B 淋巴细胞刺激因子（BLyS）调控相关蛋白的研究进展

B淋巴细胞刺激因子是肿瘤坏死因子超家族的成员之一,它同自身免疫疾病的发生发展密切相关,BLyS过度表达会促使B淋巴细胞增殖并分泌自身抗体。BLyS也是治疗自身免疫疾病的重要靶点。关于BLyS的调控涉及了许多蛋白。本文主要就G-CSF、TGF-β1、IFN-γ、IL-10和Poly(I:C)等对BLyS的调控作用作一综述。     

B淋巴细胞刺激因子（BLyS）调控相关蛋白的研究进展

B淋巴细胞刺激因子是肿瘤坏死因子超家族的成员之一,它同自身免疫疾病的发生发展密切相关,BLys过度表达会促使B淋巴细胞增殖并分泌自身抗体。BLys也是治疗自身免疫疾病的重要靶点。关于BLyS的调控涉及了许多蛋白。本文主要就G—CSF、TGF-β1、IFN-γ、IL-10和Voly（I：C）等对BLyS的调控作用作一综述。     

B淋巴细胞表面分子靶向治疗类风湿关节炎的研究进展

类风湿关节炎(rheumatoid arthritis,RA)是一种慢性、系统性的自身免疫性疾病,迄今病因尚不明确,且缺乏针对其安全有效的治疗药物.由于B淋巴细胞在RA致病机制中的重要作用,近年来不断有针对B淋巴细胞上不同靶点的治疗药物推出.这些B淋巴细胞靶向生物制剂包括针对CD20分子的抗CD20单克隆抗体,如rituximab、ocrelizumab和ofatumumab等;针对B淋巴细胞刺激因子(B lymphocyte stimulator,BLyS)及其受体的belimumab和atacicept等以及处于初期研究阶段的抗CD22单克隆抗体和B、T淋巴细胞之间CD40/CD40L共刺激反应阻断剂等.上述靶向制剂的疗效在对RA及其动物模型的治疗中得到了证实,提示将B淋巴细胞作为RA治疗靶点是一项非常有前景的治疗策略.     

人B淋巴细胞刺激因子C端肽的免疫增强作用

用套式PCR从人胎脑cDNA文库中克隆了B淋巴细胞刺激因子C端肽 (C terminalpeptideofBlymphocytestimulator,C BLyS)的cDNA。在大肠杆菌BL2 1CodonPlus (DE3)RIL中以包含体形式表达了C BLyS。对包含体的复性条件进行了摸索 ,建立了C BLyS的透析复性与纯化方法。经复性和纯化的C BLyS可结合其受体B细胞成熟抗原 (Bcellmaturationantigen ,BCMA) 人IgG1Fc融合蛋白 ,刺激体外培养的小鼠脾脏细胞增殖 ,并且可明显增强小鼠对溶菌酶的免疫应答水平。     

BLyS受体TACI、BCMA与人IgG1 Fc融合蛋白在哺乳动物细胞中的表达与鉴定

从人肝脏和肾脏cDNA文库中扩增了人IgG1Fc与B淋巴细胞刺激因子 (BLyS)的受体TACI及BCMA的胞外编码区 ,构建了TACI Fc及BCMA Fc融合表达质粒pSec1 Fc TACI与 pSec1 Fc BCMA ,并使用电穿孔法转染COS 7细胞 ,从 1L无血清培养上清中可纯化得到分泌表达的TACI Fc与BCMA Fc融合蛋白约 2mg。为获得TACI Fc与BCMA Fc的稳定来源 ,构建了TACI Fc与BCMA Fc的CHO稳定表达细胞株。免疫沉淀和ELISA结果显示 ,TACI Fc及BCMA Fc能特异性地结合其配体BLyS而不与BLyS同家族的TNF结合。TACI Fc及BCMA Fc可阻断BLyS对体外培养的小鼠B淋巴细胞的促增殖作用     

BLyS研究进展

B淋巴细胞刺激因子是新发现的属于TNF超家族成员的细胞因子,它能刺激B细胞增殖,分化并分泌IgG,IgA和IgM等免疫球蛋白,在体液免疫反应中发挥着重要作用,过表达BlyS的转基因小鼠呈现自身免疫性疾病症状。本文对B淋巴细胞刺激因子及受体的基因和蛋白结构、生物学功能,可能的作用机理及临床应用前景进行综述。     

rhsBLyS及其突变体对小鼠血清免疫球蛋白分泌及B细胞增殖分化的影响

为了进一步研究所构建的人可溶性B淋巴细胞刺激因子 (hsBLyS)及其突变体hsBY A(Cys14 6→Ala14 6 )和hsBY V(Cys14 6→Val14 6 )在体内的生物活性 ,将 3种重组蛋白注射入BALb C小鼠体内 ,进而分析重组蛋白对小鼠血清免疫球蛋白分泌和脾脏B细胞增殖分化的影响 ,并对 3种蛋白的体内生物活性进行了比较 .结果表明 ,3种蛋白均能促进小鼠血清总IgG和IgM的分泌明显增加 ,统计学检验显示突变体rhsBY V诱导这 2种免疫球蛋白分泌的能力最强 ;3种蛋白还诱导小鼠脾脏增大、脾脏成熟B细胞的相对数量增加 .该高活性突变体hsBY V的获得提示 ,Cys14 6并非BLyS发挥正常功能所必需 ,将Cys14 6突变成Val14 6有可能提高BLyS的生物活性 .     

BLyS对噬菌体随机12肽库的筛选

用B淋巴细胞刺激因子（BLyS）对噬菌体随机12肽库进行亲和淘洗,3轮筛选后阳性噬菌体得到富集。用ELISA鉴定噬菌体克隆,多个阳性克隆测序后得到了同一个小肽序列(RHKIQLRQNIIT)。将该小肽与GST融合,在大肠杆菌中进行表达及纯化,ELISA实验进一步验证了其具有与BLyS特异结合的活性。该小肽有可能成为其天然受体的拮抗剂。      

计算机辅助设计可溶性BLyS受体, eBCMA-Fc 融合蛋白及其在大肠杆菌中的表达

B 细胞成熟抗原 (BCMA)是 B淋巴细胞刺激因子(BLyS)的受体之一．它的胞外区与人IgG1 Fc的融合蛋白eBCMA-Fc,又称为诱饵受体,具有拮抗BLyS的活性．为了设计新的拮抗肽,基于BCMA和Fc的晶体结构,通过计算机图形学技术、分子模拟方法,建立了eBCMA-Fc融合蛋白的三维理论结构．利用均方根位移（root mean square distance, RMSD）对eBCMA-Fc融合蛋白与单体eBCMA、Fc构象差异进行分析．融合蛋白eBCMA-Fc中的eBCMA段与单体eBCMA的主链碳原子间RMSD值为0.036 nm,Fc段与单体Fc的主链碳原子间RMSD值为0.064 nm．结果表明,对比单体,融合蛋白eBCMA-Fc并未因eBCMA与Fc直接连接而发生构象的变化．分子对接方法显示,融合蛋白eBCMA-Fc中的BCMA与BLyS作用,而Fc扮演着稳定BCMA构象的支架作用．为进一步验证上述理论分析,构建eBCMA-Fc融合基因,并将载有eBCMA-Fc融合基因的原核表达质粒转化BL21 (DE3)菌、在细菌中表达．目的蛋白经蛋白A亲和柱纯化大约为36 kD,与理论预测值34 kD相近．免疫印迹表明抗人IgG抗体能够识别eBCMA-Fc融合蛋白．ELISA证实,eBCMA-Fc融合蛋白能够结合BLyS．随着eBCMA-Fc融合蛋白增加,结合BLyS的融合蛋白也相应增加．而对照人IgG,即使在高浓度条件下,也不结合BLyS．此外,eBCMA-Fc 融合蛋白能够抑制BLyS对B细胞肿瘤Daudi细胞的作用．这些研究为下一步设计和筛选BLyS拮抗肽提供了实验基础．     

B淋巴细胞刺激因子

B淋巴细胞刺激因子是新发现的属肿瘤坏死因子族的细胞因子。它是由单核细胞和巨噬细胞持续合成、分泌的,具有特异的激活B淋巴细胞,诱导B淋巴细胞增殖、分化为浆母细胞、分泌抗体,以及促进成熟B淋巴细胞在体内的存活等功能。在缺少B淋巴细胞刺激因子的情况下,可导致成熟B淋巴细胞缺乏。而异常过高表达B淋巴细胞刺激因子引起自身免疫性疾病,系统性红斑狼疮样症状。     

B lymphocyte stimulator regulates adaptive immune responses by directly promoting dendritic cell maturation

B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.     

Construction and function of two Cys146-mutants with high activity, derived from recombinant human soluble B lymphocyte stimulator

B lymphocyte stimulator (BLyS) is a novel member of tumor necrosis factor (TNF) ligand family that is important in B cell maturation and survival. Previous studies were almost related to the function or mechanism of its wild type. Here, we constructed two site-directed mutants of the recombinant human soluble BLyS, the BY-A and BY-V, and found that BY-V ranked the highest whenever in the process of promoting proliferation of B lymphocytes in vitro or stimulating total serum IgG and IgM secretion in vivo. Besides, assays for the biological responses of human leukemic cell lines to BLyS, BY-A and BY-V demonstrated that they could suppress the proliferation of Raji cells but promote the growth of THP-1. The discovery of BY-V with high activity will help come to a conclusion that the mutation of Cys146 to Val146 might improve the biological activity of BLyS.     

Enhanced immunogenicity of pneumococcal surface adhesin A (PsaA) in mice via fusion to recombinant human B lymphocyte stimulator (BLyS)

Background  

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of ligands that mediates its action through three known receptors. BLyS has been shown to enhance the production of antibodies against heterologous antigens when present at elevated concentrations, supporting an immunostimulatory role for BLyS in vivo.     

Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice

The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.     

Immune opsonins modulate BLyS/BAFF release in a receptor-specific fashion

TNF ligand superfamily member 13B (B lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and Ig production. While the soluble form of BLyS/BAFF is thought to be the primary biologically active form, little is known about the regulation of its cleavage and processing. We provide evidence that Fcgamma receptor cross-linking triggers a rapid release of soluble, biologically active BLyS/BAFF from myeloid cells. Surprisingly, this function is primarily mediated by FcgammaRI, but not FcgammaRIIa as defined by specific mAb, and can be initiated by both IgG and C reactive protein as ligands. The generation of a B cell proliferation and survival factor by both innate and adaptive immune opsonins through engagement of an Fcgamma receptor, which can also enhance Ag uptake and presentation, provides a unique opportunity to facilitate Ab production. These results provide a mechanism by which Fcgamma receptors can elevate circulating BLyS levels and promote autoantibody production in immune complex-mediated autoimmune diseases.     

B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels

Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and ΔBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and ΔBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and ΔBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and ΔBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.     

Structural basis of BLyS receptor recognition

B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor (TNF) superfamily, is a cytokine that induces B-cell proliferation and immunoglobulin secretion. We have determined the three-dimensional structure of BLyS to 2.0 A resolution and identified receptor recognition segments using limited proteolysis coupled with mass spectrometry. Similar to other structurally determined TNF-like ligands, the BLyS monomer is a beta-sandwich and oligomerizes to form a homotrimer. The receptor-binding region in BLyS is a deeper, more pronounced groove than in other cytokines. The conserved elements on the 'floor' of this groove allow for cytokine recognition of several structurally related receptors, whereas variations on the 'walls' and outer rims of the groove confer receptor specificity.     

Effects of human soluble BAFF synthesized in Escherichia coli on CD4+ and CD8+ T lymphocytes as well as NK cells in mice

B cell-activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, zTNF-4, or THANK) is an important survival factor for B lymphocytes. In this study, we injected mouse abdominal cavity with human soluble BAFF (hsBAFF, 0.01, 0.1, 0.5, 2 mg/kg body mass) synthesized in Escherichia coli. On the 8th day after injection, we investigated the effects of hsBAFF on immune functional activities of splenic B lymphocytes, CD4(+) and CD8(+) T lymphocytes and natural killer (NK) cells in mice. The results showed that B lymphocyte proliferation significantly increased in hsBAFF-treated groups with dosages of 0.1 mg/kg (p<0.05), 0.5 and 2 mg/kg (p<0.01). We observed a dose-dependent increase of CD4(+) T lymphocyte percentage and significantly higher values in 0.5 and 2 mg/kg hsBAFF-treated groups (p<0.05 and p<0.001, respectively) compared to control group, but CD8(+) T lymphocyte percentage remained unchanged. The ratio of CD4(+) to CD8(+) T lymphocytes rose with increasing hsBAFF dosage (p<0.05 for 2 mg/kg hsBAFF vs. control). Significantly stronger NK cell activities were found in 0.5 and 2 mg/kg hsBAFF-treated groups (p<0.05). The main finding of this study is that the hsBAFF can enhance immune responses in the body by increasing B lymphocyte and CD4(+) T lymphocyte function as well as elevating NK cell activity.     

Cutting edge: BLyS enables survival of transitional and mature B cells through distinct mediators

These studies characterize BLyS responsiveness and receptor expression among transitional and mature peripheral B cells. The results show a maturation-associated increase in BLyS binding capacity that reflects differential expression patterns of the three BLyS receptors. Accordingly, BLyS administration enlarges only late transitional and mature peripheral B (MB) cell compartments. Furthermore, bromodeoxyuridine labeling and cell cycle analyses show these effects are mediated through enhanced proportional survival of cells traversing the T2, T3, and MB cell stages, rather than by causing proliferation or slowing transit within these subsets. Despite similar effects on survival, BLyS up-regulates the antiapoptotic genes A1 and bcl-x(L) in MB cells but not immature B cells. Together, these findings show that, while BLyS influences B cell survival in several peripheral differentiation subsets, the downstream mediators differ, thus providing the first direct evidence for an established B lineage survival system whose intermediates change as B cells mature.