Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.
Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.
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DOI：
10.1016/j.jaci.2015.05.029
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发表时间：
2016-01
期刊：
影响因子：
--
通讯作者：
Boyd SD
中科院分区：
文献类型：
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作者：
Hoh RA;Joshi SA;Liu Y;Wang C;Roskin KM;Lee JY;Pham T;Looney TJ;Jackson KJL;Dixit VP;King J;Lyu SC;Jenks J;Hamilton RG;Nadeau KC;Boyd SD
食物过敏患者中过敏原特异性B细胞的频率、细胞表型、表位特异性和克隆多样性尚未完全了解，但具有重要的致病和治疗意义。在免疫治疗前和免疫治疗期间，对花生过敏原特异性B细胞群及其抗体的序列和结合活性进行表征。结合荧光标记的Ara h 1或Ara h 2的B细胞通过流式细胞术分选从基线时的18名患者和治疗期间的13名患者中进行表型分析和分离。采用ELISA、Western blotting和肽表位作图法对57株来源于变应原结合单B细胞的单克隆抗体进行了鉴定。B细胞库的深度测序鉴定了变应原特异性B细胞克隆的其他成员。在基线过敏患者血液中，中位过敏原结合B细胞频率为B细胞的0.0097%（Ara h 1）或0.029%（Ara h 2），在免疫治疗期间约高出3倍。57个过敏原特异性细胞中有5个属于含有IgE表达成员的克隆。几乎所有的过敏原特异性抗体突变，并结合构象和线性过敏原表位进行检测。在免疫治疗中观察到克隆的IgG4成员的体细胞突变增加，而克隆中的IgE突变水平没有增加。通过抗原特异性流式分选分离的大多数花生变应原结合B细胞表达突变的和同种型转换的抗体。免疫疗法增加了它们在血液中的频率，甚至狭义的过敏原表位也被患者体内许多不同的B细胞克隆识别。结果还表明，口服免疫治疗可以刺激过敏原特异性IgG4的体细胞突变。
The frequencies, cellular phenotypes, epitope specificity and clonal diversity of allergen-specific B cells in food allergic patients are not fully understood, but are of major pathogenic and therapeutic significance. To characterize peanut allergen-specific B cell populations, and the sequences and binding activities of their antibodies, before and during immunotherapy. B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by flow cytometric sorting from 18 patients at baseline and 13 during therapy. 57 monoclonal antibodies derived from allergen-binding single B cells were evaluated by ELISA, Western blotting and peptide epitope mapping. Deep sequencing of B cell repertoires identified additional members of the allergen-specific B cell clones. Median allergen-binding B cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline allergic patient blood, and were approximately three-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, while IgE mutation levels in the clone did not increase. Most peanut allergen-binding B cells isolated by antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly-defined allergen epitopes are recognized by numerous distinct B cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.