The Src-family kinase Lyn plays a critical role in establishing and maintaining B cell anergy by suppressing PI3K-dependent signaling.

Although the Src family kinase (SFK) Lyn is known to be involved in induction and maintenance of peripheral B cell tolerance, the molecular basis of its action in this context remains unclear. This question has been approached using conventional as well as B cell-targeted knockouts of Lyn, with varied conclusions likely confused by collateral loss of Lyn functions in B cell and myeloid cell development and activation. Here we utilized a system in which Lyn gene deletion is tamoxifen inducible and B cell restricted. This system allows acute elimination of Lyn in B cells without off-target effects. This genetic tool was employed in conjunction with immunoglobulin transgenic mice in which peripheral B cells are autoreactive. DNA reactive Ars/A1 B cells require continuous inhibitory signaling, mediated by the inositol phosphatase SHIP-1 and the tyrosine phosphatase SHP-1, to maintain an unresponsive (anergic) state. Here we show that Ars/A1 B cells require Lyn to establish and maintain B cell unresponsiveness. Lyn primarily functions by restricting PI3K-dependent signaling pathways. This Lyn-dependent mechanism complements the impact of reduced mIgM BCR expression to restrict BCR signaling in Ars/A1 B cells. Our findings suggest that a subset of autoreactive B cells requires Lyn to become anergic and that the autoimmunity associated with dysregulated Lyn function may, in part, be due to an inability of these autoreactive B cells to become tolerized.

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.

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.

Impaired B cell function during viral infections due to PTEN-mediated inhibition of the PI3K pathway.

Transient suppression of B cell function often accompanies acute viral infection. However, the molecular signaling circuitry that enforces this hyporesponsiveness is undefined. In this study, experiments identify up-regulation of the inositol phosphatase PTEN (phosphatase and tensin homolog) as primarily responsible for defects in B lymphocyte migration and antibody responses that accompany acute viral infection. B cells from mice acutely infected with gammaherpesvirus 68 are defective in BCR- and CXCR4-mediated activation of the PI3K pathway, and this, we show, is associated with increased PTEN expression. This viral infection-induced PTEN overexpression appears responsible for the suppression of antibody responses observed in infected mice because PTEN deficiency or expression of a constitutively active PI3K rescued function of B cells in infected mice. Conversely, induced overexpression of PTEN in B cells in uninfected mice led to suppression of antibody responses. Finally, we demonstrate that PTEN up-regulation is a common mechanism by which infection induces suppression of antibody responses. Collectively, these findings identify a novel role for PTEN during infection and identify regulation of the PI3K pathway, a mechanism previously shown to silence autoreactive B cells, as a key physiological target to control antibody responses.