Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.
ABSTRACT: Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion.
Project description:Patients and rodents with Goodpasture's syndrome (GPS) develop severe autoimmune crescentic glomerulonephritis, kidney failure, and lung hemorrhage due to binding of pathogenic autoantibodies to the NC1 domain of the alpha3 chain of type IV collagen. Target epitopes are cryptic, normally hidden from circulating Abs by protein-protein interactions and the highly tissue-restricted expression of the alpha3(IV) collagen chain. Based on this limited Ag exposure, it has been suggested that target epitopes are not available as B cell tolerogens. To determine how pathogenic anti-GPS autoantibody responses are regulated, we generated an Ig transgenic (Tg) mouse model that expresses an Ig that binds alpha3(IV)NC1 collagen epitopes recognized by serum IgG of patients with GPS. Phenotypic analysis reveals B cell depletion and L chain editing in Tg mice. To determine the default tolerance phenotype in the absence of receptor editing and endogenous lymphocyte populations, we crossed Tg mice two generations with mice deficient in Rag. Resulting Tg Rag-deficient mice have central B cell deletion. Thus, development of Tg anti-alpha3(IV)NC1 collagen B cells is halted in the bone marrow, at which point the cells are deleted unless rescued by a Rag enzyme-dependent process, such as editing. The central tolerance phenotype implies that tolerizing self-Ag is expressed in bone marrow.
Project description: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.
Project description:Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.
Project description:Organ deposition of autoantibodies against the noncollagenous-1 domain of the ?3 chain of type IV collagen leads to severe kidney and lung injury in anti-glomerular basement membrane disease. The origin and regulation of these highly pathogenic autoantibodies remains unknown. Anti-?3(IV) collagen B lymphocytes are predicted to mature in vivo ignorant of target antigen because ?3(IV) collagen expression is highly tissue restricted and pathogenic epitopes are cryptic. However, a recent analysis of an anti-?3(IV)NC1 collagen autoantibody transgenic mouse model revealed that developing B cells are rapidly silenced by deletion and editing in the bone marrow. To dissect the role of collagen as central tolerogen in this model, we determined B cell fate in autoantibody transgenic mice genetically lacking ?3(IV) collagen. We found that absence of the tissue target autoantigen has little impact on the fate of anti-?3(IV)NC1 B cells. This implies a more complex regulatory mechanism for preventing anti-glomerular basement membrane disease than has been previously considered, including the possibility that a second antigen present in bone marrow engages and tolerizes anti-?3(IV)NC1 collagen B cells.
Project description:The autoantigen in Goodpasture disease is the noncollagenous domain of alpha3 type IV collagen [alpha3(IV)NC1]. We previously demonstrated that IL-12p40(-/-) mice are protected from experimental autoimmune anti-glomerular basement membrane (anti-GBM) glomerulonephritis, seemingly defining a role for IL-12 in this disease; however, the recent identification of IL-23, a heterodimer composed of IL-12p40 and IL-23p19 subunits, raises the possibility that IL-23, rather than IL-12, may modulate this disease instead. We immunized wild-type, IL-12p35(-/-) (IL-12 deficient, IL-23 intact), IL-12p40(-/-) (deficient in both IL-12 and IL-23), and IL-23p19(-/-) (IL-12 intact, IL-23 deficient) mice with recombinant mouse alpha3(IV)NC1. Wild-type mice developed autoreactivity to alpha3(IV)NC1: Humoral responses, cellular responses, renal histologic abnormalities, leukocyte accumulation, autoantibody deposition, and IL-17A mRNA expression (a cytokine produced by the IL-23-maintained Th17 subset). IL-23 but not IL-12 was detected in the immune system. Regardless of the presence of IL-12, mice deficient in IL-23 were protected, but mice with IL-23 were not. Both IL-23-deficient strains exhibited lower autoantibody titers, reduced cellular reactivity, diminished cytokine production (IFN-gamma [Th1], IL-17A [Th17], TNF, and monocyte chemoattractant protein 1), and less renal disease and glomerular IgG deposition. The deficient responses in the absence of IL-23 were not due to increased regulatory T cells; IL-12p40(-/-) and IL-23p19(-/-) mice did not show increased proportions of CD4(+)CD25(+)FoxP3(+) cells or IL-10 levels early in the immune response. In conclusion, autoreactivity to the Goodpasture antigen is directed primarily by IL-23, absence of which results in hyporeactivity including but extending beyond a deficient Th17 response.
Project description:Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA.
Project description:Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient's plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.
Project description:Background: Goodpasture's disease (GP) is mediated by autoantibodies that bind the glomerular and alveolar basement membrane, causing rapidly progressive glomerulonephritis with or without pulmonary hemorrhage. The autoantibodies bind neoepitopes formed upon disruption of the quaternary structure of ?345NC1 hexamer, a critical structural domain of ?345 collagen IV scaffolds. Hexamer disruption leads to a conformational changes that transitions ?3 and ?5NC1 subunits into immunogens, however, the trigger remains unknown. This contrasts with another anti-GBM disease, Alports' post-transplant nephritis (APTN), where the pathogenic alloantibody binds directly to native NC1 hexamer. The current report includes the first study of antigenic specificity and allo-incompatability in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). Results: The anti-GBM antibodies were found to be directed predominantly against the EA epitope of the ?3 NC1 monomer of collagen IV and developed rapidly in patient serum reaching peak level within 5 weeks. Autoantibody binding to native ?345NC1 hexamer was minimal; however, binding was greatly increased upon dissociation of the native hexamer. There were no polymorphic genetic differences between donor and recipient collagen IV genes which would be predicted to cause a significant NC1 conformational change or to provide a target for antibody binding. Both patient and donor possessed the Goodpasture's susceptibility HLA-allele DRB1 * 1501. Conclusions: The current report includes the first in-depth study of allo-incompatability and antigenic specificity in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). No polymorphic genetic differences were identified between donor and recipient collagen IV genes which would be predicted to provide a target for antibody binding. Furthermore, autoantibody binding to native ?345NC1 hexamer was minimal, increasing greatly upon dissociation of the native hexamer, resembling wild-type GP diseases and marking this as the first example of a post-HSCT conformeropathy.
Project description:We examined the chronic lymphocytic leukemia (CLL) cells of 2457 patients evaluated by the CLL Research Consortium (CRC) and found that 63 (2.6%) expressed immunoglobulin (Ig) encoded by the Ig heavy-chain-variable-region gene (IGHV), IGHV3-21. We identified the amino acid sequence DANGMDV (motif-1) or DPSFYSSSWTLFDY (motif-2) in the Ig heavy-chain (IgH) third complementarity-determining region (HCDR3) of IgH, respectively, used by 25 or 3 cases. The IgH with HCDR3 motif-1 or motif-2, respectively, was paired with Ig light chains (IgL) encoded by IGLV3-21 or IGKV3-20, suggesting that these Ig had been selected for binding to conventional antigen(s). Cases that had HCDR3 motif-1 had a median time from diagnosis to initial therapy comparable with that of cases without a defined HCDR3 motif, as did cases that used mutated IGHV3-21 (n = 27) versus unmutated IGHV3-21 (n = 30). Of 7 examined cases that used Ig encoded by IGHV3-21/IGLV3-21, we found that 5 had a functionally rearranged IGKV allele that apparently had incurred antigendriven somatic mutations and subsequent rearrangement with KDE. This study reveals that CLL cells expressing IGHV3-21/IGLV3-21 most likely were derived from B cells that had experienced somatic mutation and germinal-center maturation in an apparent antigen-driven immune response before undergoing Ig-receptor editing and after germinal-center leukemogenic selection.
Project description:Vaccine induction of broadly neutralizing antibodies (bnAbs) to HIV remains a major challenge. Germline-targeting immunogens hold promise for initiating the induction of certain bnAb classes; yet for most bnAbs, a strong dependence on antibody heavy chain complementarity-determining region 3 (HCDR3) is a major barrier. Exploiting ultradeep human antibody sequencing data, we identified a diverse set of potential antibody precursors for a bnAb with dominant HCDR3 contacts. We then developed HIV envelope trimer-based immunogens that primed responses from rare bnAb-precursor B cells in a mouse model and bound a range of potential bnAb-precursor human naïve B cells in ex vivo screens. Our repertoire-guided germline-targeting approach provides a framework for priming the induction of many HIV bnAbs and could be applied to most HCDR3-dominant antibodies from other pathogens.