Recovery of a human natural antibody against the noncollagenous-1 domain of type IV collagen using humanized modelsReport as inadecuate

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Journal of Translational Medicine

, 13:185

Immunobiology andimmunotherapy


BackgroundAnti-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 alpha3IV 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.

MethodsNOD-scid-gamma immunodeficient mice were engrafted either with human CD34+ hematopoietic stem cells HSC Hu-HSC mice and immunized with alpha3IVNC1 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.

ResultsSequence analysis of a human Ab derived from an immunized Hu-HSC mouse and reactive with alpha3IVNC1 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.

ConclusionsHu-HSC mice reveal that potentially pathogenic B cells bearing unmutated Ig receptors reactive with the NC1 domain on alpha3IV 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.

Electronic supplementary materialThe 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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Author: Inge M Worni-Schudel - Amy G Clark - Tiffany Chien - Kwan-Ki Hwang - Benny J Chen - Mary H Foster


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