Mechanisms of protective versus autoreactive B cells in systemic lupus erythematosus

由抗原特异性记忆B细胞（mBC）和浆母细胞/浆细胞（PB/PC）介导的B谱系记忆在保护性免疫应答和自身免疫性疾病（如系统性红斑狼疮（SLE））中起关键作用。两者还形成了一种防御策略，提供稳定性和可塑性，以适应新的抗原和免疫效应（调节）功能。它还没有研究是否抗原特异性的mBC和PB/PC在自身免疫中遵循类似的原则，保护性免疫，如果一个次要的免疫反应的原则适用于flares.Previously，我们的特点是特定的mBC和PB/PC所产生的主要和次要的人体免疫反应，分别使用钥孔血蓝蛋白（KLH）和破伤风类毒素（TT）作为模型。初次KLH免疫导致初始和交叉反应性mBC的募集，其在免疫后早期分别产生低和高度IG突变的KLH特异性PB。循环KLH特异性PB主要为IgA同种型，同时也存在IgG和IgM抗KLH抗体。这些特征与二次免疫应答不同，我们使用TT疫苗接种进行建模，其中mBC和PB主要是IgG同种型，表现出不同的动力学，是克隆相关的，并且存在与GC反应中的再活化一致的IG突变水平增加。表型和分子数据提供了解决自身反应性B谱系细胞是否遵循接近初次或二次激发的原则的基础。本申请的另一个目标是描述检查点分子对抗原特异性B细胞的作用。为了解决这一问题，本研究将使用（自身）抗原的已建立的多抗原、多色FACS（Ro52、TT、突变瓜氨酸波形蛋白/MCV）来表征SLE患者a）发作期间，b）TT疫苗接种后和c）抗BAFF（贝利木单抗）干预后的特征。离体表型分析将确定（自身）反应性的频率，包括在不同条件下的多反应性细胞，包括检查点分子表达的检测和单细胞水平上VDJ体细胞超突变的分子研究。来自患者与对照的mBC的体外T细胞和BCR非依赖性分化实验将比较性地评估mBC分化成具有其分子和检查点分子特征的（自身）抗原特异性浆母细胞的频率和能力。进一步的研究将使用BCR依赖性体外疫苗接种来确定BCR刺激在自身反应性和保护性PC分化中的作用。这些研究将为SLE患者与对照组中自身反应性和保护性B细胞的调节机制提供新的见解，并有望对（自身）反应性细胞与保护性细胞的作用有新的理解，并可能指导新的治疗选择。

B lineage memory, mediated by antigen-specific memory B cells (mBC) and plasmablasts/plasma cells (PB/PC), plays a key role in both protective immune responses and autoimmune diseases, such as systemic lupus erythematosus (SLE). Both also form a defense strategy with providing stability and plasticity to allow adaptation against new antigens and immune effector (regulatory) functions. It has not been studied whether antigen-specific mBC and PB/PC in autoimmunity follow similar principles as those of protective immunity and if principles of a secondary immune response apply during flares.Previously, we characterized specific mBC and PB/PC arising from a primary and a secondary human immune response using Keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT), respectively, as models. Primary KLH immunization resulted in recruitment of naive and cross-reactive mBC that generated low and highly Ig mutated KLH-specific PB, respectively, early after immunization. Circulating KLH-specific PB were mainly of IgA isotype, while IgG and IgM anti-KLH antibodies were also present. These features are distinct of a secondary immune response, that we model using TT vaccination, in which mBC and PB were mainly of the IgG isotype, appear with different kinetics, are clonally related and present increase levels of Ig mutations consistent with reactivation in GC reaction. The phenotypic and molecular data provide the basis to address whether autoreactive B lineage cells follow principles close to a primary or secondary challenge. Another goal of our application will be to delineate the role of checkpoint molecules on antigen-specific B cells. To address this, the study will use an established multiantigenic, polychromatic FACS (Ro52, TT, mutated citrullinated vimentin/MCV) of (auto)antigens to characterize patients with SLE a) during flares, b) after TT vaccination and c) upon anti-BAFF(belimumab) interventions. Ex vivo phenotypic analyses will identify the frequency of (auto)reactive incl. polyreactive cells under different conditions including the detection of checkpoint molecule expression and molecular studies of VDJ somatic hypermutations on the single cell level. In vitro T cell and BCR-independent differentiation experiments of mBC from patients versus controls will comparatively assess the frequency and capacity of mBC to differentiate into (auto)antigen-specific plasmablasts with their molecular and checkpoint molecule characteristics. Further studies will use BCR-dependent in vitro vaccination to identify the role of BCR stimulation in the differentiation of autoreactive and protective PC. These studies will provide fresh insights into mechanisms regulating autoreactive and protective B cells in SLE patients versus controls and hold promise of a new understanding of the role of (auto)reactive versus protective cells and may guide new treatment options.