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The TNF family plays a critical role in immune regulation. Here, we present high-resolution structures of clusters formed by two TNF receptor family proteins, TNFR1 and BAFFR. Using a lipid monolayer method to mimic their membrane-bound state, we observe that the TNFα-TNFR1 complex forms highly ordered clusters of trimers on the lipid membrane. A non-competitive TNFR1 antagonist that inhibits receptor activation disrupted these clusters without blocking ligand binding or receptor trimerization. Furthermore, we find that the BAFF-BAFFR, BAFF-TACI, and BAFF-BCMA receptor-ligand complexes predominantly form pentagonal clusters of trimers on the lipid membrane. Notably, the binding of the intracellular adaptor TRAF3 to the BAFF-BAFFR complex induces a structural transition from a pentagonal to a flat hexagonal cluster. Mutations in BAFF that impair BAFFR activation prevented cluster formation. Our findings demonstrate that ligand binding induces the formation of highly ordered clusters of TNFR1 and BAFFR receptors on the lipid membrane, which is essential for their activation. The TNF family regulates immune responses. Here, the authors used cryo-EM and a lipid membrane system to reveal how TNFR1 and BAFFR receptors form clusters, demonstrating that this higher-order assembly is essential for proper receptor signaling.

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.

Animal models of autoimmunity and human genetic association studies indicate that the dysregulation of B-cell receptor (BCR) signaling is an important driver of autoimmunity. We previously showed that in circulating B cells from primary Sjögren’s syndrome (pSS) patients with high systemic disease activity, protein expression of the BCR signaling molecule Bruton’s tyrosine kinase (BTK) was increased and correlated with T-cell infiltration in the target organ. We hypothesized that these alterations could be driven by increased B-cell activating factor (BAFF) levels in pSS. Here, we investigated whether altered BCR signaling was already present at diagnosis and distinguished pSS from non-SS sicca patients. Using (phospho-)flow cytometry, we quantified the phosphorylation of BCR signaling molecules, and investigated BTK and BAFF receptor (BAFFR) expression in circulating B cell subsets in an inception cohort of non-SS sicca and pSS patients, as well as healthy controls (HCs). We found that both BTK protein levels and BCR signaling activity were comparable among groups. Interestingly, BAFFR expression was significantly downregulated in pSS, but not in non-SS sicca patients, compared with HCs, and correlated with pSS-associated alterations in B cell subsets. These data indicate reduced BAFFR expression as a possible sign of early B cell involvement and a diagnostic marker for pSS.

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE(-/-) mouse. To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE(-/-) mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93(-) CD19(+) B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19(+) B cells, CD4(+) and CD8(+) T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE(-/-) mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93(-) CD19(+) B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment. Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE(-/-) mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.

B cell activating factor (BAFF) is a member of the tumor necrosis factor family that is known to play an important role in B cell activation, proliferation, and differentiation in mammals. However, studies of BAFF in teleosts are very limited and its function, in particular that under in vivo conditions, is essentially unknown. In this study, we conducted in vivo as well as in vitro functional analyses of a BAFF homologue (CsBAFF) from the teleost fish tongue sole (Cynoglossus semilaevis). CsBAFF is composed of 261 residues and shares moderate sequence identities with known BAFFs of other teleosts. CsBAFF expression was most abundant in immune organs and was upregulated during bacterial infection. Purified recombinant CsBAFF (rCsBAFF) bound to tongue sole lymphocytes and promoted cellular proliferation and survival. The results of an in vivo study showed that CsBAFF overexpression in tongue sole significantly enhanced macrophage activation and reduced bacterial infection in fish tissues, whereas knockdown of CsBAFF expression resulted in increased bacterial dissemination and colonization in fish tissues. Furthermore, vaccination studies showed that CsBAFF enhanced the immunoprotection of a DNA vaccine and augmented the production of specific serum antibodies. Taken together, these results provide the first in vivo evidence to indicate that teleost BAFF is an immunostimulator that significantly contributes to the innate antibacterial immune response and vaccine-induced adaptive immune response.

B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers. Antigen escape represents a potential drawback of chimeric antigen receptor T cell (CAR-T) therapy targeting a single tumor-associated antigen. To reduce the risk of antigen escape, here the authors report the design and characterization of a BAFF ligand CAR-T that can recognize three different receptors (BAFF-R, BCMA and TACI), demonstrating in vitro and in vivo cytotoxicity against multiple B cell cancer models.

Various neuroprotective factors have been shown to help prevention of neuronal cell death, which is responsible for the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, most of these therapeutic potentials have been tested by administration of recombinant proteins, transgenic expression or virus vector-mediated gene transfer. Therefore, it remains to be clarified whether any endogenous factors has advantage for neuroprotection in a pathological nervous system. Here we show the role of BAFF-R signaling pathway in the control of neural cell survival. Both B cell-activating factor (BAFF) and its receptor (BAFF-R) are expressed in mouse neurons and BAFF-R deficiency reduces the survival of primary cultured neurons. Although many studies have so far addressed the functional role of BAFF-R on the differentiation of B cells, impaired BAFF-R signaling resulted in accelerated disease progression in an animal model of inherited ALS. We further demonstrate that BAFF-R deficient bone marrow cells or genetic depletion of B cells does not affect the disease progression, indicating that BAFF-mediated signals on neurons, not on B cells, support neural cell survival. These findings suggest opportunities to improve therapeutic outcome for patients with neurodegenerative diseases by synthesized BAFF treatment.

The recent understanding of plasma cell (PC) biology has been obtained mainly from murine models. The current concept is that plasmablasts home to the BM and further differentiate into long-lived PCs (LLPCs). These LLPCs survive for months in contact with a complex niche comprising stromal cells (SCs) and hematopoietic cells, both producing recruitment and survival factors. Using a multi-step culture system, we show here the possibility to differentiate human memory B cells into LLPCs surviving for at least 4 months in vitro and producing immunoglobulins continuously. A remarkable feature is that IL-6 is mandatory to generate LLPCs in vitro together with either APRIL or soluble factors produced by SCs, unrelated to APRIL/BAFF, SDF-1, or IGF-1. These LLPCs are out of the cell cycle, express highly PC transcription factors and surface markers. This model shows a remarkable robustness of human LLPCs, which can survive and produce highly immunoglobulins for months in vitro without the contact with niche cells, providing the presence of a minimal cocktail of growth factors and nutrients. This model should be useful to understand further normal PC biology and its deregulation in premalignant or malignant PC disorders.

Chronic inflammation is an important contributor to the development and progression of metabolic syndrome. Recent evidence indicates that, in addition to innate immune cells, adaptive immune cells have an important role in this process. We previously showed that the serum level of B-cell-activating factor (BAFF) was increased in patients with nonalcoholic steatohepatitis. However, it is currently unknown whether BAFF and BAFF-R (BAFF-R) have a role in lipid metabolism in the liver. To address this issue, the role played by BAFF and BAFF-R signaling in the development of insulin resistance and hepatic steatosis was examined in BAFF-R −/− mice fed a high-fat diet (HFD). Furthermore, the effect of BAFF on lipid metabolism in hepatocytes was analyzed in vitro . BAFF-R −/− mice showed improvements in HFD-induced obesity and insulin resistance. In addition, the number of B cells, levels of serum IgG, and inflammation of visceral fat were reduced in these mice. However, the expression of steatogenic genes and fatty acid deposition in the liver was higher in these mice than in control mice. BAFF was also found to downregulate the expression of steatogenesis genes and enhance steatosis in hepatocytes through BAFF-R. Collectively, these data indicated that, in addition to its known functions in inflammation and glucose metabolism, BAFF has a protective role in hepatic steatosis by regulating lipid metabolism in the liver.

ObjectivesTo evaluate the efficacy and safety of ianalumab (VAY736), a B cell-depleting, B cell activating factor receptor-blocking, monoclonal antibody, in patients with active primary Sjögren’s syndrome (pSS) in a double-blind, placebo-controlled, phase II, single-centre study.MethodsPatients with pSS, EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) ≥6, were randomised to ianalumab single infusion at either 3 mg/kg (n=6), 10 mg/kg (n=12) or placebo (n=9). Outcomes were measured blinded at baseline and weeks 6, 12, 24, and unblinded at end of study (EoS) when B cell numbers had recovered. Clinical outcomes included ESSDAI, EULAR Sjögren’s Syndrome Patient Reported Index (ESSPRI), salivary flow rate, ocular staining score, physician global assessment and patient assessments of fatigue and general quality of life. Laboratory-based measures included circulating leucocyte subsets and markers of B cell activity.ResultsA similar trend showing positive therapeutic effect by ianalumab was observed across the primary clinical outcome (ESSDAI) and all secondary clinical outcomes (ESSPRI, Multidimensional Fatigue Inventory, Short Form-36, global assessments by physician and patient) versus the placebo-treated group. Rapid and profound B cell depletion of long-lasting duration occurred after a single infusion of ianalumab at either dose. Serum Ig light chains decreased, with return to baseline levels at EoS. Changes in some clinical outcomes persisted through to EoS in the higher dose group. Adverse effects were largely limited to mild to moderate infusion reactions within 24 hours of ianalumab administration.ConclusionsOverall results in this single-dose study suggest potent and sustained B cell depletion by ianalumab could provide therapeutic benefits in patients with pSS without major side effects.