Regulatory B cells that secrete IL-10 (IL-10+ Bregs) represent a suppressive subset of the B cell compartment with prominent anti-inflammatory capacity, capable of suppressing cellular and humoral responses to cancer and vaccines. B lymphocyte stimulator (BLyS) is a key regulatory molecule in IL-10+ Breg biology with tightly controlled serum levels. However, BLyS levels can be drastically altered upon chemotherapeutic intervention. We have previously shown that serum BLyS levels are elevated, and directly associated, with increased antigen-specific antibody titers in patients with glioblastoma (GBM) undergoing lymphodepletive temozolomide (TMZ) chemotherapy and vaccination. In this study, we examined corresponding IL-10+ Breg responses within this patient population and demonstrate that the IL-10+ Breg compartment remains constant before and after administration of the vaccine, despite elevated BLyS levels in circulation. IL-10+ Breg frequencies were not associated with serum BLyS levels and ex vivo stimulation with a physiologically relevant concentration of BLyS did not increase IL-10+ Breg frequency. However, BLyS stimulation did increase the frequency of the overall B cell compartment and promoted B cell proliferation upon B cell receptor engagement. Therefore, using BLyS as an adjuvant with therapeutic peptide vaccination could promote humoral immunity with no increase in immunosuppressive IL-10+ Bregs. These results have implications for modulating humoral responses in human peptide vaccine trials in patients with GBM.

IntroductionPatients with systemic lupus erythematosus (SLE) with B-lymphocyte stimulator (BLyS) levels ≥ 2 ng/mL are at increased risk of flare. A regression analysis was undertaken to identify routine clinical measures that correlate with BLyS ≥ 2 ng/mL. Efficacy and safety of belimumab 10 mg/kg were examined in patients with BLyS ≥ 2 ng/mL and < 2 ng/mL.MethodsData from BLISS-52 and -76 (N = 1684) were pooled post hoc. A univariate logistic regression was employed to identify factors predictive of baseline BLyS ≥ 2 ng/mL. Factors significant at the 0.05 level then entered a stepwise logistic regression as covariates. Efficacy endpoints included SLE responder index (SRI), ≥ 4-point reduction in Safety of Estrogens in Lupus National Assessment–Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) and risk of severe flare over 52 weeks. Adverse events (AEs) were analyzed for each treatment arm and BLyS subgroup.ResultsBaseline predictors of BLyS ≥ 2 ng/mL included positive anti-Smith (≥ 15 U/mL), low complement (C) 3 (< 900 mg/L), anti-double-stranded DNA (anti-dsDNA) 80–200 and ≥ 200 IU/mL, immunosuppressant usage, proteinuria, elevated C-reactive protein (CRP), and low total lymphocyte count for all patients. Belimumab 10 mg/kg led to significantly greater SRI responses over 52 weeks versus placebo in both BLyS subgroups, though treatment differences were numerically greater at Week 52 in the BLyS ≥ 2 ng/mL group (24.1%, p < 0.0001) compared with BLyS < 2 ng/mL (8.2%, p = 0.0158). Results were similar for ≥ 4-point reduction in SELENA-SLEDAI. Risk of severe flare over 52 weeks was significantly reduced with belimumab 10 mg/kg versus placebo in the BLyS ≥ 2 ng/mL group (p = 0.0002). AEs were similar across treatment arms and BLyS subgroups.ConclusionsPositive anti-Smith, low C3, anti-dsDNA ≥ 80 IU/mL, immunosuppressant usage, proteinuria, elevated CRP, and low total lymphocyte count were predictors of BLyS ≥ 2 ng/mL. Monitoring these factors could identify patients with BLyS ≥ 2 ng/mL who are at risk of flare.

AB0620 B lymphocyte stimulator(BLYS) levels are higher in younger caucasian patients with SLE

The overall aim of this study is to identify clinical and serological features that are associated with B lymphocyte stimulator (BLyS) elevation in a homogeneous Caucasian SLE population and thereby identify patients who are most likely to benefit from BLyS blockade. Patients with SLE (as per ACR criteria) were recruited. Clinical history, disease activity measures and laboratory measures of disease were recorded. BLyS levels were determined by ELISA. BLyS elevation was defined as being higher than the 95th percentile of BLyS levels measured in controls. Patients were divided into two groups: those with elevated BLyS levels (group 1, n = 23) and those with normal BLyS levels (group 2, n = 22). Elevated BLyS levels were significantly associated with patients of younger age and shorter disease duration. In keeping with previous reports, patients with elevated BLyS levels had more active disease (SLEDAI 5.1 vs 0.86, P < 0.001); however, our analysis also demonstrates that BLyS elevation was significantly associated with increased organ damage at 5-year follow-up [Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC/ACR DI) 0.53 vs 0.13, P = 0.012]. Furthermore, the presence of Sm autoantibody significantly predicted elevated BLyS levels in a Caucasian population. BLyS levels were significantly higher in those with musculoskeletal involvement, malar rash, renal disease and evidence of immunological activity. BLyS blockade may be most beneficial if introduced early in the course of disease in young Caucasian patients presenting with renal, musculoskeletal and skin disease in an effort to reduce long-term damage.

To examine the relationship between circulating B lymphocyte stimulator (BLyS) levels and humoral responses to influenza vaccination in systemic lupus erythematosus (SLE) patients, as well as the effect of vaccination on BLyS levels, and to investigate clinical and serologic features of SLE that are associated with elevated BLyS levels. Clinical history, disease activity measurements, and blood specimens were collected from 60 SLE patients at baseline and after influenza vaccination. Sera were tested for BLyS levels, lupus-associated autoantibodies, serum interferon-α (IFNα) activity, 25-hydroxyvitamin D (25[OH]D), and humoral responses to influenza vaccination. Thirty percent of the SLE patients had elevated BLyS levels, with African American patients having higher BLyS levels than white patients (P = 0.006). Baseline BLyS levels in patients were not correlated with humoral responses to influenza vaccination (P = 0.863), and BLyS levels increased postvaccination only in the subset of patients with BLyS levels in the lowest quartile (P = 0.0003). Elevated BLyS levels were associated with increased disease activity, as measured by the SLE Disease Activity Index, physician's global assessment, and Systemic Lupus Activity Measure in white patients (P = 0.035, P = 0.016, and P = 0.018, respectively), but not in African Americans. Elevated BLyS levels were also associated with anti-nuclear RNP (P = 0.0003) and decreased 25(OH)D (P = 0.018). Serum IFNα activity was a significant predictor of elevated BLyS in a multivariate analysis (P = 0.002). Our findings indicate that African American patients with SLE have higher BLyS levels regardless of disease activity. Humoral response to influenza vaccination is not correlated with baseline BLyS levels in SLE patients, and only those patients with low baseline BLyS levels demonstrate an increased BLyS response after vaccination.

Abstract Rheumatoid factor (RF) autoantibodies are associated with Systemic Lupus Erythematosus and other autoimmune disorders. Previous research using AM14 mice, which have a transgene encoding a B cell antigen receptor (BCR) typical of pathogenic RF producing B cells, has demonstrated that these cells are induced to divide by chromatin immune complexes (chromatin-ICs). This work revealed a combined requisite for BCR and Toll-like receptor (TLR) signaling in the onset of RF production., We have recently found that although AM14 B cells stimulated with chromatin-ICs indeed divide, they die rapidly within 48-72 hr of the initial stimulus; by contrast, anti-IgM, CpG, or anti-IgM+CpG stimulated cells continue to proliferate within this time frame. Importantly, we have found that the cytokine BLyS (B lymphocyte stimulator) rescues survival of AM14 B cells proliferating in response to chromatin-ICs. We believe our observations may link the immune complex mediated activation of RF producing B cells with the finding that elevated BLyS levels are often seen in SLE. The ability of BLyS to rescue AM14 B cells may involve signaling through either TACI or BR3, as we have found that both of these BLyS binding receptors are upregulated after chromatin-IC stimulation. Finally, we have begun to examine the apoptotic pathway downstream of chromatin-IC stimulation, and have found that it is cell intrinsic and involves caspases 9, 3, and PARP.

B-lymphocyte stimulator (BLyS) stimulates immunoglobulin production and malignant B-cell growth in Waldenström macroglobulinemia

Serum B-lymphocyte stimulator (BLyS) levels have been found to be elevated in a number of immune disease models. Therefore, we sought to establish whether BLyS levels were elevated in patients with B-cell lymphoproliferative disorders and to determine whether elevated BLyS levels correlated with clinical characteristics of the disease. Specimens were collected from the peripheral blood of individuals diagnosed with B-cell chronic lymphocytic leukemia (B-CLL; n = 70) or from age- and sex-matched patients seen at the same institution (n = 41). Serum BLyS levels were determined by enzyme-linked immunosorbent assay, and sequencing of the BLyS promoter was performed by conventional methods and confirmed by restriction fragment length polymorphism analysis. We found that elevated BLyS levels were more common in patients with familial B-CLL than individuals with sporadic B-CLL or normal controls. Because of this association, we sequenced the BLyS promoter in patients with B-CLL and normal controls and identified a polymorphic site, -871 C/T. We found that the wild-type sequence was significantly underrepresented in patients with familial B-CLL (4%) compared with patients with sporadic B-CLL (30%; P = .01) or controls (24%; P = .04). Furthermore, using a luciferase reporter under control of the BLyS promoter containing either a C or a T at position -871, we found that the reporter construct containing a T at -871 had a 2.6-fold increase in activity (P = .004). Our data suggest serum BLyS levels are elevated in patients with familial B-CLL and that elevated BLyS levels correlate with the presence of a T at -871 in the BLyS promoter.

Role of B-Lymphocyte Stimulator (BLyS) in Waldenstrom's Macroglobulinemia.

To assess the overexpression of B lymphocyte stimulator (BLyS) over time in patients with systemic lupus erythematosus (SLE). Sixty-eight SLE patients were followed up longitudinally for a median 369 days. At each physician encounter, disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index, and blood was collected for determination of the serum BLyS level, blood BLyS messenger RNA (mRNA) level, and cell surface BLyS expression. Twenty normal control subjects underwent similar laboratory evaluations. In contrast to the uniformly normal serum BLyS and blood BLyS mRNA phenotypes in control subjects, SLE patients displayed marked heterogeneity, with 50% and 61% of patients manifesting persistently or intermittently elevated serum BLyS and blood BLyS mRNA phenotypes, respectively. Surface BLyS expression by SLE peripheral blood mononuclear cells was also often increased. Treatment of patients who had elevated serum BLyS levels with intensive courses of high-dose corticosteroids resulted in marked reductions in serum BLyS levels, and tapering of the corticosteroid dosage often resulted in increases in serum BLyS levels. Serum BLyS levels generally correlated with anti-double-stranded DNA (anti-dsDNA) titers (in those with detectable anti-dsDNA titers), but changes in serum BLyS levels did not correlate with changes in disease activity in individual patients. Serum BLyS phenotype did not associate with specific organ system involvement. Dysregulation of BLyS over extended periods of time is common in patients with SLE. Neutralization of BLyS activity with an appropriate BLyS antagonist may be therapeutically beneficial.

To determine whether serum levels of B lymphocyte stimulator (BLyS) are elevated in patients with systemic immune-based rheumatic diseases and correlate with serum Ig levels and/or autoantibody titers. Sera from 185 patients with various systemic immune-based rheumatic diseases (95 with systemic lupus erythematosus [SLE], 67 with rheumatoid arthritis [RA], 23 with other diagnoses) were assayed for BLyS and Ig. In 7 patients who required arthrocentesis of a swollen knee, coincident serum and synovial fluid samples were assayed for BLyS. Medical charts were retrospectively reviewed for elevated autoantibody titers and proteinuria within a 1-month period before or after collection of sera for BLyS and Ig determination. Sera concurrently collected from 48 normal healthy subjects served as controls. Serum BLyS levels were elevated in 38 of 185 patients (21%) and correlated significantly with serum IgG levels. Serum BLyS levels did not correlate with the patients' age, sex, race, or medications, but correlated positively with anti-double-stranded DNA antibody titers among SLE patients and with rheumatoid factor titers among seropositive RA patients. In contrast, serum BLyS levels correlated inversely with nephrotic-range proteinuria among SLE patients. In every case tested, BLyS levels in clinically inflamed synovial fluids were greater than those in simultaneously obtained sera. BLyS may be an important factor in driving polyclonal hypergammaglobulinemia and elevated autoantibody titers in patients with systemic immune-based rheumatic diseases. Local production of BLyS in the joints may contribute to joint pathology. Patients with elevated serum BLyS levels may be ideal candidates for therapeutic targeting of BLyS.