Cell Activating Factor Research Articles

Distinct binding mode of BAFF antagonist antibodies belimumab and tabalumab, analyzed by computer simulation.

B cell-activating factor (BAFF) can bind with specific receptors to activate signalling pathways associated with the B cell activation. Belimumab and tabalumab are anti-BAFF (B cell depleting) monoclonal antibodies, with therapeutic efficacy demonstrated for the treatment of autoimmune disorders, while belimumab was approved by FDA in 2011 as a targeted therapy for systemic lupus erythematosus (SLE) and exhibited better clinical outcome than tabalumab. In this investigation, the combination modes of BAFF-belimumab and BAFF-tabalumab complexes were simulated in silico to better understand the reason for the comparative inhibitory difference between belimumab and tabalumab. The structures of belimumab and tabalumab were constructed through homology modelling. The combination mode of BAFF-belimumab complex was analyzed by molecular dynamics simulation, while that of BAFF-tabalumab complex was analyzed by protein-protein docking following the molecular dynamics simulation. Both belimumab and tabalumab were bound with BAFF at the same hydrophobic center to which the natural receptors of BAFF bind as well. Belimumab heavy chain components I51, F54, K58, D100, D101, L102, L103, and P105 and R27, Y30, K49, and S65 of belimumab light chain contribute to the BAFF-belimumab interaction mainly via hydrogen bonds, salt bridges, and hydrophobic interactions. More importantly, belimumab could bind to L83 of BAFF and produce steric hindrance with the adjacent BAFF trimers, while tabalumab could not. Therefore, our results indicated that belimumab has a better clinical outcome compared with tabalumab mainly because belimumab could bind to L83 of BAFF and interfere the formation of a BAFF 60-mer, besides mediating inhibition of the interaction of BAFF with its receptors.

B cells in systemic lupus erythematosus: Targets of new therapies and surveillance tools.

B cell hyperactivity is a hallmark of the complex autoimmune disease systemic lupus erythematosus (SLE), which has justified drug development focusing on B cell altering agents during the last decades, as well as the off-label use of B cell targeting biologics. About a decade ago, the anti-B cell activating factor (BAFF) belimumab was the first biological agent to be licensed for the treatment of adult patients with active yet non-renal and non-neuropsychiatric SLE, to later be expanded to include treatment of pediatric SLE and, recently, lupus nephritis. B cell depletion is recommended as an off-label option in refractory cases, with the anti-CD20 rituximab having been the most used B cell depleting agent to date while agents with a slightly different binding specificity to CD20 such as obinutuzumab have also shown promise, forming a part of the current pipeline. In addition, terminally differentiated B cells have also been the targets of experimental therapies, with the proteasome inhibitor bortezomib being one example. Apart from being promising drug targets, B and plasma cells have also shown promise in the surveillance of patients with SLE, especially for monitoring B cell depleting or B cell altering therapies. Inadequate B cell depletion may signify poor expected clinical response to rituximab, for example, while prominent reductions in certain B cell subsets may signify a protection against flare development in patients treated with belimumab. Toward an era with a richer therapeutic armamentarium in SLE, including to a large extent B cell altering treatments, the challenge that emerges is to determine diagnostic means for evidence-based therapeutic decision-making, that uses clinical information, serological markers, and gene expression patterns to guide individualized precision strategies.

Lymphoid follicular hyperplasia in patients with systemic lupus erythematosus after multiple cycles of rituximab.

Rituximab is indicated in some patients with refractory systemic lupus erythematosus (SLE). Occasionally, this medication is required in chronic form to maintain control of the disease. We described two patients who developed lymphoid follicular hyperplasia (LFH) after multiple cycles of rituximab and evaluated the expression of B cell activating factor belonging to the tumor necrosis factor (TNF) family (BAFF) and its receptors [BAFF-receptor (BAFF-R) and B cell maturation antigen (BCMA)], as possible factors related to lymphoid node enlargement. Two patients with SLE completed six and nine cycles of rituximab (1 g every 2 weeks) indicated each 9 months, achieving remission for 5 and 7 years, respectively, when developed prominent lymphadenopathies. Biopsies showed LFH. Haematological neoplasms were ruled out. Immunohistochemistry showed BAFF overexpression in the follicles, and moderate expression of BAFF-R confined to the mantle zone and BCMA to the germinal centre. Belimumab B cell activating factor belonging to the TNF family (anti-BAFF therapy) was started with positive effects on the clinical condition. LFH can develop in patients with SLE who received multiple cycles of rituximab. BAFF overexpression and moderate expression of BAFF-R and BCMA in lymph nodes were seen. These findings added to the improvement with the change to belimumab could suggest that LFH after cluster of differentiation (CD20) depletion therapy may be associated with a compensatory overexpression of BAFF and its receptors.

Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an ‘experiment of nature’ to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent ‘domino effect’ that impacts stringency of tolerance and B cell fate in the periphery.

Safety and Biological Activity of Rozibafusp alfa, a Bispecific Inhibitor of Inducible Costimulator Ligand and B Cell Activating Factor, in Patients With Rheumatoid Arthritis: Results of a Phase 1b, Randomized, Double-Blind, Placebo-Controlled, Multiple Ascending Dose Study.

ObjectiveTo assess the safety and biological activity of rozibafusp alfa, a first‐in‐class bispecific antibody–peptide conjugate targeting inducible costimulator ligand (ICOSL) and B cell activating factor (BAFF), in patients with rheumatoid arthritis (RA).MethodsThis phase 1b, double‐blind, placebo‐controlled, multiple ascending dose study included 34 patients (18–75 years; 82.4% female) with active RA (Disease Activity Score of 28 joints–C‐reactive protein [DAS28‐CRP] >2.6, on stable methotrexate) randomized 3:1 to receive rozibafusp alfa (n = 26, in four ascending dose cohorts of 70, 140, 210, and 420 mg) or a placebo (n = 8) subcutaneously once every 2 weeks for 10 weeks (six total doses), with 24 weeks of follow‐up. The primary end point was the incidence of treatment‐emergent adverse events (TEAEs). Additional assessments included serum pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity, and RA disease activity measures (DAS28‐CRP, Patient Global Assessment of Disease, and Physician Global Assessment of Disease).ResultsTEAEs occurred in 96.2% and 87.5% of patients receiving rozibafusp alfa and the placebo, respectively; most were mild or moderate in severity. Two (7.7%) patients treated with rozibafusp alfa reported serious TEAEs; none were considered treatment related. Multiple doses of rozibafusp alfa showed nonlinear PK (mean t 1/2 = 4.6–9.5 days) and dose‐related, reversible PD (>90% ICOSL receptor occupancy in 210‐ and 420‐mg cohorts; reduction in naïve B cells and increase in memory B cells in all cohorts). Five (20%) patients developed anti–rozibafusp alfa antibodies, with no apparent impact on safety. RA disease activity showed greater numerical improvement from baseline with rozibafusp alfa versus the placebo in the 210‐ and 420‐mg cohorts.ConclusionMultiple ascending doses of rozibafusp alfa were well tolerated, with PK and PD reflecting dual ICOSL and BAFF blockade. Findings support further clinical evaluation of rozibafusp alfa in autoimmune disease.

Serum B cell activating factor (BAFF) as a biomarker for induction of remission with rituximab in ANCA-associated vasculitis

We examined whether serum B cell activating factor (BAFF) is useful for predicting the remission of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) following rituximab treatment. We used the Birmingham Vasculitis Activity Score (BVAS) 2008 version 3 for the evaluation of 27 patients with AAV 6 months after rituximab treatment. Those with BVAS = 0 achieved remission, whereas those with BVAS score > 0 did not achieve remission. We considered changes in serum BAFF before rituximab treatment, 1 month after treatment, and 6 months after treatment. In the remission group, the serum BAFF increased consistently. In the non-achieved group, serum BAFF was within the normal range. In addition, there was no statistically significant difference between the two groups in terms of serum BAFF before and 1 month after rituximab treatment. However, the serum BAFF level at 6 months after rituximab treatment was significantly higher in the remission group than in the non-achieved group. If serum BAFF does not increase after 6 months of rituximab in AAV, it may be assumed that there are residual B cells and plasma cells in the tissues. Enhanced treatment targeting B cells, including re-administration of rituximab or the addition of other immunosuppressive drugs, should be considered.

Anti-EBNA1 IgG titre is not associated with fatigue in multiple sclerosis patients.

Fatigue is the most frequent symptom in multiple sclerosis (MS), although it is still poorly understood due to its complexity and subjective nature. There is an urgent need to identify reliable biomarkers to improve disease prognosis and therapeutic strategies. Epstein-Barr virus (EBV) is the major environmental risk factor associated with MS aetiology, and trials with EBV-targeted T cell therapies have reduced fatigue severity in MS patients. We investigated whether the serum amount of immunoglobulin (Ig)G-specific for EBV antigens could be a suitable prognostic marker for the assessment of MS-related fatigue. A total of 194 MS patients were enrolled. We quantified EBV nuclear antigen 1 (EBNA1) and EBV viral capsid antigen (VCA) immunoglobulin (Ig) G levels and B cell-activating factor of the tumour necrosis factor family (BAFF) concentration in the serum of patients with relapsing-remitting MS (RRMS) and chronic progressive MS (CPMS), and we analysed their correlation with aspects of fatigue and other clinical disease parameters. A complete EBV seropositivity could be detected in our cohort. After adjusting for confounding variables and covariates, neither EBNA1 nor VCA antibody titres were associated with levels of fatigue, sleepiness, depression, or with any of the clinical values such as expanded disability status scale, lesion count, annual relapse rate, or disease duration. However, patients with RRMS had significantly higher EBNA1 IgG titre than those with CPMS, whereas this was not the case under therapies targeting CD20+ cells. BAFF levels in serum were inversely proportional to anti-EBNA1 IgG. Our results show that EBNA1 IgG titre is not associated with the presence or level of fatigue. Whether the increased EBNA1 titre in RRMS plays a direct role in disease progression, or is only a consequence of excessive B cell activation, remains to be answered in future studies.

Myeloid-derived suppressor cells cross-talk with B10 cells by BAFF/BAFF-R pathway to promote immunosuppression in cervical cancer

Immunosuppression induced by myeloid-derived suppressor cells (MDSCs) is one of the main obstacles to the efficacy of immunotherapy for cervical cancer. Recent studies on the immunosuppressive ability of MDSCs have primarily focused on T cells, but the effect of MDSCs on B cells function is still unclear. In a study of clinical specimens, we found that the accumulation of MDSCs in patients with cervical cancer was accompanied by high expression of B cell activating factor (BAFF) on the surface and high expression of interleukin (IL)-10-producing B cells (B10) in vivo. We found that the absence of BAFF could significantly inhibit tumor growth in a cervical cancer model using BAFF KO mice. Further studies showed that abundant MDSCs in cervical cancer induced B cells to differentiate into B10 cells by regulating BAFF which acted on the BAFF receptor (BAFF-R) of them. In this process, we found that a large amount of IL-10 secreted by B10 cells can activate STAT3 signaling pathway in MDSCs, and then form a positive feedback loop to promote the differentiation of B10 cells. Therefore, this study reveals a new mechanism of BAFF-mediated mutual immune regulation between MDSCs and B cells in the occurrence and development of cervical cancer.

Efficacy and Safety of Masitinib in Corticosteroid-Dependent Severe Asthma: A Randomized Placebo-Controlled Trial

BackgroundMasitinib is an oral tyrosine kinase inhibitor that selectively targets mast cell activity and platelet-derived growth factor receptor (PDGFR) signaling, both of which are implicated in various mechanisms of asthma pathogenesis.ObjectiveAssessment of masitinib as an add-on to standard maintenance therapy as compared with placebo in the treatment of oral corticosteroid-dependent severe asthma.MethodsWe conducted a randomized (2:1), placebo-controlled study of masitinib (6 mg/kg/d) in adults with severe asthma uncontrolled by high dose inhaled corticosteroids and long-acting beta-adrenoreceptor agonists plus oral corticosteroids (OCS) (≥7.5 mg/d). No minimum baseline blood eosinophil count was specified. Following a protocol amendment, the primary endpoint was reduction of annualized severe asthma exacerbation rate adjusted for the overall time on treatment (SAER). Subgroup analysis according to yearly cumulative OCS intake was also performed, a higher OCS dose indicating more severe asthma that is harder to control.ResultsFollowing an average exposure of approximately 13 months, masitinib (n = 240) reduced the SAER by 35% relative to placebo (n = 115) (rate ratio (RR) 0.65 (95% CI [0.47–0.90]; P = 0.010)). For patients with eosinophil ≥150 cell/µL, masitinib (n = 181) reduced SAER by 38% relative to placebo (n = 87); RR 0.62 (95% CI [0.42–0.91]; P = 0.016). Benefit of masitinib was shown to increase in the most severely affected patients (OCS intake of >1000 mg/year), with a significant (P < 0.01) reduction in SAER of 50%–70%. Safety was consistent with the known masitinib profile.ConclusionOrally administered masitinib reduces the risk of asthma exacerbations in severe asthma patients, with an acceptable safety profile. Masitinib may potentially provide a new treatment option for oral corticosteroid-dependent severe asthma.

Single-Cell Sequencing of Immune Cell Heterogeneity in IgG4-Related Disease.

BackgroundThe IgG4-related disease (IgG4-RD) is an immune-mediated disorder with fibrotic manifestations. However, the transcriptional profiles of immune cell subsets at single-cell level are unknown. Herein, single-cell sequencing was used to assess the specific cell subpopulations and pathways in peripheral blood mononuclear cells (PBMCs) of IgG4-RD.MethodsSingle-cell sequencing was performed using the PBMCs from four patients with IgG4-RD and three healthy controls (HCs). Functional enrichment and cell analysis were performed through re-clustering of PBMCs to assess functional pathways and intercellular communication networks in IgG4-RD. Western blot and flow cytometry were used to verify sequencing and functional enrichment results.ResultsFour major cell types and 21 subtypes were identified. Further subclustering demonstrated that plasma B-cell proportions increased with increasing glycolysis/gluconeogenesis activity in IgG4-RD. Re-clustering of myeloid cells showed that EGR1 and CD36 expressions were significantly increased in CD14+ monocytes of IgG4-RD, as validated by Western blot analysis. Moreover, tumor necrosis factor (TNF) production pathways were positively regulated in CD14+ monocytes of IgG4-RD. In vitro stimulation showed that CD14+ monocytes of IgG4-RD could secrete higher levels of TNF-α . Notably, the proportions of CD8 central memory T (TCM) and TIGIT+ CD8 cytotoxic T (CTL) increased in patients with IgG4-RD compared with HCs. Further interaction analysis showed that B cell activation factor (BAFF) signaling pathways were enriched from myeloid cells subsets to B cells.ConclusionThis study enhances the understanding of the cellular heterogeneity and transcriptional features involved in the pathogenesis of IgG4-RD, providing key clinical implications.

Neutrophils recruited to immunization sites initiating vaccine-induced antibody responses by locally expressing BAFF

SummaryNeutrophils played a key role in the innate immune responses. Less is known about whether and how the neutrophils recruited in the immunization sites affecting the vaccine-induced antibody responses. In the process of evaluating the efficacy of an oil-in-water emulsion-formulated vaccine in mice, we found that neutrophils were rapidly and massively recruited to immunization sites but were barely detected in the draining lymph nodes. Interestingly, B cell-activating factor (BAFF) was abundantly expressed in the recruiting neutrophils at a very early stage. The initial neutrophil-derived BAFF firstly brought about the B cell responses in the local part, then subsequently in lymphoid organs. Activated B cells produced more BAFF through TLR9-IRF5 signaling pathway, thereby amplifying the vaccine-induced antibody responses. Suppressing BAFF in the neutrophils could weaken the B cell activation and reduce the antibody production. The data indicate that vaccines endow neutrophils with the potential to orchestrate antibody responses at immunization sites.

B-Cell-Directed Therapies: A New Era in Multiple Sclerosis Treatment.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS) that often progresses to severe disability. Previous studies have highlighted the role of T cells in disease pathophysiology; however, the success of B-cell-targeted therapies has led to an increased interest in how B cells contribute to disease immunopathology. In this review, we summarize evidence of B-cell involvement in MS disease mechanisms, starting with pathology and moving on to review aspects of B cell immunobiology potentially relevant to MS. We describe current theories of critical B cell contributions to the inflammatory CNS milieu in MS, namely (i) production of autoantibodies, (ii) antigen presentation, (iii) production of proinflammatory cytokines (bystander activation), and (iv) EBV involvement. In the second part of the review, we summarize medications that have targeted B cells in patients with MS and their current position in the therapeutic armamentarium based on clinical trials and real-world data. Covered therapeutic strategies include the targeting of surface molecules such as CD20 (rituximab, ocrelizumab, ofatumumab, ublituximab) and CD19 (inebilizumab), and molecules necessary for B-cell activation such as B cell activating factor (BAFF) (belimumab) and Bruton's Tyrosine Kinase (BTK) (evobrutinib). We finally discuss the use of B-cell-targeted therapeutics in pregnancy.

Simultaneous electrochemical immunosensing of relevant cytokines to diagnose and track cancer and autoimmune diseases

This work reports the first dual magnetic beads (MBs) assisted immunoplatform for the simultaneous determination of BAFF (B cell activation factor) and APRIL (a proliferation-induced ligand), two cytokines related to immunity and tumour invasion, growth and metastasis. The electrochemical immunoplatform involves sandwich-type immunoassays implemented on magnetic microparticles functionalized with neutravidin (NA-MBs) or carboxylic groups (HOOC-MBs), and amperometric detection (Eapp = − 0.20 V vs. Ag pseudo-reference electrode) at screen-printed dual carbon electrodes (SPdCE) using the H2O2/hydroquinone (HQ) system. The developed immunosensors provide improved sensitivity, with LOD values of 0.33 and 16.4 pg mL−1 for BAFF and APRIL, respectively, and much shorter assay time that those claimed for ELISA kits and allow their simultaneous determination. The dual immunosensor permits discrimination between healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) or colorectal cancer (CRC) through the determination of both cytokines in 100-times diluted human sera with results in agreement with those provided by the individual ELISA methodologies.

Coding joint: kappa-deleting recombination excision circle ratio and B cell activating factor level: predicting juvenile dermatomyositis rituximab response, a proof-of-concept study

BackgroundThis pilot study’s primary aim was to determine if oligoclonal B cell expansion in children with Juvenile Dermatomyositis (JDM) predicts response to Rituximab therapy. We evaluated: (1) tissue B cell depletion efficacy by measuring the ratio of Coding joint (CJ) to Kappa-deleting recombination excision circle (KREC) DNA, and (2) serum BAFF level upon B cell recovery.MethodsCJ and KREC values were measured via qPCR assessment of serial PBMC stored (− 80 °C) in the CureJM Center’s BioRepository. Serum BAFF was quantitated by Mesoscale® technology. Oligoclonal B cell expansion was defined as a CJ:KREC ≥ 8 prior to Rituximab therapy. Detection of a CJ:KREC ratio ≤ 2.5 in the first sample after Rituximab was designated as adequate B cell depletion. A significant clinical response to therapy was defined as improvement in Disease Activity Score (DAS) by at least 2 points on consecutive visits within the first 12 months of therapy.ResultsSix out of nine children with JDM showed oligoclonal B cell expansion prior to Rituximab (CJ:KREC ≥ 8). Of those 6 patients, 4 had evidence of effective B cell depletion after Rituximab (CJ:KREC ≤ 2.5), and all 4 of those subjects displayed a significant clinical response to Rituximab. Serum BAFF level increased in 8/9 children after Rituximab.ConclusionsIn this proof-of-concept study, JDM patients with oligoclonal B cell expansion prior to Rituximab have more favorable clinical outcomes after Rituximab. We speculate: (1) B cell depletion post-Rituximab predicts JDM clinical response; (2) increased BAFF post-Rituximab may contribute to disease flare.

Circulating C-X-C Motif Ligand 13 as a Biomarker for Early Predicting Efficacy of Subcutaneous Immunotherapy in Children With Chronic Allergic Rhinitis.

BackgroundC-X-C motif ligand 13 (CXCL13) and B cell-activating factor (BAFF) are proven to be involved in inflammatory diseases, but their role in allergic rhinitis (AR) remains unclear. The aim of this study was to investigate the role of serum CXCL13 and BAFF in AR and their clinical values as objective biomarkers to predict the efficacy of subcutaneous immunotherapy (SCIT).MethodsWe prospectively recruited 90 children with AR treated with SCIT and collected their serum specimens before SCIT. One-year follow-up was conducted for all patients, and they were categorized into effective and ineffective groups based on efficacy. The serum concentrations of CXCL13 and BAFF were detected and compared between the two groups. A validation cohort of 52 responders and 26 non-responders were further assessed for both cytokines and serum CXCL13 and BAFF levels were assayed by enzyme-linked immunosorbent assay (ELISA).ResultsEighty children completed the follow-up schedule, and 56 children were categorized into the effective group and 24 children into the ineffective group. The serum levels of CXCL13 in the effective group were clearly higher than those in the ineffective group (P < 0.05). Receiver operating characteristic (ROC) curves revealed the potential values of CXCL13 as a biomarker in predicting the response of SCIT. Further, in the validation cohort, ELISA results demonstrated that serum CXCL13 levels were increased in responders than non-responders (P < 0.05). ROC curves showed good accuracy of serum CXCL13 in predicting the efficacy of SCIT.ConclusionOur discovery–validation study demonstrated that circulating CXCL13 might serve as a novel biomarker to predict the outcome of SCIT in childhood AR. The findings indicated that CXCL13 was involved in the pathological mechanisms of AR and made help to the fundamental therapeutic mechanism of SCIT.

BAFF 60‐mer binding to BAFF receptor 3 utilizes the NF‐κB1 signaling pathway to hyperactivate B cells

B cell activating factor (BAFF) is a critical cytokine for survival, differentiation, proliferation, and antibody production of B cells. Abnormal B cell activation by BAFF has been implicated in diseases such as Systemic Lupus Erythematous (SLE) and rheumatoid arthritis. BAFF is expressed as a membrane bound 3‐mer and then cleaved to its soluble form where it can form a 60‐mer. It is not completely understood how the 3‐mer versus 60‐mer form can differentially activate B cells. Using surface plasmon resonance (SPR), we determined that human BAFF 60‐mer bound strongly to soluble murine BAFF receptor 3 (BR3), Transmembrane activator and CAML interactor (TACI), and B‐cell maturation antigen (BCMA), while human BAFF 3‐mer only bound to soluble murine BR3. We treated purified mouse splenic B cells with BAFF 3‐mer, BAFF 60‐mer, or left untreated in the presence of a control antibody or mBAFFR‐Fc, which only blocks BAFF binding to BR3. A global transcriptomics study revealed that BAFF 60‐mer upregulated genes involved in B cell activation and NF‐κB signaling, compared to BAFF 3‐mer treatment. mBAFFR‐Fc treatment reduced the expression of many of the genes involved in B cell activation and NF‐κB signaling. We next assessed B cell energy metabolism utilizing the Seahorse mitochondrial stress test and glycolysis stress test. B cells treated with BAFF 60‐mer had significantly increased glucose oxidation by oxidative phosphorylation (OXPHOS) and aerobic glycolysis compared to the 3‐mer treated B cells, and this result was attenuated by the addition of mBAFFR‐Fc. Treatment with inhibitors that block the NF‐κB pathway (using BMS) or only the NF‐κB1 pathway (using BI 605906) attenuated OXPHOS and aerobic glycolysis. BAFF 60‐mer treatment increased mitochondrial density and mitochondrial membrane potential (MMP), while decreasing cellular reactive oxygen species (ROS) production indicating generation of healthier mitochondria. Treatment with mBAFFR‐Fc or BMS impaired the BAFF 60‐mer mediated changes to the mitochondrial density, MPP, and ROS. Interestingly, a mitochondrial substrate metabolism assay found that glycerol 3‐PO4 and succinate were significantly utilized by the B cells. While the addition of BAFF, mBAFFR‐Fc, or BMS did not significantly affect glycerol 3‐PO4 usage, succinate utilization significantly increased after BAFF 60‐mer treatment. BAFF 60‐mer treatment increased glucose uptake which was reduced after mBAFFR‐Fc treatment. In contrast, BMS attenuated both the basal and BAFF 60‐mer induced glucose uptake. Altogether, these results show that BAFF 60‐mer binding, via the BR3, hyperactivates B cells by increasing metabolic activity, mitochondrial health, succinate utilization, and glucose uptake as a result of NF‐κB1 activation. Furthermore, our research suggests that BAFF 60‐mer formation may be a potential target for future anti‐BAFF biologics for diseases such as SLE.

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.

P247 Novel therapeutic peptides targeting CD206 modulate ATP-induced release of inflammatory and B cell activating factors in lupus macrophages

Abstract Background/Aims Whilst plasmacytoid dendritic cells, B-cells and damaged tissue resident cells are established as key players in lupus pathogenesis, macrophages have received limited attention. However, in the disease microenvironment these cells might release inflammatory cytokines, thus contributing to the tissue damage. Moreover, macrophages have the potential to synthesise factors that stimulate B-cell proliferation and promote polyclonal immunoglobulin synthesis, underlying the autoantibody component. There is evidence that anti-inflammatory peptides derived from endogenous host defence proteins can modulate the macrophage activation state. Here, we measure the capacity for RP832c, one such peptide that targets cells via the CD206 receptor, to inhibit ATP-stimulated lupus macrophages. Methods Peripheral blood monocytes from SLE patients and healthy controls (HC) (both n = 4) were cultured with 4ng/ml of M-CSF for 7 days, in order to derive macrophages. These cells were stimulated with 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) 0.1mM, a selective P2X7 receptor agonist, to model the effect of damaged tissue, with or without RP832c (10µM). After 24 hours media were removed for assay of inflammatory factors (IL-6), and B-cell promoting growth factors (IL-10 and BAFF) by ELISA, and cells lysed for RNA extraction, profiled by qPCR for CD206 (M2-like), CD86 (classical M1-like), relative to TBP. Results In SLE macrophages there was lower basal IL-6 release than in healthy controls (Mean±SEM: HC 74±44, SLE 4±0.7, P-value &amp;lt;0.029). BzATP induced IL-6 release (basal HC 74±44 vs BzATP stimulated HC 187±63, p-value NS; basal SLE 4±0.7 vs BzATP stimulated SLE 104±46, p-value 0.037). The addition of RP832c treatment inhibted BzATP induced IL-6 (BzATP stimulated HC 187±63 vs BzATP+RP832c HC 126±45, p NS; BzATP stimulated SLE 104±46 vs BzATP+RP832c SLE 27±10, p &amp;lt; 0.078). IL-10 release in SLE lines followed a similar pattern, induced by BzATP and suppressed by co-added RP832c whereas IL-10 release in HC lines did not change with the various treatments. Most interestingly, BAFF was detectable only in BzATP treated lupus macrophage media, and was subsequently suppressed in 3 out of 4 lines by co-addition of RP832c. In certain lupus macrophage lines, BzATP treatment led to induction of CD206, inhibited by RP832c (basal 6.84±0.29, BzATP treated 12.41±1.8, BzATP+RP832c 9.08±1.48, RP832c alone 9.11±1.48 relative expression P &amp;lt; 0.025 for effect of BzATP, P &amp;lt; 0.13 for effect of RP832c), whereas the RP832c treatment appeared to enhance CD86 expression in lupus (enhanced in 3 out of 4 lupus lines, 0 of 4 HC lines). Conclusion These data confirm macrophages as a potential source of inflammatory mediators and B cell-activating factors in lupus. These properties are enhanced by exposure to an ATP-like stimulus. In particular, lupus but not control macrophages had the capacity to synthesis BAFF when exposed to the DAMP stimulus. Moreover, we observed a trend towards suppression of pathogenic responses by RP832c, of potential benefit to patients. Disclosure S. Shah: None. R. Smillie: None. B. Ahmed Abdi: None. R.J. Stratton: None.

Modulation of Immune Responses Against HA1 Influenza Vaccine Candidate by B-lymphocyte Stimulator Cytokine in Mice.

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied. The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 μg/μL of rHA1 were mixed with 2 μg/μL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain. Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose. The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection.

Serum Soluble B Cell-Activating Factor Is a Non-Invasive Biomarker of Antibody-Mediated Rejection in Kidney Allograft With Satisfactory Risk Stratification Performance But Negligible Diagnostic Value.

ObjectivesB cell-activating factor (BAFF), which is critical in the activation and differentiation of B cells, is a candidate diagnostic and predictive biomarker for antibody-mediated rejection (ABMR). We aimed to investigate the value of serum soluble BAFF (sBAFF) for the diagnosis and risk stratification of ABMR after kidney transplantation.MethodsIn the diagnostic study, sBAFF level among ABMR (n = 25), T cell-mediated rejection (TCMR) (n = 14), 4 other pathological lesions (n = 21), and stable allograft function group (n = 15) were compared. In the nested case-control study, kidney allograft recipients with de novo donor-specific antibody (DSA) or ABMR (n = 16) vs. stable allograft function (n = 7) were enrolled, and sBAFF was measured preoperatively, at D7, M1, M3, M6, M9, M12, M18 posttransplant and at allograft biopsy.ResultsThere was no significant difference in sBAFF level at biopsy between ABMR and non-ABMR groups. Longitudinal study showed that the sBAFF levels decreased dramatically at D7 in both groups. The sBAFF level in the DSA group started to increase within M1, while in the stable group, it maintained a low level until M3 and M6. The sBAFF levels of the DSA group were significantly higher than that of the stable group at M1 [1,013.23 (633.97, 1,277.38) pg/ml vs. 462.69 (438.77, 586.48) pg/ml, P = 0.005], M3 [1,472.07 (912.79, 1,922.08) pg/ml vs. 561.63 (489.77, 630.00) pg/ml, P = 0.002], and M6 [1,217.95 (965.25, 1,321.43) pg/ml vs. 726.93 (604.77, 924.60) pg/ml, P = 0.027]. sBAFF levels at M3 had the best predictive value for the DSA/ABMR with the area under the receiver operating characteristic (AUROC) curve value of 0.908. The predictive performance of the maximum (max) change rate from D7 to the peak within M3 was also excellent (AUROC 0.949, P = 0.580).ConclusionWe clarified by a diagnostic study that sBAFF is not a diagnostic biomarker for ABMR in kidney transplantation and revealed by a nested case-control study that sBAFF values at M3 posttransplant and dynamic changes in sBAFF within M3 posttransplant have a good predictive value for the DSA/ABMR. It provides a useful tool for early screening of low-risk patients with negative preoperative DSA for the risk of developing postoperative DSA in kidney allograft recipients.