Abstract
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 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.MethodsNOD-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.ResultsSequence 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.ConclusionsHu-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0539-4) contains supplementary material, which is available to authorized users.
Highlights
Anti-glomerular basement membrane nephritis and Goodpasture syndrome result from autoantibody (Ab)-mediated destruction of kidney and lung
We find that human anti-alpha3(IV)noncollagenous 1 (NC1) collagen autoantibodies can be recovered from immunized Hu-hematopoietic stem cells (HSC) mice, and that the antigen binding site in at least a subset of these autoantibodies is generated using uniquely human Ig gene elements
Blood was collected from two subjects with anti-GBM glomerulonephritis within 6 days of renal biopsy and from healthy donors, after informed consent as approved by the Institutional Review Board of Duke University, and peripheral blood leukocytes (PBL) isolated by density gradient (Ficoll-PaqueTM PLUS, GE Healthcare)
Summary
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. Anti-glomerular basement membrane (anti-GBM) glomerulonephritis is a human autoimmune disease that typically presents with acute kidney injury or hematuria. Anti-GBM glomerulonephritis is considered the prototype human autoimmune nephritis because the target antigen is well characterized [5]. Despite considerable advances in defining antigenic epitopes, little is known about the origins and molecular basis of human anti-GBM Ab or their regulatory control. Low titers of anti-GBM IgG that recognize the same epitopes as patient anti-GBM IgG can be identified in serum of healthy individuals using sensitive techniques [7, 8], suggesting their presence in the healthy immune repertoire
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