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

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.

A murine Ig light chain transgene reveals IGKV3 gene contributions to anti-collagen types IV and II specificities

“…Sections of kidneys frozen in OCT medium were stained for mouse kappa Ig and images acquired essentially as described (direct IF) (Worni-Schudel et al, 2015), with the following modification: sections were blocked with the avidin-biotin blocking kit (Life Technologies, Frederick, MD, USA), and deposited mLCV3-Tg detected using biotin-conjugated goat-anti-mouse-Ig-kappa (Southern Biotech) and streptavidin-Alexa Fluor 488 (Life Technologies). For indirect IF, sections of kidney from NOD-scid-gamma immunodeficient mice (Jackson Laboratory) were incubated with supernatant from mLCV3-Tg/kKO mouse splenocytes cultured with R848 + CpG as described above, or with positive control mouse anti-alpha3(IV)NC1 IgG MAB3 (Eurodiagnostica), and mouse kappa detected as described above.…”

Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen

“…Hu-HSC mice were generated essentially as described (Worni-Schudel et al, 2015), with the following modifications; 3–4 hours after irradiation (100 cGy, X-RAD 320, Precision X-ray, North Branford, CT) 9.5–10 week old NSG recipients were intravenously injected with purified human CD34+ HSC (AllCells LLC, Alameda, CA), using 2.5–10×10 4 CD34+ HSC per mouse (Pearson et al, 2008; Shultz et al, 2005). All studies were approved by the Duke University Institutional Review Board and Animal Care and Use Committee and conform to institutional standards and to the National Institutes of Health guide for the care and use of Laboratory animals.…”

“…Single-cell suspensions of cells were analyzed by flow cytometry as described (Clark et al, 2011; Worni-Schudel et al, 2015), using fluorescent-conjugated antibodies (Becton Dickinson-Pharmingen, San Jose, CA). Cells were analyzed on a FACSCalibur or BD CANTO apparatus (Becton Dickinson-Pharmingen) and results analyzed using FlowJo (Treestar, Ashland, OR, USA), with live cells gated on forward and side scatter.…”

“…Harvested human B cells were transformed and fused essentially as described (Bonsignori et al, 2011; Worni-Schudel et al, 2015). Briefly, cells were cultured in bulk overnight in complete medium supplemented with Cyclosporin A, CpG oligonucleotides, checkpoint 2 kinase inhibitor, and EBV B95-8 virus suspension (ATCC, Manassas, VA, USA), and then seeded into 96-well plates and refed weekly, with cyclosporin A and CpG oligonucleotides withdrawal at two weeks.…”

“…Human immunoglobulin (Ig) class, light chain type, Ig concentration, and anti-alpha3(IV)NC1 collagen Ab in serum and cell supernatants were detected by ELISA, as described (Worni-Schudel et al, 2015; Zhang et al, 2008). Results for binding to antigen [bovine alpha3(IV)NC1 collagen (Eurodiagnostica, Malmo, Sweden)] were recorded as OD on antigen minus OD on wells coated with diluent only (6M guanidine-HCL), using as controls mouse anti-alpha3(IV)NC1 collagen IgG (Mab3) (Eurodiagnostica) and Goodpasture patient serum.…”