Susan L. Kalled

Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren’s syndrome

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sjögrens syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.

Reduced Competitiveness of Autoantigen-Engaged B Cells due to Increased Dependence on BAFF

Peripheral autoantigen binding B cells are poorly competitive with naive B cells for survival and undergo rapid cell death. However, in monoclonal Ig-transgenic mice lacking competitor B cells, autoantigen binding B cells can survive for extended periods. The basis for competitive elimination of autoantigen binding B cells has been unknown. Here we demonstrate that autoantigen binding B cells have increased dependence on BAFF for survival. In monoclonal Ig-transgenic mice, each autoantigen binding B cell receives elevated amounts of BAFF, exhibiting increased levels of NFkappaB p52 and of the prosurvival kinase Pim2. When placed in a diverse B cell compartment, BAFF receptor engagement and signaling are reduced and the autoantigen binding cells are unable to protect themselves from Bim and possibly other death-promoting factors induced by chronic BCR signaling. These findings indicate that under conditions where BAFF levels are elevated, autoantigen-engaged cells will be rescued from rapid competitive elimination, predisposing to the development of autoimmune disease.

BAFF selectively enhances the survival of plasmablasts generated from human memory B cells

The generation of Ig-secreting cells (ISCs) from memory B cells requires interactions between antigen-specific (Ag-specific) B cells, T cells, and dendritic cells. This process must be strictly regulated to ensure sufficient humoral immunity while avoiding production of pathogenic autoantibodies. BAFF, a member of the TNF family, is a key regulator of B cell homeostasis. BAFF exerts its effect by binding to three receptors - transmembrane activator of and CAML interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R). To elucidate the contribution of BAFF to the differentiation of B cells into ISCs, we tracked the fate of human memory B cells stimulated with BAFF or CD40L. BAFF and CD40L significantly increased the overall number of surviving B cells. This was achieved via distinct mechanisms. CD40L induced proliferation of nondifferentiated blasts, while BAFF prevented apoptosis of ISCs without enhancing proliferation. The altered responsiveness of activated memory B cells to CD40L and BAFF correlated with changes in surface phenotype such that expression of CD40 and BAFF-R were reduced on ISCs while BCMA was induced. These results suggest BAFF may enhance humoral immunity in vivo by promoting survival of ISCs via a BCMA-dependent mechanism. These findings have wide-ranging implications for the treatment of human immunodeficiencies as well as autoimmune diseases.

B Cell-Activating Factor Belonging to the TNF Family (BAFF)-R Is the Principal BAFF Receptor Facilitating BAFF Costimulation of Circulating T and B Cells

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using specific mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti-μ responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells.

Identification of proteoglycans as the APRIL-specific binding partners

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors—transmembrane activator and calcium signal–modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)—that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific “receptor.” This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRILs basic sequence. Moreover, syndecan-1–positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Normal B Cell Homeostasis Requires B Cell Activation Factor Production by Radiation-resistant Cells

The cellular source of B cell activation factor (BAFF) required for peripheral B cell survival/maturation is unknown. To determine the nature of BAFF-producing cells we established and analyzed reciprocal bone marrow (BM) chimeras with wild-type (WT) and BAFF-deficient mice. The results revealed that BAFF production by radiation-resistant stromal cells is completely sufficient to provide a necessary signal for B cell survival/maturation, as BAFF−/− BM cells transferred into lethally irradiated WT mice gave rise to normal numbers of follicular (FO) and marginal zone (MZ) B cell subpopulations. On the other hand, transfer of WT BM into BAFF−/−lethally irradiated mice resulted only in minimal reconstitution of mature FO B cells and no restoration of MZ B cells. Thus, in the absence of BAFF+/+stromal cells, BAFF production by BM-derived cells, presumably by macrophages, dendritic cells, and/or neutrophils, was not at all sufficient to support normal B cell homeostasis. Interestingly, immunization of both types of chimeras stimulated high levels of antigen-specific antibody secretion, indicating that either stromal cell– or hematopoietic cell–derived BAFF is sufficient for B cell antibody responses.

B Cell-Activating Factor Belonging to the TNF Family Acts through Separate Receptors to Support B Cell Survival and T Cell-Independent Antibody Formation

The TNF-related ligand, B cell-activating factor belonging to the TNF family (BAFF), is necessary for normal B cell development and survival, and specifically binds the receptors transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R. Similarities between mice completely lacking BAFF and A/WySnJ strain mice that express a naturally occurring mutant form of BAFF-R suggest that BAFF acts primarily through BAFF-R. However, the nearly full-length BAFF-R protein expressed by A/WySnJ mice makes unambiguous interpretation of receptor function in these animals impossible. Using homologous recombination we created mice completely lacking BAFF-R and compared them directly to A/WySnJ mice and to mice lacking BAFF. BAFF-R-null mice exhibit loss of mature B cells similar to that observed in BAFF−/− and A/WySnJ mice. Also, mice lacking both TACI and BCMA simultaneously exhibit no B cell loss, thus confirming that BAFF-R is the primary receptor for transmitting the BAFF-dependent B cell survival signal. However, while BAFF-R-null mice cannot carry out T cell-dependent Ab formation, they differ from BAFF-deficient mice in generating normal levels of Ab to at least some T cell-independent Ags. These studies clearly demonstrate that BAFF regulates Ab responses in vivo through receptors in addition to BAFF-R.

Normal Induction but Attenuated Progression of Germinal Center Responses in BAFF and BAFF-R Signaling–Deficient Mice

The factors regulating germinal center (GC) B cell fate are poorly understood. Recent studies have defined a crucial role for the B cell–activating factor belonging to TNF family (BAFF; also called BLyS) in promoting primary B cell survival and development. A role for this cytokine in antigen-driven B cell responses has been suggested but current data in this regard are limited. A BAFF receptor expressed by B cells (BAFF-R/BR3) is defective in A/WySnJ mice which exhibit a phenotype similar to BAFF-deficient (BAFF−/−) animals. Here, we show that although GC responses can be efficiently induced in both A/WySnJ and BAFF−/− mice, these responses are not sustained. In BAFF−/− mice, this response is rapidly attenuated and accompanied by perturbed follicular dendritic cell development and immune complex trapping. In contrast, analysis of the A/WySnJ GC response revealed a B cell autonomous proliferative defect associated with reduced or undetectable Ki67 nuclear proliferation antigen expression by GC B cells at all stages of the response. These data demonstrate a multifaceted role for the BAFF pathway in regulating GC progression.

Fibroblast-like synoviocytes of mesenchymal origin express functional B cell-activating factor of the TNF family in response to proinflammatory cytokines.

Immunohistochemical analysis revealed that the intimal lining cells of synovial tissue of inflamed joints of patients with rheumatoid arthritis differed from that of normal joints or of diseased joints in osteoarthritis in that they stained with mAb specific for the B cell-activating factor of the TNF family (BAFF; also called BLyS). We generated fibroblast-like synoviocytes (FLS) cell lines that were bereft of myelomonocytic cells to examine whether mesenchymal-derived FLS could express this critical B cell survival factor. We found that FLS expressed low amounts of BAFF mRNA relative to that of myelomonocytic cells. However, when various cytokines/factors were added to such FLS cell lines, we found that IFN-γ or TNF-α were unique in that they could induce significant increases in BAFF mRNA and protein. Even minute amounts of IFN-γ primed FLS for TNF-α, allowing the latter to stimulate significantly higher levels of BAFF mRNA and protein than could TNF-α alone. Consistent with this, B cells cocultured with IFN-γ and/or TNF-α-treated FLS had a significantly greater viability than B cells cocultured with nontreated FLS. The enhanced protection of B cells afforded by IFN-γ/TNF-α-treated FLS was inhibited by the addition of BAFF-R:Fc fusion protein. We conclude that the proinflammatory cytokines IFN-γ and TNF-α can induce mesenchymal-derived FLS to express functional BAFF in vitro. The induced expression of BAFF on FLS by proinflammatory cytokines may enhance the capacity of such cells to protect B cells from apoptosis in inflammatory microenvironments in vivo.

Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function

Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.