title: Recovery of a human natural antibody against the noncollagenous-1 domain of type IV collagen using humanized models.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. ingeschudel@gmail.com.
authors.forename: Inge M
authors.initials: IM
authors.lastname: Worni-Schudel
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. amy.clark@duke.edu.|Durham VA Medical Center, Durham, NC, USA. amy.clark@duke.edu.
authors.forename: Amy G
authors.initials: AG
authors.lastname: Clark
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. tiffany.chien@duke.edu.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Chien
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. kwan-ki.hwang@dm.duke.edu.|Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, USA. kwan-ki.hwang@dm.duke.edu.
authors.forename: Kwan-Ki
authors.initials: KK
authors.lastname: Hwang
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. benny.chen@dm.duke.edu.|Duke Cancer Institute, Duke University Medical Center, Durham, NC, USA. benny.chen@dm.duke.edu.
authors.forename: Benny J
authors.initials: BJ
authors.lastname: Chen
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, NC, USA. mhfoster@duke.edu.|Durham VA Medical Center, Durham, NC, USA. mhfoster@duke.edu.|Duke Cancer Institute, Duke University Medical Center, Durham, NC, USA. mhfoster@duke.edu.
authors.forename: Mary H
authors.initials: MH
authors.lastname: Foster
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D024141:Collagen Type IV / Q000737:chemistry* / Q000276:immunology*; D005260:Female; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D016131:Lymphocyte Subsets / Q000276:immunology; D051379:Mice; D023421:Models, Animal; D008969:Molecular Sequence Data; D009393:Nephritis / Q000276:immunology; D017434:Protein Structure, Tertiary; D017464:Receptors, Complement 3d / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014019:Tissue Donors
chemical_list: D000911:Antibodies, Monoclonal; D024141:Collagen Type IV; D017464:Receptors, Complement 3d