Recovery of a human natural antibody against the noncollagenous-1 domain of type IV collagen using humanized models.
Recovery of a human natural antibody against the noncollagenous-1 domain of type IV collagen using humanized models.
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DOI：
10.1186/s12967-015-0539-4
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发表时间：
2015-06-06
影响因子：
7.4
通讯作者：
Foster MH
中科院分区：
文献类型：
--
作者：
Worni-Schudel IM;Clark AG;Chien T;Hwang KK;Chen BJ;Foster MH
抗肾小球基底膜肾炎和GoodPasure综合征是由自身抗体(Ab)介导的肾和肺的破坏引起的。AB靶向于α3(IV)胶原的非胶原性1(NC1)结构域，但对AB的来源或结构知之甚少。这种忽视在一定程度上是由于无法通过转化患者的血细胞来恢复单抗。这项研究的目的是评估两种人性化模型对这一目的的适用性。将NOD-SCID-γ免疫缺陷小鼠植入人CD34+造血干细胞(HSC)(HU-HSC)，并用含有Goodstraure表位的α3(IV)NC1胶原免疫肾炎患者外周血白细胞(PBL)(HU-PBL小鼠)。经体内免疫细胞培养和/或扩增后，回收的人B细胞经EB病毒(EBV)转化，筛选抗原(Ag)结合，与人-鼠杂交瘤电融合，亚克隆，逆转录成cDNA后，用聚合酶链式反应(PCR)测定人AbRNA序列。用流式细胞仪检测人B细胞在Hu-PBL小鼠体内的分化情况。对免疫的HU-HSC小鼠产生的与α3(IV)NC1胶原蛋白反应的人源抗体的序列分析表明，它是由未突变的重链和轻链基因编码的。重链互补决定区3是Ag结合的主要决定因素，它含有一些不常见的基序，包括一个N区的高度疏水的四肽，以及由一个独特的人IGHD2-2Ab基因片段编码的双半胱氨酸，而该基因片段缺乏小鼠的相应片段。人类和小鼠自身抗体的比较表明，通过趋同选择可能产生结构相似的小鼠抗体。与HU-HSC模型相比，转化的人B细胞很少从HU-PBL小鼠中恢复，在这种情况下，人B细胞终末分化并失去EBV受体CD21的表达，从而排除了其转化和恢复。HU-HSC小鼠发现，携带未突变的Ig受体的潜在致病B细胞可以在人类前免疫谱系中产生，而不是从人类前免疫谱系中清除。独特的人类基因元件被招募来在这些自身抗体的至少一个子集中产生抗原结合部位，这表明人源化模型可能提供使用传统小鼠模型无法获得的见解。本文的在线版本(doi：10.1186/s12967-0150539-4)包含补充材料，授权用户可以使用。
Anti-glomerular basement membrane nephritis and Goodpasture syndrome result from autoantibody (Ab)-mediated destruction of kidney and lung. Ab target the noncollagenous 1 (NC1) domain of alpha3(IV) collagen, but little is known about Ab origins or structure. This ignorance is due in part to the inability to recover monoclonal Ab by transformation of patients’ blood cells. The aim of this study was to assess the suitability of two humanized models for this purpose. NOD-scid-gamma immunodeficient mice were engrafted either with human CD34+ hematopoietic stem cells (HSC) (Hu-HSC mice) and immunized with alpha3(IV)NC1 collagen containing the Goodpasture epitopes or with nephritis patients’ peripheral blood leukocytes (PBL) (Hu-PBL mice). After in vivo immune cell development and/or expansion, recovered human B cells were Epstein Barr virus (EBV)-transformed, screened for antigen (Ag) binding, electrofused with a mouse–human heterohybridoma, subcloned, and human Ab RNA sequenced by PCR after reverse transcription to cDNA. Flow cytometry was used to assess human B cell markers and differentiation in Hu-PBL mice. Sequence analysis of a human Ab derived from an immunized Hu-HSC mouse and reactive with alpha3(IV)NC1 collagen reveals that it is encoded by unmutated heavy and light chain genes. The heavy chain complementarity determining region 3, a major determinant of Ag binding, contains uncommon motifs, including an N-region somatically-introduced highly hydrophobic tetrapeptide and dual cysteines encoded by a uniquely human IGHD2-2 Ab gene segment that lacks a murine counterpart. Comparison of human and mouse autoantibodies suggests that structurally similar murine Ab may arise by convergent selection. In contrast to the Hu-HSC model, transformed human B cells are rarely recovered from Hu-PBL mice, in which human B cells terminally differentiate and lose expression of EBV receptor CD21, thus precluding their transformation and recovery. Hu-HSC mice reveal that potentially pathogenic B cells bearing unmutated Ig receptors reactive with the NC1 domain on alpha3(IV) collagen can be generated in, and not purged from, the human preimmune repertoire. Uniquely human gene elements are recruited to generate the antigen binding site in at least a subset of these autoantibodies, indicating that humanized models may provide insights inaccessible using conventional mouse models. The online version of this article (doi:10.1186/s12967-015-0539-4) contains supplementary material, which is available to authorized users.
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影响因子：
6.4
作者：
Biswas, Subhabrata;Chang, Hong;Marasco, Wayne A.
通讯作者：
Marasco, Wayne A.
影响因子：
2.7
作者：
Rajesh D;Zhou Y;Jankowska-Gan E;Roenneburg DA;Dart ML;Torrealba J;Burlingham WJ
通讯作者：
Burlingham WJ
影响因子：
4.8
作者：
Hopfer, H;Maron, R;Kalluri, R
通讯作者：
Kalluri, R
影响因子：
4.4
作者：
Clark, Amy G.;Mackin, Katherine M.;Foster, Mary H.
通讯作者：
Foster, Mary H.
DOI：
10.4049/jimmunol.1001152
发表时间：
2010-09-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Luo W;Wang XP;Kashtan CE;Borza DB
通讯作者：
Borza DB