Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.
Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.
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DOI：
10.1136/annrheumdis-2015-208356
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发表时间：
2016-10
影响因子：
27.4
通讯作者：
Pitzalis C
中科院分区：
文献类型：
--
作者：
Corsiero E;Bombardieri M;Carlotti E;Pratesi F;Robinson W;Migliorini P;Pitzalis C
风湿性关节炎（RA）的特征是对瓜氨酸抗原的自身耐受性破坏，产生抗瓜氨酸肽/蛋白抗体（ACPA）。目前，瓜氨酸化抗原的性质和来源驱动的体液自身免疫反应内滑膜异位淋巴结构（ELS）是一个关键的未知方面的RA发病机制。在这里，我们描述了从ELS+RA滑膜中分离的病变B细胞的自身反应性B细胞反应。对单个滑膜组织CD 19+细胞进行荧光激活细胞分选（FACS）分选，并克隆VH/VL IG基因以产生来自ELS+/ACPA+RA患者的重组单克隆抗体（rmAb）。RA-rmAbs免疫反应性分析提供了以下关键发现：（1）在含有300种自身抗原的基于chIP的阵列和“瓜氨酸组”多重测定中，对瓜氨酸化组蛋白H2 A/H2 B的强反应性（citH 2A/H2 B）在约40%的RA-rmAbs中观察到，其次是cit-fibrinogen和cit-vimentin;（2）抗citH 2A/H2 B反应性RA-rmAb（但非抗citH 2A/H2 B阴性）选择性识别来自外周血和/或RA关节中性粒细胞的中性粒细胞胞外陷阱（NET）;（3）抗citH 2A/citH 2B和抗NET免疫结合依赖于亲和力成熟，并且在高突变IgVH/VL基因回复到种系序列后完全消失;（4）移植到严重联合免疫缺陷（SCID）小鼠中的ELS+（而非ELS−）RA滑膜组织释放了人抗citH 2A/citH 2B和抗NET抗体，这与ELS的两种主要调节因子CXCL 13和光敏素（LT）-β的移植物内表达有关。我们提供了新的证据表明，在RA关节滑膜ELS内分化的B细胞经常靶向脱亚氨基蛋白，这些蛋白可以在RA滑膜中性粒细胞的NETosis期间产生，包括组蛋白。因此，NET可能代表瓜氨酸化抗原的来源，从而在RA滑膜内激发ACPA自身免疫反应。
Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a ‘citrullinome’ multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS−) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS. We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.
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影响因子：
2.3
作者：
Lee JY;Choi IA;Kim JH;Kim KH;Lee EY;Lee EB;Lee YM;Song YW
通讯作者：
Song YW
影响因子：
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27.4
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27.4
作者：
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通讯作者：
Pitzalis, Costantino
影响因子：
15.9
作者：
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通讯作者：
Holers, VM