Karin Reif

Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position

B lymphocytes re-circulate between B-cell-rich compartments (follicles or B zones) in secondary lymphoid organs, surveying for antigen. After antigen binding, B cells move to the boundary of B and T zones to interact with T-helper cells. Despite the importance of B–T-cell interactions for the induction of antibody responses, the mechanism causing B-cell movement to the T zone has not been defined. Here we show that antigen-engaged B cells have increased expression of CCR7, the receptor for the T-zone chemokines CCL19 and CCL21, and that they exhibit increased responsiveness to both chemoattractants. In mice lacking lymphoid CCL19 and CCL21 chemokines, or with B cells that lack CCR7, antigen engagement fails to cause movement to the T zone. Using retroviral-mediated gene transfer we demonstrate that increased expression of CCR7 is sufficient to direct B cells to the T zone. Reciprocally, overexpression of CXCR5, the receptor for the B-zone chemokine CXCL13, is sufficient to overcome antigen-induced B-cell movement to the T zone. These findings define the mechanism of B-cell relocalization in response to antigen, and establish that cell position in vivo can be determined by the balance of responsiveness to chemoattractants made in separate but adjacent zones.

Cutting Edge: Differential Roles for Phosphoinositide 3-Kinases, p110γ and p110δ, in Lymphocyte Chemotaxis and Homing

Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110γ-deficient T cells, but not B cells, show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110δ, a major PI3K isoform in lymphocytes. B cells deficient in p110δ showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110δ-deficient B cells revealed diminished homing to Peyer’s patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer’s patches. These observations establish that p110γ and p110δ function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.

RGS Molecule Expression in Murine B Lymphocytes and Ability to Down-Regulate Chemotaxis to Lymphoid Chemokines

Ag-mediated changes in B lymphocyte migration are important for normal immune function, yet the mechanisms by which these changes occur are poorly defined. Because chemokines direct many lymphocyte movements, molecules that regulate signaling by G protein-coupled chemokine receptors are likely to participate in Ag receptor-induced changes in cell migration. In this study, we have investigated the expression pattern and activity in murine B cells of members of the regulators of G protein signaling (RGS) family of molecules. We present the sequence of mouse RGS1 and describe a novel short isoform of RGS3 that we term RGS3s. Following in vivo activation by Ag, B cells rapidly up-regulate expression of RGS1 and RGS2 while simultaneously decreasing expression of RGS3 and RGS14. Anergic hen egg lysozyme autoantigen-binding B cells are also shown to have slightly elevated RGS1 and RGS2 expression. CD40 signaling, by contrast, fails to cause rapid up-regulation of RGS1 or RGS2. Using a transient transfection approach in a mature B cell line, 2PK3, we demonstrate that RGS1 and RGS3s are effective inhibitors of chemotaxis toward the lymphoid tissue chemokines stromal cell-derived factor-1, B lymphocyte chemoattractant, and EBV-induced molecule 1 ligand chemokine, whereas RGS2 has a minimal effect on migration to these chemokines. Together these findings support the conclusion that Ag-mediated changes in RGS molecule expression are part of the mechanism by which Ag receptor signaling regulates B cell migration within lymphoid tissues. The findings also suggest important roles for additional G protein-mediated events in B cell activation and tolerance.

The CDM protein DOCK2 in lymphocyte migration

T and B lymphocytes migrate hundreds of micrometers each day to survey the bodys lymphoid tissues for antigens. No other mammalian cell type undergoes such extensive and continual movement, raising the question of whether lymphocytes have specializations to support their migratory behavior. This possibility has recently gained support from studies of mice deficient in DOCK2, a member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of scaffolding proteins. Migration of lymphocytes, but not other cell types, is severely disrupted in DOCK2-deficient mice. Despite the conserved role of CDM molecules in regulating Rac activation and actin assembly, relatively little is known about how these molecules function. Here, we review the role of DOCK2 in lymphocyte homing to lymphoid tissues and discuss recent findings for other CDM family molecules that provide a basis for understanding how DOCK2 might function in lymphocytes.

Antiarthritis Effect of a Novel Bruton's Tyrosine Kinase (BTK) Inhibitor in Rat Collagen-Induced Arthritis and Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling: Relationships between Inhibition of BTK Phosphorylation and Efficacy

Brutons tyrosine kinase (BTK) plays a critical role in the development, differentiation, and proliferation of B-lineage cells, making it an attractive target for the treatment of rheumatoid arthritis. The objective of this study was to evaluate the antiarthritis effect of GDC-0834 [R-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide], a potent and selective BTK inhibitor, and characterize the relationship between inhibition of BTK phosphorylation (pBTK) and efficacy. GDC-0834 inhibited BTK with an in vitro IC50 of 5.9 and 6.4 nM in biochemical and cellular assays, respectively, and in vivo IC50 of 1.1 and 5.6 μM in mouse and rat, respectively. Administration of GDC-0834 (30–100 mg/kg) in a rat collagen-induced arthritis (CIA) model resulted in a dose-dependent decrease of ankle swelling and reduction of morphologic pathology. An integrated disease progression pharmacokinetic/pharmacodynamic model where efficacy is driven by pBTK inhibition was fit to ankle-diameter time-course data. This model incorporated a transit model to characterize nondrug-related decreases in ankle swelling occurring at later stages of disease progression in CIA rats. The time course of ankle swelling in vehicle animals was described well by the base model. Simultaneous fitting of data from vehicle- and GDC-0834-treated groups showed that overall 73% inhibition of pBTK was needed to decrease the rate constant describing the ankle swelling increase (kin) by half. These findings suggest a high degree of pBTK inhibition is required for maximal activity of the pathway on inflammatory arthritis in rats.

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell–associated damage in IFNα-driven lupus nephritis

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Brutons tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.

Depletion of major pathogenic cells in asthma by targeting CRTh2

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.

Discovery of GDC-0853: A Potent, Selective, and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development

Brutons tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.