insulin-binding B cells (IBCs). Extensive biophysical characterization was performed for the SAgA molecules. SAgAIns molecules were successfully used to affect the biologic activity of IBCs by inducing desensitization of the B cell antigen receptors (BCR). SAgAIns bound specifically to insulin-reactive B cells without blocking epitopes recognized by antibodies against the Fc regions of membrane immunoglobulin or CD79 transducer components of the BCR. Preincubation of IBCs (125Tg) with SAgAIns, but not HA alone, rendered the IBCs refractory to restimulation. SAgAIns induced a decrease in BCR expression and IP3R-mediated intracellular calcium release. Surprisingly, SAgAIns binding to BCR on the surface of IBCs induced the observed effects at both high and low SAgAIns valency. Future studies aim to test the effects of SAgAIns on disease progression in the VH125.NOD mouse model of T1D.

Of ITIMs, ITAMs, and ITAMis: revisiting immunoglobulin Fc receptor signaling.

Receptors for immunoglobulin Fc regions play multiple critical roles in the immune system, mediating functions as diverse as phagocytosis, triggering degranulation of basophils and mast cells, promoting immunoglobulin class switching, and preventing excessive activation. Transmembrane signaling associated with these functions is mediated primarily by two amino acid sequence motifs, ITAMs (immunoreceptor tyrosine-based activation motifs) and ITIMs (immunoreceptor tyrosine-based inhibition motifs) that act as the receptors' interface with activating and inhibitory signaling pathways, respectively. While ITAMs mobilize activating tyrosine kinases and their consorts, ITIMs mobilize opposing tyrosine and inositol-lipid phosphatases. In this review, we will discuss our current understanding of signaling by these receptors/motifs and their sometimes blurred lines of function.

Silencing of high-affinity insulin-reactive B lymphocytes by anergy and impact of the NOD genetic background in mice.

AIMS/HYPOTHESIS: Previous studies have demonstrated that high-affinity insulin-binding B cells (IBCs) silenced by anergy in healthy humans lose their anergy in islet autoantibody-positive individuals with recent-onset type 1 diabetes, and in autoantibody-negative first-degree relatives carrying certain risk alleles. Here we explore the hypothesis that IBCs are found in the immune periphery of disease-resistant C57BL/6-H2g7 mice, where, as in healthy humans, they are anergic, but that in disease-prone genetic backgrounds (NOD) they become activated and migrate to the pancreas and pancreatic lymph nodes, where they participate in the development of type 1 diabetes. METHODS: We compared the status of high-affinity IBCs in disease-resistant VH125.C57BL/6-H2g7 and disease-prone VH125.NOD mice. RESULTS: Consistent with findings in healthy humans, high-affinity IBCs reach the periphery in disease-resistant mice and are anergic, as indicated by a reduced expression of membrane IgM, unresponsiveness to antigen and failure to become activated or accumulate in the pancreatic lymph nodes or pancreas. In NOD mice, high-affinity IBCs reach the periphery early in life and increase in number prior to the onset of hyperglycaemia. These cells are not anergic; they become activated, produce autoantibodies and accumulate in the pancreas and pancreatic lymph nodes prior to disease development. CONCLUSIONS/INTERPRETATION: These findings are consistent with genetic determination of the escape of high-affinity IBCs from anergy and their early contribution to the development of type 1 diabetes.

The cGAS/STING Pathway Detects Streptococcus pneumoniae but Appears Dispensable for Antipneumococcal Defense in Mice and Humans.

Streptococcus pneumoniae is a frequent colonizer of the upper respiratory tract and a leading cause of bacterial pneumonia. The innate immune system senses pneumococcal cell wall components, toxin, and nucleic acids, which leads to production of inflammatory mediators to initiate and control antibacterial defense. Here, we show that the cGAS (cyclic GMP-AMP [cGAMP] synthase)-STING pathway mediates detection of pneumococcal DNA in mouse macrophages to primarily stimulate type I interferon (IFN) responses. Cells of human individuals carrying HAQ TMEM173, which encodes a common hypomorphic variant of STING, were largely or partly defective in inducing type I IFNs and proinflammatory cytokines upon infection. Subsequent analyses, however, revealed that STING was dispensable for restricting S. pneumoniae during acute pneumonia in mice. Moreover, explorative analyses did not find differences in the allele frequency of HAQ TMEM173 in nonvaccinated pneumococcal pneumonia patients and healthy controls or an association of HAQ TMEM173 carriage with disease severity. Together, our results indicate that the cGAS/STING pathway senses S. pneumoniae but plays no major role in antipneumococcal immunity in mice and humans.

Activation of thyroid antigen-reactive B cells in recent onset autoimmune thyroid disease patients.

Autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), is the most common autoimmune disorder in the United States, affecting over 20 million people. At the time of diagnosis, both HD and GD are characterized by the accumulation of B and T lymphocytes in the thyroid gland and production of autoantibodies targeting the thyroid, indicating that a breach in tolerance of autoreactive lymphocytes has occurred. However, few studies have sought to understand the underlying pathogenesis of AITD that ultimately leads to production of autoantibodies and loss of thyroid function. In this study, we analyzed the phenotype of thyroid antigen-reactive B cells in the peripheral blood of recent onset and long standing AITD patients. We found that in recent onset patients thyroid antigen-reactive B cells in blood no longer appear anergic, rather they express CD86, a marker of activation. This likely reflects activation of these cells leading to their production of autoantibodies. Hence, this study reports the early loss of anergy in thyroid antigen-reactive B cells, an event that contributes to development of AITD.

Contamination of DNase Preparations Confounds Analysis of the Role of DNA in Alum-Adjuvanted Vaccines.

Aluminum salt (alum) adjuvants have been used for many years as adjuvants for human vaccines because they are safe and effective. Despite its widespread use, the means by which alum acts as an adjuvant remains poorly understood. Recently, it was shown that injected alum is rapidly coated with host chromatin within mice. Experiments suggested that the host DNA in the coating chromatin contributed to alum's adjuvant activity. Some of the experiments used commercially purchased DNase and showed that coinjection of these DNase preparations with alum and Ag reduced the host's immune response to the vaccine. In this study, we report that some commercial DNase preparations are contaminated with proteases. These proteases are responsible for most of the ability of DNase preparations to inhibit alum's adjuvant activity. Nevertheless, DNase somewhat reduces responses to some Ags with alum. The effect of DNase is independent of its ability to cleave DNA, suggesting that alum improves CD4 responses to Ag via a pathway other than host DNA sensing.

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.

γδ T cells affect IL-4 production and B-cell tolerance.

γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αß T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αß T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

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.

Imbalanced PTEN and PI3K Signaling Impairs Class Switch Recombination.

Class switch recombination (CSR) generates isotype-switched Abs with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA PI3K catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. In this article, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role for p110α in CSR. Furthermore, we identify a cooperative role for p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN inversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.

A balance between B cell receptor and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels.

Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.

Anti-CD79 antibody induces B cell anergy that protects against autoimmunity.

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.