B Quiet: Autoantigen-Specific Strategies to Silence Raucous B Lymphocytes and Halt Cross-Talk with T Cells in Type 1 Diabetes

Islet autoantibodies are the primary biomarkers used to predict type 1 diabetes (T1D) disease risk. They signal immune tolerance breach by islet autoantigen-specific B lymphocytes. T-B lymphocyte interactions that lead to expansion of pathogenic T cells underlie T1D development. Promising strategies to broadly prevent this T-B crosstalk include T cell elimination (anti-CD3, teplizumab), B cell elimination (anti-CD20, rituximab), and disruption of T cell costimulation/activation (CTLA-4/Fc fusion, abatacept). However, global disruption or depletion of immune cell subsets is associated with significant risk, particularly in children. Therefore, antigen-specific therapy is an area of active investigation for T1D prevention. We provide an overview of strategies to eliminate antigen-specific B lymphocytes as a means to limit pathogenic T cell expansion to prevent beta cell attack in T1D. Such approaches could be used to prevent T1D in at-risk individuals. Patients with established T1D would also benefit from such targeted therapies if endogenous beta cell function can be recovered or islet transplant becomes clinically feasible for T1D treatment

Bruton\u27s tyrosine kinase supports gut mucosal immunity and commensal microbiome recognition in autoimmune arthritis

Bruton\u27s tyrosine kinase

High-dimensional analysis reveals distinct endotypes in patients with idiopathic inflammatory myopathies

The idiopathic inflammatory myopathies (IIM) are a rare clinically heterogeneous group of conditions affecting the skin, muscle, joint, and lung in various combinations. While myositis specific autoantibodies are well described, we postulate that broader immune endotypes exist in IIM spanning B cell, T cell, and monocyte compartments. This study aims to identify immune endotypes through detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) in IIM patients compared to healthy controls. We collected PBMCs from 17 patients with a clinical diagnosis of inflammatory myositis and characterized the B, T, and myeloid cell subsets using mass cytometry by time of flight (CyTOF). Data were analyzed using a combination of the dimensionality reduction algorithm t-distributed stochastic neighbor embedding (t-SNE), cluster identification, characterization, and regression (CITRUS), and marker enrichment modeling (MEM); supervised biaxial gating validated populations identified by these methods to be differentially abundant between groups. Using these approaches, we identified shared immunologic features across all IIM patients, despite different clinical features, as well as two distinct immune endotypes. All IIM patients had decreased surface expression of RP105/CD180 on B cells and a reduction in circulating CD3+CXCR3+ subsets relative to healthy controls. One IIM endotype featured CXCR4 upregulation across all cellular compartments. The second endotype was hallmarked by an increased frequency of CD19+CD21loCD11c+ and CD3+CD4+PD1+ subsets. The experimental and analytical methods we describe here are broadly applicable to studying other immune-mediated diseases (e.g., autoimmunity, immunodeficiency) or protective immune responses (e.g., infection, vaccination)

High-Throughput Detection of Autoantigen-Specific B Cells Among Distinct Functional Subsets in Autoimmune Donors

Antigen-specific B cells (ASBCs) can drive autoimmune disease by presenting autoantigen to cognate T cells to drive their activation, proliferation, and effector cell differentiation and/or by differentiating into autoantibody-secreting cells. Autoantibodies are frequently used to predict risk and diagnose several autoimmune diseases. ASBCs can drive type 1 diabetes even when immune tolerance mechanisms block their differentiation into antibody-secreting cells. Furthermore, anti-histidyl tRNA synthetase syndrome patients have expanded IgM+ Jo-1-binding B cells, which clinically diagnostic IgG Jo-1 autoantibodies may not fully reflect. Given the potential disconnect between the pathologic function of ASBCs and autoantibody secretion, direct study of ASBCs is a necessary step towards developing better therapies for autoimmune diseases, which often have no available cure. We therefore developed a high-throughput screening pipeline to 1) phenotypically identify specific B cell subsets, 2) expand them in vitro, 3) drive them to secrete BCRs as antibody, and 4) identify wells enriched for ASBCs through ELISA detection of antibody. We tested the capacity of several B cell subset(s) to differentiate into antibody-secreting cells following this robust stimulation. IgM+ and/or IgD+, CD27- memory, memory, switched memory, and BND B cells secreted B cell receptor (BCR) as antibody following in vitro stimulation, whereas few plasmablasts responded. Bimodal responses were observed across autoimmune donors for IgM+ CD21lo and IgM- CD21lo B cells, consistent with documented heterogeneity within the CD21lo subset. Using this approach, we detected insulin-binding B cell bias towards CD27- memory and CD27+ memory subsets in pre-symptomatic type 1 diabetes donors. We took advantage of routine detection of Jo-1-binding B cells in Jo-1+ anti-histidyl tRNA synthetase syndrome patients to show that Jo-1-binding B cells and total B cells expanded 20-30-fold using this culture system. Overall, these studies highlight technology that is amenable to small numbers of cryopreserved peripheral blood mononuclear cells that enables interrogation of phenotypic and repertoire attributes of ASBCs derived from autoimmune patients

CD19 + CD21lo/neg cells are increased in systemic sclerosis-associated interstitial lung disease

Interstitial lung disease (ILD) represents a significant cause of morbidity and mortality in systemic sclerosis (SSc). The purpose of this study was to examine recirculating lymphocytes from SSc patients for potential biomarkers of interstitial lung disease (ILD). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with SSc and healthy controls enrolled in the Vanderbilt University Myositis and Scleroderma Treatment Initiative Center cohort between 9/2017-6/2019. Clinical phenotyping was performed by chart abstraction. Immunophenotyping was performed using both mass cytometry and fluorescence cytometry combined with t-distributed stochastic neighbor embedding analysis and traditional biaxial gating. This study included 34 patients with SSc-ILD, 14 patients without SSc-ILD, and 25 healthy controls. CD2

An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes

IntroductionThe antigen-presenting cell function of insulin-reactive B cells promotes type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by stimulating pathogenic T cells leading to destruction of insulin-producing β-cells of pancreatic islets.Methods/ResultsTo target insulin-reactive B cells, AKS-107, a human IgG1 Fc molecule fused with human insulin A and B chains, was engineered to retain conformational insulin epitopes that bound mouse and human B cell receptors but prevented binding to the insulin metabolic receptor. AKS-107 Fc-mediated deletion of insulin-reactive B cells was demonstrated via ex vivo and in vivo experiments with insulin-reactive B cell receptor transgenic mouse strains, VH125Tg/NOD and Tg125(H+L)/NOD. As an additional immune tolerance feature, the Y16A mutation of the insulin B(9-23) dominant T cell epitope was engineered into AKS-107 to suppress activation of insulin-specific T cells. In mice and non-human primates, AKS-107 was well-tolerated, non-immunogenic, did not cause hypoglycemia even at high doses, and showed an expectedly protracted pharmacokinetic profile. AKS-107 reproducibly prevented spontaneous diabetes from developing in NOD and VH125Tg/NOD mice that persisted for months after cessation of treatment, demonstrating durable immune tolerance.DiscussionThese preclinical outcomes position AKS-107 for clinical development in T1D prevention settings

Bruton's tyrosine kinase promotes persistence of mature anti-insulin B cells

Autoreactive B lymphocytes are essential for the development of T cell-mediated type 1 diabetes (T1D). Cytoplasmic Bruton's tyrosine kinase (BTK) is a key component of B cell signaling, and its deletion in T1D-prone NOD mice significantly reduces diabetes. However, the role of BTK in the survival and function of autoreactive B cells is not clear. To evaluate the contributions of BTK, we used mice in which B cells express an anti-insulin BCR (125Tg) and promote T1D, despite being anergic. Crossing Btk deficiency onto 125Tg mice reveals that, in contrast to immature B cells, mature anti-insulin B cells are exquisitely dependent upon BTK, because their numbers are reduced by 95%. BTK kinase domain inhibition reproduces this effect in mature anti-insulin B cells, with less impact at transitional stages. The increased dependence of anti-insulin B cells on BTK became particularly evident in an Igκ locus site-directed model, in which 50% of B cells edit their BCRs to noninsulin specificities; Btk deficiency preferentially depletes insulin binders from the follicular and marginal zone B cell subsets. The persistent few Btk-deficient anti-insulin B cells remain competent to internalize Ag and invade pancreatic islets. As such, loss of BTK does not significantly reduce diabetes incidence in 125Tg/NOD mice as it does in NOD mice with a normal B cell repertoire. Thus, BTK targeting may not impair autoreactive anti-insulin B cell function, yet it may provide protection in an endogenous repertoire by decreasing the relative availability of mature autoreactive B cells

Bruton’s Tyrosine Kinase Is Not Essential for B Cell Survival beyond Early Developmental Stages

Bruton's tyrosine kinase (Btk) is a crucial regulator of B cell signaling and is a therapeutic target for lymphoma and autoimmune disease. BTK-deficient patients suffer from humoral immunodeficiency, as their B cells fail to progress beyond the bone marrow. However, the role of Btk in fully developed, mature peripheral B cells is not well understood. Analysis using BTK inhibitors is complicated by suboptimal inhibition, off-target effects, or failure to eliminate BTK's adaptor function. Therefore a mouse model was developed and used to excise after B cell populations were established. Mice lacking from birth are known to have reduced follicular (FO) compartments, with expanded transitional populations, suggesting a block in development. In adult mice, excision did not reduce FO B cells, which persisted for weeks. Autoimmune-prone B1 cells also survived conditional excision, contrasting their near absence in global -deficient mice. Therefore, Btk supports BCR signaling during selection into the FO and B1 compartments, but is not needed to maintain these cell populations. B1-related natural IgM levels remained normal, contrasting global deficiency, but B cell proliferation and T-independent type II immunization responses were blunted. Thus, B cells have nuanced signaling responses that are differentially regulated by Btk for development, survival, and function. These findings raise the possibility that Btk may also be expendable for survival of mature human B cells, therefore requiring prolonged dosing to be effective, and that success of BTK inhibitors may depend in part on off-target effects