BAFF Transgenic Mice Research Articles

Efficacy evaluation of B cell targeting drugs and the involved mechanism of action study in humanized BAFF transgenic SLE mice model

Abstract The B cell activating factor (BAFF) functions as a crucial factor in B cell survival, and heightened BAFF expression can augment the presence of autoreactive B cells, thereby fostering autoimmunity like Systemic Lupus Erythematosus (SLE). This study introduces transgenic mouse strains with human BAFF overexpression, offering insights into SLE mechanisms and B cell-targeting therapies. Human BAFF overexpression in B6-hBAFF mice led to a substantial rise in Igs and anti-dsDNA levels from 6 weeks of age, persistently escalating with time compared to wild-type mice. A heightened population of B cells but a reduced ratio of T cells was observed. At week 25 of age, B6-hBAFF mice developed lupus nephritis. Belimumab, a BAFF antibody administrated to BAFF transgenic mice demonstrated the effectively reduced serum antibody levels after 16 weeks of dosing. Belimumab ameliorated kidney function by decreasing urine albumin-to-creatinine ratio and IgA deposition after 20 weeks of treatment. Mice were also treated with Enpatoran (TLR7/8 inhibitors) and BTK inhibitor Orelabrutinib. Only the BTK inhibitor Orelabrutinib significantly eliminated serum Igs levels. Enpatoran exhibited a similar ability to Orelabrutinib in reducing T follicular helper cell and plasma cell populations spleen and lymph nodes. In summary, the humanized BAFF transgenic SLE model proves to be a valuable tool for preclinical efficacy studies in SLE treatment and understanding pathogenesis.

Humanized BAFF transgenic mice develop autoimmune manifestations

Abstract The B cell activating factor(BAFF) is a B cell survival factor that supports autoreactive B cells’ survival and prevents their depletion. Excessive BAFF can increase autoreactive B cells, driving autoimmunity. BAFF is commonly overexpressed in Systemic Lupus Erythematosus(SLE) and is strongly involved in the pathogenesis of the disease. Mice with BAFF deficiency results in the lack of mature B cells, whereas mice that overproduce BAFF have high numbers of mature B cells and antibodies, including autoantibodies, and develop an autoimmune disease similar to SLE in humans. BAFF has been shown to be the pivotal target in autoimmune diseases. In this study, we report the generation of two transgenic mouse strains with overexpression of human BAFF (B6-hBAFF, T036791 and T036794), which could be used to study anti-BAFF therapies. Overexpression of human BAFF led to significantly increased IgG, IgA, and IgM levels since 6 weeks of age compared with wild-type mice, which continued to increase gradually as the transgenic mice aged. These mice secreted a high level of anti-DNA autoantibodies, along with an increased ratio of B cells/CD45 +in the spleen and lymph node compared with wild-type mice, indicating overreactive B cells. Proteinuria started at 6 weeks of age with an increase in protein/creatinine ratio. This was accompanied by the infiltration of inflammatory leukocytes in the kidney at 15 weeks of age, suggesting local kidney inflammation. Taken together, the humanized BAFF transgenic mice autoimmune manifestations with enhanced overreactive B cells, providing a valuable tool supporting preclinical efficacy studies of drugs targeting B cells for the development of autoimmune disease therapies.

The effects of B-cell-activating factor on the population size, maturation and function of murine natural killer cells.

The role of B-cell-activating factor (BAFF) in B-lymphocyte biology has been comprehensively studied, but its contributions to innate immunity remain unclear. Natural killer (NK) cells form the first line of defense against viruses and tumors, and have been shown to be defective in patients with systemic lupus erythematosus (SLE). The link between BAFF and NK cells in the development and progression of SLE remains unstudied. By assessing NK cell numbers in wild-type (WT), BAFF-/- (BAFF deficient), BAFF-R-/- (BAFF receptor deficient), TACI-/- (transmembrane activator and calcium modulator and cyclophilin ligand interactor deficient), BCMA-/- (B-cell maturation antigen deficient) and BAFF transgenic (Tg) mice, we observed that BAFF signaling through BAFF-R was essential for sustaining NK cell numbers in the spleen. However, according to the cell surface expression of CD27 and CD11b on NK cells, we found that BAFF was dispensable for NK cell maturation. Ex vivo and in vivo models showed that NK cells from BAFF-/- and BAFF Tg mice produced interferon-γ and killed tumor cells at a level similar to that in WT mice. Finally, we established that NK cells do not express receptors that interact with BAFF in the steady state or in the BAFF Tg mouse model of SLE. Our findings demonstrate that BAFF has an indirect effect on NK cell homeostasis and no effect on NK cell function.

Interleukin-33 Exacerbates IgA Glomerulonephritis in Transgenic Mice Overexpressing B Cell Activating Factor.

The cytokine IL-33 is an activator of innate lymphoid cells 2 (ILC2s) in innate immunity and allergic inflammation. B cell activating factor (BAFF) plays a central role in B cell proliferation and differentiation, and high levels of this protein cause excess antibody production, including IgA. BAFF-transgenic mice overexpress BAFF and spontaneously develop glomerulonephritis that resembles human IgA nephropathy. We administered IL-33 or PBS to wild-type and BAFF-transgenic mice. After treating Rag1-deficient mice with IL-33, with or without anti-CD90.2 to preferentially deplete ILC2s, we isolated splenocytes, which were adoptively transferred into BAFF-transgenic mice. BAFF-transgenic mice treated with IL-33 developed more severe kidney dysfunction and proteinuria, glomerular sclerosis, tubulointerstitial damage, and glomerular deposition of IgA and C3. Compared with wild-type mice, BAFF-transgenic mice exhibited increases of CD19+ B cells in spleen and kidney and ILC2s in kidney and intestine, which were further increased by administration of IL-33. Administering IL-33 to wild-type mice had no effect on kidney function or histology, nor did it alter the number of ILC2s in spleen, kidney, or intestine. To understand the role of ILC2s, splenocytes were transferred from IL-33-treated Rag1-deficient mice into BAFF-transgenic mice. Glomerulonephritis and IgA deposition were exacerbated by transfer of IL-33-stimulated Rag1-deficient splenocytes, but not by ILC2 (anti-CD90.2)-depleted splenocytes. Wild-type mice infused with IL-33-treated Rag1-deficient splenocytes showed no change in kidney function or ILC2 numbers or distribution. IL-33-expanded ILC2s exacerbated IgA glomerulonephritis in a mouse model. These findings indicate that IL-33 and ILC2s warrant evaluation as possible mediators of human IgA nephropathy.

Immunoglobulin A nephropathy is characterized by anticommensal humoral immune responses.

IgA nephropathy (IgAN) is a leading cause of kidney failure, yet little is known about the immunopathogenesis of this disease. IgAN is characterized by deposition of IgA in the kidney glomeruli, but the source and stimulus for IgA production are not known. Clinical and experimental data suggest a role for aberrant immune responses to mucosal microbiota in IgAN, and in some countries with high disease prevalence, tonsillectomy is regarded as standard-of-care therapy. To evaluate the relationship between microbiota and mucosal immune responses, we characterized the tonsil microbiota in patients with IgAN versus nonrelated household-matched control group participants and identified increased carriage of the genus Neisseria and elevated Neisseria-targeted serum IgA in IgAN patients. We reverse-translated these findings in experimental IgAN driven by BAFF overexpression in BAFF-transgenic mice rendered susceptible to Neisseria infection by introduction of a humanized CEACAM-1 transgene (B × hC-Tg). Colonization of B × hC-Tg mice with Neisseria yielded augmented levels of systemic Neisseria-specific IgA. Using a custom ELISPOT assay, we discovered anti-Neisseria–specific IgA-secreting cells within the kidneys of these mice. These findings suggest a role for cytokine-driven aberrant mucosal immune responses to oropharyngeal pathobionts, such as Neisseria, in the immunopathogenesis of IgAN. Furthermore, in the presence of excess BAFF, pathobiont-specific IgA can be produced in situ within the kidney.

Bob.1 Blocks a SYK Protein Degradation Pathway in Mice That Develop Chronic Graft Versus Host Disease Manifestations

After elucidating a role for aberrant B Cell Receptor (BCR) signaling in chronic graft versus host disease (cGVHD) (Allen et al, Blood. 2014;123:2108), we showed that the proximal BCR molecule SYK is a viable therapeutic target in mice and patients (Flynn et al, Blood 2015; 125:4085. Poe et al, JCI Insight. 2018;3:e122430. NCT02611063). Increased SYK protein levels associate with enhance phosphorylation of SYK activation sites, leading to immediate BCR responsiveness in cGVHD. In mice that develop cGVHD manifestations after allogeneic bone marrow transplantation (BMT), BCR responsiveness is promoted by B Cell Activating Factor (BAFF) (Jia et al, Blood. 2021;137:2544). Instead of SYK protein levels decreasing upon engagement with surrogate antigen to regulate excessive BCR-signaling, SYK protein was maintained when BAFF and alloantigen were present. In the present study, we address a potential molecular mechanism underpinning SYK protein maintenance in cGVHD B cells.In an unbiased scRNAseq study examining purified B cells from active cGVHD patients, we found that Pou2af1 was significantly altered within a BCR-activated B cell subset (Poe et al, Blood. 2019; 134; Suppl1:874-874). Pou2af1 encodes B cell Oct binding factor 1 (Bob.1), a prime candidate for further study. Bob.1 is a transcriptional coactivator that is also able to bind to SYK and regulate SYK protein stability (Siegel et al, Cell. 2006; 125:761). To determine whether BAFF promoted increased Bob.1 expression we examined B cells from BAFF transgenic (Tg) mice. Consistent with a role for this molecule in blocking SYK degradation, Bob.1 protein levels were notably higher in BAFF-Tg B cells. Intracellular flow cytometry analyses of B cells at day 40 after BMT (cGVHD n=9 vs control n=5 mice) confirmed that Bob.1 is significantly higher in cGVHD mouse B cells (p=0.0002, data not shown). We then investigated whether phosphorylation of a SYK negative regulatory site after BCR-activation was affected by BAFF. Since tyrosine 317 (SYK-Y317) phosphorylation is known to initiate ubiquitination and degradation of SYK, we stimulated purified B cells through the BCR before preparing cell lysates and performing Western blot analyses to measure SYK-Y317 phosphorylation, total SYK, LYN, Bob.1 and BLNK. As shown in a representative blot in Fig 1A, phosphorylation of SYK-Y317 (pSYK-Y317) was significantly decreased in B cells from BAFF transgenic (BAFF-Tg) mice compared to those from wild type (WT) mice. These data suggest that BAFF promotes SYK through Bob.1.Hypothesizing that Bob.1 is required to maintain SYK protein after allogeneic BMT, we employed Bob.1 knockout mice. After verifying that heterozygous mice (Bob.1 +/-) had lower Bob.1 protein levels and comparable B cell numbers to WT mice, we investigated the effect of Bob.1 reduction on SYK levels. Bob.1 +/- mice were crossed with BAFF-Tg mice and then SYK expression in B cells was determined using intracellular flow cytometry. We found that SYK protein levels were significantly increased in B cells exposed to a high BAFF environment and that SYK levels were significantly lower when Bob.1 protein was decreased (Fig 1B). These data demonstrate that Bob.1 is needed in vivo to maintain BAFF-mediated SYK protein maintenance after BCR engagement. Finally, we evaluated whether Bob.1 affected phosphorylation at SYK-Y317 in B cells from cGVHD mice. Splenic B cells were purified 41 days after BMT from recipients of B6 syngeneic (Syn), BM alone (BM only, control) or BM plus splenocytes (BM+Sp, cGVHD) and then stimulated with anti-IgM for 30 seconds before pSyk-Y317 and total SYK were detected by Western blot (Fig 1C). Using Image Studio Lite software, we normalized the intensity of pSyk-Y317 to actin in n=4 (Syn-BM+Sp), n=5 (BM only) and n=4 (BM+Sp) and found a significant decrease in pSyk-Y317 in cGVHD mice. We are currently investigating potential pathologic effects of Bob.1 after BMT in our mouse model and in patient cells.In summary, we have now demonstrated that, Bob.1 is required to maintain SYK protein in B cells after allogeneic BMT in mice that develop cGVHD. Our data suggest that Bob.1 maintains the stability of BCR proximal SYK by blocking a known SYK degradation pathway (Fig 1D). Improving our understanding of Bob.1 and SYK protein homeostasis after HCT will impact how we implement SYK inhibitors in patients.This work was supported the National Institutes of Health (NHLBI R01 HL 129061-06). [Display omitted] DisclosuresSarantopoulos: Rigel: Other: Advisory Board.

Long-term exposure to monoclonal anti-TNF is associated with an increased risk of lymphoma in BAFF-transgenic mice.

The impact of treatment on the risk of lymphoma in patients with rheumatoid arthritis (RA) is unclear. Here, we aimed to assess if the risk of lymphoma differs according to the type of tumor necrosis factor inhibitor (TNFi), comparing monoclonal anti-TNF antibodies to the soluble TNF receptor. We used B cell activating factor belonging to the TNF family (BAFF)-transgenic (Tg) mice as a model of autoimmunity-associated lymphoma. Six-month-old BAFF-Tg mice were treated with TNFi for 12months. Histological examination of the spleen, assessment of the cellular composition of the spleen by flow cytometry and assessment of B cell clonality were performed at euthanasia. Crude mortality and incidence of lymphoma were significantly higher in mice treated with monoclonal anti-TNF antibodies compared to both controls and mice treated with the soluble TNF receptor, even at a high dose. Flow cytometry analysis revealed decreased splenic macrophage infiltration in mice treated with monoclonal anti-TNF antibodies. Overall, this study demonstrates, for the first time, that a very prolonged treatment with monoclonal anti-TNF antibodies increase the risk of lymphoma in B cell-driven autoimmunity. These data suggest a closer monitoring for lymphoma development in patients suffering from B cell-driven autoimmune disease with long-term exposure to monoclonal anti-TNF antibodies.

BAFF promotes heightened BCR responsiveness and manifestations of chronic GVHD after allogeneic stem cell transplantation

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell–activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex–mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic μMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)–activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell–depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.

P0344NEFECON® (BUDESONIDE) SELECTIVELY REDUCES CIRCULATING LEVELS OF BAFF (BLYS) AND SOLUBLE BCMA AND TACI IN IGA NEPHROPATHY

Abstract Background and Aims There is increasing evidence to support a pivotal role for the gut-associated lymphoid tissue (GALT) as a major source of the poorly O-galactosylated immunoglobulin A1 (IgA1) that triggers the formation of nephritogenic immune complexes in IgA nephropathy (IgAN). Critical to the regulation of IgA synthesis in GALT are two cytokines, which regulate B-cell maturation, function and survival: BAFF (B-cell activating factor [also known as BLyS (B-lymphocyte stimulator)]) and APRIL (A PRoliferation-inducing ligand). BAFF and APRIL bind to specific cell-surface receptors: TACI (transmembrane activator and calcium modulator and cyclophilin-ligand interactor), BCMA (B-cell maturation antigen) and the BAFF receptor. Several studies have shown elevated levels of BAFF and APRIL in IgAN linked to worse clinical outcomes. The therapeutic potential of selectively targeting GALT was demonstrated in the NEFIGAN trial (NCT01738035), which assessed the safety and efficacy of a novel targeted-release formulation of budesonide (NEFECON®), designed to deliver budesonide to the GALT-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, 9-month treatment and 3-month follow-up phase: 48 patients received NEFECON® 16 mg/day, 51 patients received NEFECON® 8 mg/day and 50 patients received placebo. The headline result of the study was that NEFECON® 16 mg/day, added to optimised renin–angiotensin system blockade, reduced proteinuria and stabilised estimated glomerular filtration rate in patients with IgAN. In this study, we aimed to determine whether NEFECON® treatment altered synthesis of the BAFF and APRIL B-cell maturation modulators. Method Serum levels of BAFF, APRIL, BCMA and TACI were measured using Luminex technology. Changes in the levels of each biomarker between randomisation (T0), end of treatment (T1) and the end of study (T2) and between treatments arms were compared using a one-way analysis of variance. Significance was set at p<0.05. Results A significant, dose-dependent reduction in serum BAFF levels was seen with NEFECON®, which reversed on cessation of NEFECON®. There were similar significant reductions in the levels of soluble BCMA and TACI with treatment but no effect was seen on circulating levels of APRIL. These changes were mirrored by parallel changes in soluble CD27 levels and are consistent with our previous reports describing a dose-dependent reduction in circulating IgA–IgG immune complexes, secretory IgA and galactose-deficient IgA levels with NEFECON®. Conclusion Delivering budesonide to the GALT-rich distal ileum modulates key regulators of GALT B-cell maturation in IgAN. Although the samples available are not sufficient to determine that the observed changes in serum BAFF, BCMA and TACI levels are due to a direct action on GALT, it is intriguing to note that BAFF-transgenic mice develop an IgAN-like disease, principally due to IgA overexpression in the intestinal lamina propria.

The role of BAFF and APRIL in rheumatoid arthritis.

Development and activation of B cells quickly became clear after identifying new ligands and receptors in the tumor necrosis factor superfamily. B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are the members of membrane proteins Type 2 family released by proteolytic cleavage of furin to form active, soluble homotrimers. Except for B cells, ligands are expressed by all such immune cells like T cells, dendritic cells, monocytes, and macrophages. BAFF and APRIL have two common receptors, namely TNFR homolog transmembrane activator and Ca2+ modulator and CAML interactor (TACI) and B cell-maturation antigen. BAFF alone can also be coupled with a third receptor called BAFFR (also called BR3 or BLyS Receptor). These receptors are often expressed by immune cells in the B-cell lineage. The binding of BAFF or APRIL to their receptors supports B cells differentiation and proliferation, immunoglobulin production and the upregulation of B cell-effector molecules expression. It is possible that the overexpression of BAFF and APRIL contributes to the pathogenesis of autoimmune diseases. In BAFF transgenic mice, there is a pseudo-autoimmune manifestation, which is associated with an increase in B-lymphocytes, hyperglobulinemia, anti-single stranded DNA, and anti-double-stranded DNA antibodies, and immune complexes in their peripheral blood. Furthermore, overexpressing BAFF augments the number of peripheral B220+ B cells with a normal proliferation rate, high levels of Bcl2, and prolonged survival and hyperactivity. Therefore, in this review article, we studied BAFF and APRIL as important mediators in B-cell and discussed their role in rheumatoid arthritis.

Integrated B Cell, Toll-like, and BAFF Receptor Signals Promote Autoantibody Production by Transitional B Cells.

The B cell survival cytokine BAFF has been linked with the pathogenesis of systemic lupus erythematosus (SLE). BAFF binds distinct BAFF-family surface receptors, including the BAFF-R and transmembrane activator and CAML interactor (TACI). Although originally characterized as a negative regulator of B cell activation, TACI signals are critical for class-switched autoantibody (autoAb) production in BAFF transgenic mice. Consistent with this finding, a subset of transitional splenic B cells upregulate surface TACI expression and contribute to BAFF-driven autoAb. In the current study, we interrogated the B cell signals required for transitional B cell TACI expression and Ab production. Surprisingly, despite established roles for dual BCR and TLR signals in autoAb production in SLE, signals downstream of these receptors exerted distinct impacts on transitional B cell TACI expression and autoAb titers. Whereas loss of BCR signals prevented transitional B cell TACI expression and resulted in loss of serum autoAb across all Ig isotypes, lack of TLR signals exerted a more limited impact restricted to autoAb class-switch recombination without altering transitional B cell TACI expression. Finally, in parallel with the protective effect of TACI deletion, loss of BAFF-R signaling also protected against BAFF-driven autoimmunity. Together, these findings highlight how multiple signaling pathways integrate to promote class-switched autoAb production by transitional B cells, events that likely impact the pathogenesis of SLE and other BAFF-dependent autoimmune diseases.

Methotrexate and BAFF interaction prevents immunization against TNF inhibitors

ObjectivesTNF inhibitors (TNFi) can induce anti-drug antibodies (ADA) in patients with autoimmune diseases (AID) leading to clinical resistance. We explored a new way of using methotrexate (MTX) to decrease this...

TACI deletion protects against progressive murine lupus nephritis induced by BAFF overexpression

B cells are known to promote the pathogenesis of systemic lupus erythematosus (SLE) via the production of pathogenic anti-nuclear antibodies. However, the signals required for autoreactive B cell activation and the immune mechanisms whereby B cells impact lupus nephritis pathology remain poorly understood. The B cell survival cytokine B cell activating factor of the TNF Family (BAFF) has been implicated in the pathogenesis of SLE and lupus nephritis in both animal models and human clinical studies. Although the BAFF receptor has been predicted to be the primary BAFF family receptor responsible for BAFF-driven humoral autoimmunity, in the current study we identify a critical role for signals downstream of Transmembrane Activator and CAML Interactor (TACI) in BAFF-dependent lupus nephritis. Whereas transgenic mice overexpressing BAFF develop progressive membranoproliferative glomerulonephritis, albuminuria and renal dysfunction, TACI deletion in BAFF-transgenic mice provided long-term (about 1 year) protection from renal disease. Surprisingly, disease protection in this context was not explained by complete loss of glomerular immune complex deposits. Rather, TACI deletion specifically reduced endocapillary, but not mesangial, immune deposits. Notably, although excess BAFF promoted widespread breaks in B cell tolerance, BAFF-transgenic antibodies were enriched for RNA- relative to DNA-associated autoantigen reactivity. These RNA-associated autoantibody specificities were specifically reduced by TACI or Toll-like receptor 7 deletion. Thus, our study provides important insights into the autoantibody specificities driving proliferative lupus nephritis, and suggests that TACI inhibition may be novel and effective treatment strategy in lupus nephritis.

Cutting Edge: BAFF Promotes Autoantibody Production via TACI-Dependent Activation of Transitional B Cells.

Mice overexpressing B cell activating factor of the TNF family (BAFF) develop systemic autoimmunity characterized by class-switched anti-nuclear Abs. Transmembrane activator and CAML interactor (TACI) signals are critical for BAFF-mediated autoimmunity, but the B cell developmental subsets undergoing TACI-dependent activation in settings of excess BAFF remain unclear. We report that, although surface TACI expression is usually limited to mature B cells, excess BAFF promotes the expansion of TACI-expressing transitional B cells. TACI(+) transitional cells from BAFF-transgenic mice are characterized by an activated, cycling phenotype, and the TACI(+) cell subset is specifically enriched for autoreactivity, expresses activation-induced cytidine deaminase and T-bet, and exhibits evidence of somatic hypermutation. Consistent with a potential contribution to BAFF-mediated humoral autoimmunity, TACI(+) transitional B cells from BAFF-transgenic mice spontaneously produce class-switched autoantibodies ex vivo. These combined findings highlight a novel mechanism through which BAFF promotes humoral autoimmunity via direct, TACI-dependent activation of transitional B cells.

BAFF-driven autoimmunity requires CD19 expression

B cell activating factor of the tumor necrosis factor family (BAFF or BLyS) is a critical factor for B cell survival and maturation. BAFF-transgenic (BAFF-Tg) mice develop autoimmunity that resembles systemic lupus erythematosus (SLE) in a T cell-independent but MyD88-dependent manner, implicating toll-like receptor (TLR) signaling. The specific B cell subtypes that make pro-inflammatory autoantibodies in BAFF-Tg mice are TLR-activated innate B cells known as marginal zone (MZ) and B1 B cells. These cells infiltrate the salivary glands and kidneys of diseased BAFF-Tg mice. However, loss of B1a or MZ B cells does not protect BAFF-Tg mice against disease, suggesting that B1b B cells might be the important pathogenic B cell subset. To test this hypothesis, we have generated BAFF-Tg mice that retained follicular B cells, but are deficient in B1a, B1b and MZ B cells, by crossing BAFF-Tg mice to CD19-deficient mice (BTg-CD19−/−). The BTg-CD19−/− mice did not produce autoantibodies and were protected from splenomegaly, kidney pathology and all signs of autoimmunity. This work suggests that B1b B cells, rather than MZ or B1a B cells, are sufficient and possibly required for the development of autoimmunity. Loss of the majority of innate-like B cells was able to protect BAFF-Tg mice from developing disease, so we can now conclude that autoimmunity induced by excessive BAFF production requires B1b B cells and CD19 signaling.

Effect of TACI signaling on humoral immunity and autoimmune diseases.

Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) is one of the receptors of B cell activating factor of the tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL). TACI is a regulator in the immune responses. TACI inhibits B cell expansion and promotes the differentiation and survival of plasma cells. The mechanisms underlying these effects probably involve changed expressions of some crucial molecules, such as B lymphocyte induced maturation protein-1 (Blimp-1) and inducible T-cell costimulator ligand (ICOSL) in B cells and/or plasma cells. However, abnormal TACI signaling may relate to autoimmune disorders. Common variable immune deficiency (CVID) patients with heterozygous mutations in TACI alleles increase susceptibility to autoimmune diseases. Taci −/− mice and BAFF transgenic mice both develop signs of human SLE. These findings that indicate inappropriate levels of TACI signaling may disrupt immune system balance, thereby promoting the development of autoimmune diseases. In this review, we summarize the basic characteristics of the TACI ligands BAFF and APRIL, and detail the research findings on the role of TACI in humoral immunity. We also discuss the possible mechanisms underlying the susceptibility of CVID patients with TACI mutations to autoimmune diseases and the role of TACI in the pathogenesis of SLE.

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)

Abstract One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. Anti-EBNA-1 antibody obtained from mice from all three strains was observed to cross-react with dsDNA. However, the frequency of mice producing cross-reactive antibody was greater and the amount of cross-reactive antibody was higher in BAFF than wild type mice. This suggests that a viral protein such as EBNA-1 may trigger the development of antibodies that cross-react with dsDNA and that genetic susceptibility to lupus may enhance the development of cross-reactive antibodies upon viral exposure.

Prolonged Cardiac Allograft Survival in BAFF-Transgenic Mice Associated with Increased Tregs

Prolonged Cardiac Allograft Survival in BAFF-Transgenic Mice Associated with Increased Tregs

FcγRIIb and BAFF Differentially Regulate Peritoneal B1 Cell Survival

B1 cells produce most natural Abs in unimmunized mice and play a key role in the response to thymus-independent Ags and microbial infection. Enlargement of B1 cell number in mice is often associated with autoimmunity. However, the factors that control peripheral B1 cell survival remain poorly characterized. Mice lacking the inhibitory receptor FcγRIIb exhibit a massive expansion in peritoneal B1 cells, implicating this receptor in B1 cell homeostasis. In this study, we show that peritoneal B1 cells express the highest levels of FcγRIIb among B cell subsets and are highly susceptible to FcγRIIb-mediated apoptosis. B1 cells upregulate FcγRIIb in response to innate signals, including CpG, and the B cell homeostatic cytokine BAFF efficiently protects activated B1 cells from FcγRIIb-mediated apoptosis via receptor downregulation. BAFF-transgenic mice manifest an expansion of peritoneal B1 cells that express lower levels of FcγRIIb and exhibit reduced susceptibility to apoptosis. Whereas both peritoneal B1 cells from wild-type and BAFF-transgenic mice immunized with CpG exhibit an increase in FcγRIIb levels, this change is blunted in BAFF-transgenic animals. Our combined results demonstrate that FcγRIIb controls peritoneal B1 cell survival and this program can be modulated by the BAFF signaling axis.