Impact of B-Cell-Targeted Therapies on Cardiovascular Disease.

Atherosclerosis is a lipid-driven chronic inflammatory disease that is modulated by many immune cell subsets, including B cells. Therefore, targeting the inflammatory component of cardiovascular disease represents a promising therapeutic strategy. In the past years, immunotherapy has revolutionized the treatment of autoimmunity and cancer. Many of these clinically used strategies target B cells. Given the multifaceted role of B cells in atherogenesis, it is conceivable that B-cell-directed therapies can modulate disease development. Here, we review clinically available B-cell-targeted therapies and the possible benefits or detrimental effects on cardiovascular disease.

Exploring BAFF: its expression, receptors and contribution to the immunopathogenesis of Sjögren's syndrome.

SS is an autoimmune condition characterized by exocrine gland destruction, autoantibody production, immune complex deposition and systemic complications associated with lymphocytic infiltration of many organs. Genetic, environmental and viral factors play a role in disease aetiology, however, the exact mechanisms driving the immunopathogenesis of SS remain uncertain. Here we discuss a role for B cell activating factor (BAFF), whereby B cell hyperactivity and increased BAFF secretion observed in patients and animal models of the disease can be explained by the altered expression of cell-specific BAFF/BAFF receptor (BAFF-R) variants in several immune cell types. Understanding the role of BAFF/BAFF-R heterogeneity in SS pathogenesis could help to facilitate new treatment strategies for patients.

The role of BAFF in the progression of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common autoimmune disease that is marked by a systemic inflammatory reaction and joint erosions. Elevated levels of B cell activating factor (BAFF) have been detected in the serum and synovial fluid of RA patients. Moreover, the levels of BAFF increase in cases of autoimmune disease and are correlated with the level of disease activity. As an innate cytokine mediator, BAFF affects the immune response of the synovial microenvironment. In this review, we consider recent observations of BAFF and its receptors in RA progression, as well as the effects of BAFF on the cell-cell interactions network. We also summarize the clinical development of BAFF antagonists for the treatment of RA.

Intrinsic molecular factors cause aberrant expansion of the splenic marginal zone B cell population in nonobese diabetic mice.

Marginal zone (MZ) B cells are an innate-like population that oscillates between MZ and follicular areas of the splenic white pulp. Differentiation of B cells into the MZ subset is governed by BCR signal strength and specificity, NF-κB activation through the B cell-activating factor belonging to the TNF family (BAFF) receptor, Notch2 signaling, and migration signals mediated by chemokine, integrin, and sphingosine-1-phosphate receptors. An imbalance in splenic B cell development resulting in expansion of the MZ subset has been associated with autoimmune pathogenesis in various murine models. One example is the NOD inbred mouse strain, in which MZ B cell expansion has been linked to development of type 1 diabetes and Sjögren's syndrome. However, the cause of MZ B cell expansion in this strain remains poorly understood. We have determined that increased MZ B cell development in NOD mice is independent of T cell autoimmunity, BCR specificity, BCR signal strength, and increased exposure to BAFF. Rather, mixed bone marrow chimeras showed that the factor(s) responsible for expansion of the NOD MZ subset is B cell intrinsic. Analysis of microarray expression data indicated that NOD MZ and precursor transitional 2-MZ subsets were particularly dysregulated for genes controlling cellular trafficking, including Apoe, Ccbp2, Cxcr7, Lgals1, Pla2g7, Rgs13, S1pr3, Spn, Bid, Cd55, Prf1, and Tlr3. Furthermore, these B cell subsets exhibited an increased steady state dwell time within splenic MZ areas. Our data therefore reveal that precursors of mature B cells in NOD mice exhibit an altered migration set point, allowing increased occupation of the MZ, a niche favoring MZ B cell differentiation.

Efficient B cell responses to Borrelia hermsii infection depend on BAFF and BAFFR but not TACI.

T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by the B1b cell subset are critical for controlling bacteremia. We found that B1b cells express BAFFR and TACI and that the surface expression of both receptors is upregulated on B1b cells following exposure to whole B. hermsii cells. Surprisingly, we found that TACI(-/-) mice are not impaired either in specific antibody responses to B. hermsii or in controlling B. hermsii bacteremia. In contrast, TACI-deficient mice immunized with heat-killed type 3 serotype pneumococcus cells are impaired in generating pneumococcal polysaccharide-specific responses and succumb to challenge with live type 3 serotype pneumococcus, indicating that TACI is required for T cell-independent antibody responses to bacterial-associated polysaccharides. Although we have found that TACI is dispensable for controlling B. hermsii infection, mice deficient in BAFFR or BAFF exhibit impairment in B. hermsii-specific IgM responses and clearance of bacteremia. Collectively, these data indicate a disparity in the roles for TACI and BAFFR in primary T cell-independent antibody responses to bacterial pathogens.

BAFF receptor deficiency reduces the development of atherosclerosis in mice--brief report.

OBJECTIVE: The goal of this study was to assess the role of B-cell activating factor (BAFF) receptor in B-cell regulation of atherosclerosis. METHODS AND RESULTS: Male LDL receptor-deficient mice (Ldlr(-/-)) were lethally irradiated and reconstituted with either wild type or BAFF receptor (BAFF-R)-deficient bone marrow. After 4 weeks of recovery, mice were put on a high-fat diet for 6 or 8 weeks. BAFF-R deficiency in bone marrow cells led to a marked reduction of conventional mature B2 cells but did not affect the B1a cell subtype. This was associated with a significant reduction of dendritic cell activation and T-cell proliferation along with a reduction of IgG antibodies against malondialdehyde-modified low-density lipoprotein. In contrast, serum IgM type antibodies were preserved. Interestingly, BAFF-R deficiency was associated with a significant reduction in atherosclerotic lesion development and reduced numbers of plaque T cells. Selective BAFF-R deficiency on B cells led to a similar reduction in lesion size and T-cell infiltration but in contrast did not affect dendritic cell activation. CONCLUSIONS: BAFF-R deficiency in mice selectively alters mature B2 cell-dependent cellular and humoral immune responses and limits the development of atherosclerosis.

B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans.

B-cell survival depends on signals induced by B-cell activating factor (BAFF) binding to its receptor (BAFF-R). In mice, mutations in BAFF or BAFF-R cause B-cell lymphopenia and antibody deficiency. Analyzing BAFF-R expression and BAFF-binding to B cells in common variable immunodeficiency (CVID) patients, we identified two siblings carrying a homozygous deletion in the BAFF-R gene. Removing most of the BAFF-R transmembrane part, the deletion precludes BAFF-R expression. Without BAFF-R, B-cell development is arrested at the stage of transitional B cells and the numbers of all subsequent B-cell stages are severely reduced. Both siblings have lower IgG and IgM serum levels but, unlike most CVID patients, normal IgA concentrations. They also did not mount a T-independent immune response against pneumococcal cell wall polysaccharides but only one BAFF-R-deficient sibling developed recurrent infections. Therefore, deletion of the BAFF-R gene in humans causes a characteristic immunological phenotype but it does not necessarily lead to a clinically manifest immunodeficiency.

BAFF-R promotes cell proliferation and survival through interaction with IKKbeta and NF-kappaB/c-Rel in the nucleus of normal and neoplastic B-lymphoid cells.

BLyS and its major receptor BAFF-R have been shown to be critical for development and homeostasis of normal B lymphocytes, and for cell growth and survival of neoplastic B lymphocytes, but the biologic mechanisms of this ligand/receptor-derived intracellular signaling pathway(s) have not been completely defined. We have discovered that the BAFF-R protein was present in the cell nucleus, in addition to its integral presence in the plasma membrane and cytoplasm, in both normal and neoplastic B cells. BAFF-R interacted with histone H3 and IKKbeta in the cell nucleus, enhancing histone H3 phosphorylation through IKKbeta. Nuclear BAFF-R was also associated with NF-kappaB/c-Rel and bound to NF-kappaB targeted promoters including BLyS, CD154, Bcl-xL, IL-8, and Bfl-1/A1, promoting the transcription of these genes. These observations suggested that in addition to activating NF-kappaB pathways in the plasma membrane, BAFF-R also promotes normal B-cell and B-cell non-Hodgkin lymphoma (NHL-B) survival and proliferation by functioning as a transcriptional regulator through a chromatin remodeling mechanism(s) and NF-kappaB association. Our studies provide an expanded conceptual view of the BAFF-R signaling, which should contribute a better understanding of the physiologic mechanisms involved in normal B-cell survival and growth, as well as in the pathophysiology of aggressive B-cell malignancies and autoimmune diseases.

B cell receptor and BAFF receptor signaling regulation of B cell homeostasis.

B lymphocyte homeostasis depends on tonic and induced BCR signaling and receptors sensitive to trophic factors, such as B cell-activating factor receptor (BAFF-R or BR3) during development and maintenance. This review will discuss growing evidence suggesting that the signaling mechanisms that maintain B cell survival and metabolic fitness during selection at transitional stages and survival after maturation rely on cross-talk between BCR and BR3 signaling. Recent findings have also begun to unravel the molecular mechanisms underlying this crosstalk. In this review I also propose a model for regulating the amplitude of BCR signaling by a signal amplification loop downstream of the BCR involving Btk and NF-kappaB that may facilitate BCR-dependent B cell survival as well as its functional coupling to BR3 for the growth and survival of B lymphocytes.

Absence of mature peripheral B cell populations in mice with concomitant defects in B cell receptor and BAFF-R signaling.

Generation of mature B lymphocytes from early (T1) and late transitional (T2) precursors requires cooperative signaling through BCR and B cell-activating factor receptor 3 (BR3). Recent studies have shown that BCR signaling positively regulates NF-kappaB2, suggesting BCR regulation of BR3 signaling. To investigate the significance of signal integration from BCR and BR3 in B cell development and function, we crossed Btk-deficient mice (btk(-/-)), which are developmentally blocked between the T2 and the mature follicular B cell stage as a result of a partial defect in BCR signaling, and A/WySnJ mice, which possess a mutant BR3 defective in propagating intracellular signals that results in a severely reduced peripheral B cell compartment, although all B cell subsets are present in relatively normal ratios. A/WySnJ x btk(-/-) mice display a B cell-autonomous defect, resulting in a developmental block at an earlier stage (T1) than either mutation alone, leading to the loss of mature splenic follicular and marginal zone B cells, as well as the loss of peritoneal B1 and B2 cell populations. The competence of the double mutant T1 B cells to respond to TLR4 and CD40 survival and activation signals is further attenuated compared with single mutations as evidenced by severely reduced humoral immune responses in vivo and proliferation in response to anti-IgM, LPS, and anti-CD40 stimulation in vitro. Thus, BCR and BR3 independently and in concert regulate the survival, differentiation, and function of all B cell populations at and beyond T1, earliest transitional stage.

Cell-autonomous role for NF-kappa B in immature bone marrow B cells.

The NF-kappaB transcription factors have many essential functions in B cells, such as during differentiation and proliferation of Ag-challenged mature B cells, but also during final maturation of developing B cells in the spleen. Among the various specific functions NF-kappaB factors carry out in these biologic contexts, their ability to assure the survival of mature and maturing B cells in the periphery stands out. Less clear is what if any roles NF-kappaB factors play during earlier stages of B cell development in the bone marrow. Using mice deficient in both NF-kappaB1 and NF-kappaB2, which are thus partially compromised in both the classical and alternative activation pathways, we demonstrate a B cell-autonomous contribution of NF-kappaB to the survival of immature B cells in the bone marrow. NF-kappaB1 and NF-kappaB2 also play a role during the earlier transition from proB to late preB cells; however, in this context these factors do not act in a B cell-autonomous fashion. Although NF-kappaB1 and NF-kappaB2 are not absolutely required for survival and progression of immature B cells in the bone marrow, they nevertheless make a significant contribution that marks the beginning of the profound cell-autonomous control these factors exert during all subsequent stages of B cell development. Therefore, the lifelong dependency of B cells on NF-kappaB-mediated survival functions is set in motion at the time of first expression of a full BCR.

B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading.

Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.

Control of peripheral B-cell development.

Three subsets of mature B cells exist in mice: B-1, follicular and marginal zone B cells. The recruitment of newly formed transitional B cells into these compartments depends on signals emanating from the B-cell antigen receptor, possibly in response to (self-)antigen recognition. Recent evidence suggests that peripheral B-cell fate is controlled by B-cell antigen receptor signal strength, acting in concert with the cytokine B-cell activating factor. Other work indicates that peripheral B-cell development is orchestrated by a complex network of transcription factors, which drive B-cell subset differentiation, at the same time preventing mature B cells from undergoing trans-differentiation or premature terminal differentiation.

Homeostatic control of B lymphocyte subsets.

Lymphocyte homeostasis poses a multi-faceted biological puzzle, because steady pre-immune populations must be maintained at an acceptable steady state to yield effective protection, despite stringent selective events during their generation. In addition, activated, memory and both short- and long-term effectors must be governed by independent homeostatic mechanisms. Finally, advancing age is accompanied by substantial changes that impact the dynamics and behavior of these pools, leading to cumulative homeostatic perturbations and compensation. Our laboratory has focused on the over-arching role of BLyS family ligands and receptors in these processes. These studies have led to a conceptual framework within which distinct homeostatic niches are specified by BLyS receptor signatures, which define the BLyS family ligands that can afford survival. The cues for establishing these receptor signatures, as well as the downstream survival mechanisms involved, are integrated with cell extrinsic inputs via cross talk among downstream mediators. A refined understanding of these relationships should yield insight into the selection and maintenance of B cell subsets, as well as an appreciation of how homeostatic mechanisms may contribute to immunosenescence.

Depletion of B2 but not B1a B cells in BAFF receptor-deficient ApoE mice attenuates atherosclerosis by potently ameliorating arterial inflammation.

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE(-/-) mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE(-/-) mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R(-/-) ApoE(-/-) (BaffR.ApoE DKO) and BAFF-R(+/+)ApoE(-/-) (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE(-/-) mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-ß and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin-6 to induce the expression of recombination-activating gene in B lymphocytes.

OBJECTIVE: B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. METHODS: B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin kappa and lambda light chain expression and by ligation-mediated-PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. RESULTS: RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the kappa-to-lambda ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. CONCLUSION: Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA.