Collagen 3(IV)

Central Tolerance Regulates B Cells Reactive with Goodpasture Antigen α3(IV)NC1 Collagen

This information is current as Ying Zhang, Susan C. Su, Douglas B. Hecox, Graham F. of September 29, 2021. Brady, Katherine M. Mackin, Amy G. Clark and Mary H. Foster J Immunol 2008; 181:6092-6100; ; doi: 10.4049/jimmunol.181.9.6092

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Central Tolerance Regulates B Cells Reactive with Goodpasture Antigen ␣3(IV)NC1 Collagen1

Ying Zhang, Susan C. Su, Douglas B. Hecox, Graham F. Brady, Katherine M. Mackin, Amy G. Clark, and Mary H. Foster2

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 ␣3 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 ␣3(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 ␣3(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 Downloaded from 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-␣3(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. The Journal of Immunology, 2008, 181: 6092–6100. http://www.jimmunol.org/ atients and rodents with Goodpasture syndrome (GPS)3 tients lack the collagen epitopes targeted in GPS due to mutations develop rapidly progressive renal failure due to necrotiz- in one of the genes encoding the ␣-chains (␣3, ␣4, or ␣5) of type P ing crescentic glomerulonephritis and potentially fatal pul- IV collagen, such that introduction of the epitopes in the trans- monary hemorrhage due to alveolitis. In a subset of patients injury planted kidney leads to an antiforeign (antiallotypic) immune re- is restricted to the kidneys, referred to as anti-glomerular basement sponse. The sites of organ injury are in a large part explained by membrane (GBM) nephritis. The pathologic and diagnostic hall- the highly tissue-restricted expression of the ␣3(IV) collagen marks are the presence of circulating anti-GBM IgG autoantibod- chain, which is limited to basement membranes of the glomerulus, ies and linear deposition of IgG along the glomerular and alve- renal tubules, alveolus, cochlea, anterior lens capsule, Descemet’s by guest on September 29, 2021 olar capillary basement membranes. The mainstay of therapy is membrane, ovary, and testis (4). The exact mechanism by which an aggressive regimen of corticosteroids, cyclophosphamide, GPS Ig deposits in lung and kidney remains under investigation. and plasmapheresis to broadly suppress the immune system. The epitopes recognized by pathogenic Abs are conformational Disease-specific mechanism-based and less toxic interventions and normally buried within the native NC1 hexamer, such that are desirable, the rational design of which requires better un- patient sera IgG preferentially binds to dissociated, as compared derstanding of the origins and regulation of anti-GBM autore- with native, Ag (5–8). It is proposed that in diseased individuals Ig activity and of the genetic and environmental factors that sub- binding is facilitated by limited NC1 hexamer cross-linking and vert those mechanisms in disease. environmental toxins that promote in vivo exposure of normally Pathogenic autoantibodies from GPS patients bind the C-termi- cryptic epitopes (9–11). ␣ nal globular noncollagenous domain 1 (NC1) of the 3-chain of It is currently unclear whether potential tolerizing epitopes are type IV collagen, ␣3(IV)NC1 (1, 2). Similar epitopes are targeted accessible to developing and circulating lymphocytes in healthy by alloantibodies in a subset of patients with Alport nephritis who individuals, or whether epitope exposure is essential for lympho- receive renal allografts (1–3). The native kidneys of Alport pa- cyte activation and disease initiation in patients. Low levels of anti-␣3(IV)NC1 IgG are reported in normal serum (12), and CD4ϩ and CD8ϩ T cells reactive with ␣3(IV)NC1 can be isolated from Department of Medicine and Durham Veterans Affairs Medical Center Research Ser- vice, Duke University Medical Center, Durham, NC 27710 healthy individuals as well as patients (13–15), the latter despite Received for publication June 26, 2008. Accepted for publication August 20, 2008. the presence of Ag in the thymus (15, 16). These findings have been interpreted to support a model wherein T and B cell epitopes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance are cryptic, such that anti-␣3(IV)NC1 B and T cells are not toler- with 18 U.S.C. Section 1734 solely to indicate this fact. ized in vivo. Disease is presumed to result from activation of “ig- 1 This work was supported in part by the National Institutes of Health (T32AI07217 norant” lymphocytes by an unknown stimulant coupled with Ag to D.B.H.; R01DK47424 to M.H.F.), a Veterans Affairs Medical Center Merit Award (to M.H.F.), the Durham Veterans Affairs Medical Center Research Service and In- exposure and end organ susceptibility. stitute for Medical Research, Duke University Undergraduate Research Support To determine whether and how Goodpasture-like anti-collagen Grants (to S.C.S.), and a grant from the American Society of Nephrology (to M.H.F.). B cells are regulated in vivo and how they escape regulation to 2 Address correspondence and reprint requests to Dr. Mary H. Foster, Nephrology cause disease, we developed a novel anti-␣3(IV)NC1 collagen Ig Research, Duke University Medical Center, Box 103015, Durham, NC 27710. E-mail address: [email protected] transgenic (Tg) model. The Ig Tg model is useful to study B cell 3 Abbreviations used in this paper: GPS, Goodpasture syndrome; GBM, glomerular tolerance because it greatly increases the frequency of B cells of basement membrane; Tg, transgenic; B6, C57BL/6. the defined specificity and permits determination of their fate in www.jimmunol.org The Journal of Immunology 6093

vivo. Our strategy involved identification of a murine anti- sequences, and germline J␬ genes. The forward primer, 5Ј-AAAAAATT ␣3(IV)NC1 mAb that bound a pathogenic epitope, cloning of the GTATTTAAGAAGGGTCCTTTGA-3Ј, lies 567 nucleotides upstream of Ab H and L chain genes and regulatory sequences, generation of the Vk gene and includes the regulatory elements. This primer was de- ϩ signed based on the mouse germline Vk sequence most closely matching Ig H L Tg mice, and evaluation of the resulting B cell phenotype. the anti-␣3(IV)NC1 mAb L chain sequence, using mouse genomic BLAST The Tg BCR in this model targets a collagen epitope recognized by (http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.html). The reverse pathogenic IgG in the sera of GPS patients. Analysis of the fate of primer, 5Ј-ATAGTCGACAGACCACGCTACCTGCAGTCAGAC-3Ј, lies Tg B cells indicates that collagen-reactive cells are tolerized and 498 bp downstream of Jk5 (22) and includes a unique SalI restriction site (underlined). This 2.3-kb gene fragment was then ligated to a 4.5-kb not immunologically ignorant. Although autoantibodies are genomic fragment containing the ␬ enhancer and C␬ gene, provided by Dr. present at low levels in serum and recoverable by fusion of Tg K. Rajewsky, Harvard University, Boston, MA (22), via Dr. T. Imanishi- mouse spleens, Tg mice have significantly fewer splenic B cells Kari, Tufts-New England Medical Center, Boston, MA. than non-Tg littermates and have evidence of L chain editing. Ex vivo expression of the anti-␣3(IV)NC1 H and L chains Elimination of endogenous (non-Tg) Ab and T cells by establish- ing Tg mice deficient in Rag enzyme leads to near-complete Ig and H chain-loss variant myeloma (J558L) cells carrying the ␭1 L chain were B cell depletion that is evident in bone marrow as well as spleen. cotransfected with the coselectable marker, pSV2-neo, encoding amino- ␣ glycoside phosphotransferase and with either NotI-linearized H chain con- Collectively these findings indicate that anti- 3(IV)NC1 B cells struct or with both linearized H and L chain constructs, using electropo- are regulated in vivo, suggesting that the reactive epitopes are ex- ration with a Gene Pulsar apparatus (Bio-Rad) in HT medium (84% RPMI posed and that either ␣3(IV)NC1 collagen or a cross-reactive 1640, 10% FBS; HyClone, Thermo Fisher Scientific) and 1ϫ nonessen- self-Ag is tolerogenic. The central tolerance in Rag-deficient Ig Tg tial amino acids, hypoxanthine-thymidine, sodium pyruvate, HEPES buffer solution, L-glutamine, penicillin-streptomycin, respectively; all mice further indicates that tolerogen is encountered in bone Downloaded from reagents were obtained from Invitrogen unless otherwise noted. Trans- marrow. fected cells were selected in medium containing 400 ␮g/ml aminogly- coside G-418 (Invitrogen), screened for secreted IgM,␬ Ig by ELISA, Materials and Methods and subcloned by limiting dilution. Immunization and generation of hybridomas Animals Immunization of SJL and C57BL/6 (B6) mice with 10 ␮g of recombinant Tg mice carrying both the H and L chain constructs were generated via human ␣3(IV)NC1 Ag (17) in Freund’s adjuvant, followed by 2–8 boosts http://www.jimmunol.org/ with recombinant human or purified bovine Ag (Wieslab) in IFA, and standard techniques by the Duke Comprehensive Cancer Center Trans- generation of anti-␣3(IV) NC1 hybridomas by fusion of unmanipulated genic Mouse Facility, Duke University. Founders were generated in hybrid splenocytes with murine Sp2 myeloma cells are described elsewhere (18). strain B6SJLF1/J obtained from The Jackson Laboratory. Offspring were Hybridoma culture supernatants were screened for secreted Ig, IgM allo- genotyped by PCR using transgene-specific forward and reverse primers type, and binding to purified ␣3(IV)NC1 collagen by solid-phase ELISA as and Tg lines established on the B6 background. The experiments described described below. Described below is selection of a prototypic Goodpasture here were conducted on mice of either sex hemizygous for the introduced mAb based on inhibition of Ag binding by multiple human Goodpasture transgenes and reared under conventional specific pathogen-free condi- patients’ sera IgG. Hybridomas producing anti-␣3(IV)NC1 collagen Ab tions. B6 breeders, BALB/c (IgMa allotype) controls, and mice carrying a and hybridomas subsequently generated from Tg mice and producing Tg H targeted mutation in Rag1 on the B6 background (RAG-KO) were obtained chain IgMa allotype were subcloned either by limiting dilution or by cell from The Jackson Laboratory. The care and use of all experimental animals sorting at the Duke University flow cytometry facility. were in accordance with institutional guidelines, and all studies and pro- by guest on September 29, 2021 cedures were approved by the Animal Care and Use Committees of Duke Production of constructs containing the anti-␣3(IV)NC1 mAb University and the Durham Veterans Affairs Medical Center. VDJ or VkJk cDNAs and regulatory sequences Cell and tissue staining To identify the mAb-rearranged variable region sequences of our proto- Single cell suspensions were prepared, stained, and analyzed by flow cy- typic anti-␣3(IV)NC1 collagen Ig, cDNA was generated from hybridoma tometry as described (23, 24). Direct immunofluorescence staining of kid- RNA and rearranged VDJ and VkJk genes amplified by PCR using pro- neys was performed as described (19). miscuous upstream V gene primers and downstream IgM and ␬ constant region primers, as described (19). Complete H and L chain variable region Ab isotype, allotype, and binding specificity sequences are described in detail elsewhere (18). To produce a DNA construct encoding the anti-␣3(IV)NC1 H chain V Ig concentrations and isotype- and allotype-specific binding in serum and region with appropriate tissue-specific expression of secreted and trans- in culture supernatants from B cells, transfectants, and hybridomas were membrane IgM, we took advantage of existing genomic DNA fragments determined by ELISA as described (23). Collagen binding activity was containing Ig regulatory elements and signal sequences (20) and a strategy determined by ELISA. In brief, Immulon II plates were coated overnight at previously described in our laboratory to generate the 238H H chain Tg 4°C with bovine ␣3(IV)NC1 collagen (Wieslab) diluted in 6M guanidine- (21), with minor modifications. First, the mAb VDJ was amplified from HCl. After blocking with 3% BSA in PBS, serum or supernatant was in- cDNA using forward and reverse primers based on mouse germline J558.3 cubated at 50 ␮l per well at room temperature for 1 h, washed once with

(VH) and JH2 sequences (GenBank accessions AF303834 and X63167) and PBS/0.05% Tween and twice with PBS, followed by alkaline phosphatase- designed to insert unique SfuI(AsuII) and SauI(Bsu36I) restriction sites at conjugated anti-mouse IgM or IgG (Boehringer Mannheim) diluted in Ј the signal peptide-VH junction and within JH2, respectively: 5 VH-SFU PBS/0.1% BSA. Binding was detected using p-nitrophenyl phosphate sub- (5Ј-CGT TCG AAG TCC AGC TGC AAC AGT CTG GAC CTG-3Ј) and strate and assays were monitored at 405 nm. Control Ig included anti- 3ЈJH2-SAU (5Ј-TCA CCT GAG GAG ACT GTG AGA GTG GTG CC-3Ј). ␣3(IV)NC1 IgG mAb (Wieslab) and transfectant IgM. Results were recorded We next used a 3-kb construct containing 5Ј regulatory sequences (700 bp), as the mean sample OD after subtraction of mean OD on diluent-coated ␮ 238H VDJ (320 bp), and the JH germline genes with Ig enhancer (2 kb), plates and subtraction of OD blank (diluent without Ig). Ag binding by previously generated in our laboratory and carried in plasmid Bluescript transgene-encoded Ig was confirmed using biotin-labeled anti-allotype (Stratagene; Ref. 21). The 238H VDJ was replaced with the anti- (IgM-a)-specific second-step reagents (BD Biosciences) detected with avi- ␣3(IV)NC1 VDJ by a two-step cloning method, taking advantage of the 5Ј din-alkaline phosphatase (Southern Biotechnology Associates). For assays Sfu I site to link mAb VDJ in frame with 5Ј regulatory sequences, and then of the inhibition of Ag binding by competitive ELISA, the mAb concen- using SauI digestion to link the 1-kb promoter-VDJ fragment to a 5-kb tration (5 microg/ml) or Tg mouse serum dilution that gave 50% maximal ␮ ␣ fragment containing JH genes and C enhancer in plasmid Bluescript. A binding to 3(IV)NC1 collagen was determined by direct-binding ELISA. 3-kb NotI/EcoRI fragment from this vector was ligated to a 12-kb EcoRI/ This dilution was then preincubated with varying concentrations of inhib- NotI fragment containing the 9-kb IgMa constant region (gifted by D. itor (Goodpasture patient or normal human serum) for1hat37°C before Nemazee, The Scripps Research Institute, La Jolla, CA) to generate the incubation with Ag-coated wells. Bound mAb or serum Tg-encoded IgMa final 15-kb VDJ-C␮ construct. This construct was linearized with NotI and were detected with 1/1000 dilution alkaline phosphatase-labeled goat anti- purified for transfection experiments. mouse Ig (Southern Biotechnology Associates) or 1/500 dilution biotin- The L chain construct was generated using PCR of hybridoma cell DNA labeled anti-allotype (IgM-a)-Ig (BD Biosciences), followed by avidin-al- to amplify a 2.2-kb genomic fragment containing mAb VkJk, 5Ј regulatory kaline phosphatase and second-step reagents, respectively. Archived coded 6094 REGULATION OF ANTIGOODPASTURE AUTOREACTIVITY

FIGURE 2. Human GPS sera inhibit Ag binding by mouse mAb reactive with anti-␣3(IV) NC1 collagen. The concentration (5 ␮g/ml) of mouse mAb that gave 50% maximal binding to Ag was preincubated with varying dilutions of inhibitor (human serum, shown as reciprocal dilution on x-

axis). Results using serum from three different patients and human serum Downloaded from from a healthy control are shown. FIGURE 1. Immunofluorescence staining of kidney for Ig. A, Linear glomerular capillary wall and tubular basement membrane IgG deposits in a kidney harvested at the time of fusion from a mouse immunized with Production of functional anti-NC1 H and L chain Ig DNA ␣ 3(IV)NC1 (direct immunofluorescence with anti-mouse-IgG-FITC, constructs and anti-NC1 Ig Tg mice ϫ200). B, Background staining using isotype control (ϫ200). Similar minimal staining was observed in kidneys from nonimmunized mice (not A mAb IgM H chain construct (Fig. 3A) was generated by ligation http://www.jimmunol.org/ shown). C, Glomerular IgM in an Ig Tg mouse bearing the anti-␣3(IV)NC1 of the rearranged VDJ gene, captured by PCR from hybridoma collagen HϩLIgTg(ϫ400). D, Background staining of kidney tissue from DNA of the prototypic mAb to existing genomic fragments con- a non-Tg mouse (ϫ400). taining regulatory elements. This construct was then ligated to the 9-kb IgMa constant region, as previously described for the 238H H chain Tg (21), with minor modifications. The L chain construct was generated using PCR of hybridoma genomic DNA to isolate a Goodpasture patient sera were provided by Dr. D. Howell, Duke University 2.2-kb fragment containing the 5Ј promoter region, VkJk, and Medical Center and Durham Veterans Affairs Medical Center (Durham, NC) and purchased from Wieslab. downstream elements including Jk5 (Fig. 3B). The 2.2-kb gene

segment was ligated to a 4.5-kb fragment containing the ␬ en- by guest on September 29, 2021 L chain analysis from hybridomas or splenic cDNA hancer and C␬ gene. The integrity and successful expression of Rearranged endogenous VK-Jk genes were identified by sequence analysis of both constructs was confirmed by their cotransfection into ␭ L PCR-amplified cDNA, using a 3Ј primer complementary to C␬ and 5Ј primers chain-expressing murine myeloma cells. Culture supernatants from designed to complement disparate V␬ families, essentially as described (24). cells transfected with the Ig H and L chain constructs, but not Sequence analysis, alignment, and assignment of V families were performed supernatants from mock transfected cells, contained IgM,␬ Ig with using ClustalW (www.ebi.ac.uk/clustalw), IgBLAST (www.ncbi.nlm.nih. ␣ gov), and the international ImMunoGeneTics database (imgt.cines.fr; M.-P. anti- 3(IV)NC1 specificity (not shown). Lefranc, Montpellier, France, initiator and coordinator). Phenotypic characterization of mice expressing the anti-NC1 Ig Statistical analyses H and L chain Tg All data are shown as median values and interquartile range (25th and 75th Ig Tg mice were established by coinjection of the H and L chain percentile) unless otherwise indicated. Comparisons between two groups constructs into B6SJLF2 fertilized eggs at the Duke University were analyzed with Mann-Whitney U test and between three or more groups with the Kruskal-Wallis one-way ANOVA. Analyses were per- Transgenic Mouse Core Facility and are maintained as hemizy- formed with Analyze-it software. A value of p Ͻ 0.05 was considered to gotes. Four founders carrying both genes were identified by PCR be significant. Results Identification of ␣3(IV)NC1 collagen mAb Six SJL or B6 mice were immunized with human recombinant or bovine GBM ␣3(IV)NC1 collagen in Freund’s adjuvant. Mice developed high titer serum ␣3(IV) NC1-reactive IgG and linear renal basement membrane IgG deposits (Fig. 1A). Splenic fusions from six immunized mice yielded five anti-␣3(IV)NC1 Ig mAb as determined by ELISA. One B6-derived IgM, termed 1G6, met both criteria for reactivity with pathogenic GPS epitope(s); it bound strongly in ELISA to ␣3(IV)NC1 collagen coated in 6 M guani- FIGURE 3. Linear maps of the complete H (A)andL(B) chain DNA dine-HCl, which dissociates NC1 hexamers into monomers and constructs used to generate anti-␣3(IV)NC1 collagen HϩL Ig Tg mice. dimers (25), but not to diluent coated wells, and mAb 1G6 binding Solid boxes depict exons; R, EcoRI; X, XhoI; N, NotI; S, SalI. The restric- to ␣3(IV)NC1 collagen was inhibited by serum IgG from multiple tion fragments containing the rearranged VDJ or Vk-Jk2 genes and leader patients with GPS (Fig. 2). This mAb was subsequently designated sequences also contain the Ig transcriptional control elements (P, Ig pro- the prototypic Goodpasture mAb. moter; E, Ig H chain enhancer; Ek, ␬-chain enhancer). The Journal of Immunology 6095 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. B cell surface transgene expression of spleen (A and B) and bone marrow (C and D) from Rag enzyme-sufficient (A and C) and Rag-deficient (B and D) B6 mice. Shown are representative dot plots of log fluorescence data for stained unstimulated cells gated on lymphocytes on the basis of forward and side scatter. Mouse genotypes include mice lacking the Tg (Non-Tg), mice hemizygous for both H and L chain Tg (Tg), mice sufficient in Rag enzyme (Ragϩ), and mice homozygous for Rag enzyme deficiency (RagϪ). Cells were stained with PE- or PerCP-conjugated anti-B220 and FITC- or PE-labeled anti-IgM, anti-IgMa, or anti-IgMb. The Tg H chain is IgMa-allotype, whereas B6 mice endogenously express only IgMb.

genotyping and crossed with B6 breeders. Genotype analysis of I. In the bone marrow the frequency of IgMϩ B220 cells was lower back-cross progeny from these four lines is consistent with coseg- in Tg compared with non-Tg mice: 45.9 (43.4, 47.7) vs 52.8 (47.0, regation of the Tg H and L chain constructs in each line. 54.5) percent, Tg vs non-Tg, p Ͻ 0.05. Collectively, these findings Flow cytometric analysis of splenocytes and bone marrow lym- suggest that a large population of B cells were deleted in Tg mice. phocytes confirmed in vivo expression of Tg IgM a-allotype on the Among residual splenic B cells L chain editing was common. ␭ B cell surface (Fig. 4, A and C) from progeny of each founder line. L chains were expressed on the cell surface of up to 11% (median Tg expression was restricted to B220ϩ cells (B lymphocytes). Tg 8.8% (4.1, 10.9), n ϭ 9) of splenic B cells in Tg mice as deter- and non-Tg mice from each line were included in the analyses mined by flow cytometry, despite the presence of the productively (Table I). rearranged ␬-chain Tg. This exceeded surface ␭ expression in B cell numbers were decreased ϳ4-fold in spleens of Tg prog- non-Tg littermates (median 5.4% (4.3, 5.8), p Ͻ 0.05). Whereas eny as compared with non-Tg littermates: 9.4 (6.9, 12.0) vs 37 flow cytometry did not distinguish Tg and non-Tg (endogenous) (32.3, 42.3) million, Tgϩ vs non-Tg littermates, p Ͻ 0.0001; Table ␬-chains, ␬-chain editing was revealed by two alternative 6096 REGULATION OF ANTIGOODPASTURE AUTOREACTIVITY

Table I. Splenic proﬁles of anti-␣3(IV)NC1 collagen Ig Tg mice

Non-Tg Tg p Value

Spleen weight (mg) 73 (69, 76) 49 (44, 58) Ͻ0.01 Spleen cells (millions)a 83.8 (68.5, 98) 38 (27.4, 43.1) Ͻ0.0001 %B220ϩ 56.8 (54.5, 58.4) 32.5 (31.2, 34.4) Ͻ0.0001 B cells (millions) 37 (32.3, 42.3) 9.4 (6.9, 12) Ͻ0.0001 %CD3ϩ 35.7 (31.8, 36.1) 55.7 (53.5, 56.7) Ͻ0.0001 T cells (millions) 27.1 (20.9, 28.2) 17.5 (13.2, 18.7) Ͻ0.01

a Splenocyte suspensions from Tg and non-Tg littermates were depleted of erythrocytes and enumerated, and B220 and CD3 subpopulations were identiﬁed using ﬂow cytometry. Percentages of positive cells are derived from dot plots after gating on small lymphocytes using side and forward scatter. Values are median (25th, 75th percentile); n ϭ 9–11 mice/group.

approaches. Initial query of Tg mouse spleen cDNA by PCR using splenocytes. Among 136 hybridoma recovered by fusion, 103 su- an upstream primer to detect the Tg L chain sequence (18) and a pernatants expressed Tg IgMa. Supernatants of the 25 clones with downstream C␬ primer revealed not only Tg L chain message, but highest OD for IgMa were subsequently screened for binding to also two ␬-chains derived from productive secondary rearrange- ␣3(IV) NC1 Ag: 18/25 (72%) of hybridoma, including 69% (11/ ments at the endogenous loci and encoded by IGKV3–4*01-Jk1 16) of those derived from unmanipulated splenocytes and 78%

and IGKV3–2*01-Jk2 genes. One of these two L chains was iden- (7/9) of those derived from endotoxin-stimulated splenocytes, Downloaded from tified in spleen cDNA from each of four Tg mice, derived from bound Ag. PCR analysis of cDNA generated from mRNA of two ␬ three different Tg lineages. To further explore the extent of ed- subcloned anti-␣3(IV)NC1 collagen Tg hybridoma revealed an un- iting in this model, rearranged ␬ L chains were recovered from a ␬ ϩ ϩ mutated Tg-encoded L chain transcript, confirming in vivo ex- cDNA library generated from B220 IgMa B cells sorted by flow pression of the Ig Tg L chain. cytometry from splenocytes of a fifth young HϩL Tg mouse. PCR Ј ␬ amplification used a universal 3 C primer and a panel of three http://www.jimmunol.org/ upstream primers derived from disparate V␬ families, as previously described (24). Amplification products were cloned and three clones from each primer pair selected at random for sequencing. Three rearranged endogenous L chain sequences were recovered, each using a different V␬ family and different J␬ gene: IGKV1–133*01-J␬4, IGKV19–120*01-J␬5, and IGKV19–93* 01-J␬1. These findings confirm that L chain receptor editing con- tributes to regulation of B cells autoreactive with ␣3(IV)NC1. Sera of non-Tg littermates contained only IgMb allotype, as by guest on September 29, 2021 expected in the B6 strain. IgMb was also the dominant allotype in serum of Tg mice, despite the paucity of IgMbϩ B cells in bone marrow and peripheral lymphoid organs. Tg mice had limited spontaneous secretion of transgene-encoded IgMa. Serum total IgMa levels (range 0.64–22.06 ␮g/ml, median 9.18 ␮g/ml, n ϭ 10, excluding one outlier with level 149.6 ␮g/ml) were lower than in concurrently measured healthy normal BALB/c (IgMa allotype) mouse serum (114.8 ␮g/ml). These levels are also ϳ5- to 30-fold lower than reported in the non-self-reactive nontolerant anti- lysozyme Ig Tg model, but similar to levels in the tolerant anti- lysozyme Ig and neo-self-Ag double Tg model in which Tg B cells are regulated by anergy (range of serum Tg Ig ϳ0.5–5.0 ␮g/ml; Ref. 26). A subset of serum Tg Abs were autoreactive. ELISA revealed IgMa binding to ␣3(IV) NC1 Ag in the serum of most Tg mice (Fig. 5A). Competition ELISA using GPS patient sera confirmed binding to pathogenic epitopes by a subset of Tg serum IgMa (Fig. 5B). Immunohistochemical analysis of kidneys showed limited glomerular deposition of IgM in 4 of 10 Tg mice (Fig. 1, C–D). FIGURE 5. Anti-␣3(IV)NC1 reactivity of Tg mouse serum. A, Scat- Glomerular scores for Ig fluorescence intensity did not correlate terplot of serum reactivity. Mouse serum diluted 1/20 was incubated on with serum Ag binding or serum IgMa levels (not shown). microwells coated with ␣3(IV)NC1 collagen diluted in 6 M guanidine, T cell numbers were also decreased ϳ33% in spleens of Tg or on wells coated with diluent alone and detected with labeled anti- progeny as compared with non-Tg littermates: 17.5 (11.5, 19.1) vs allotype IgM-a-specific second step reagent. OD405 represents value for 27.1 (20.2, 28.4) million, Tgϩ vs non-Tg littermates, p Ͻ 0.01; binding to Ag minus diluent. Values for individual Tg and non-Tg mice Table I. Both CD4ϩ and CD8ϩ T cell subsets were significantly are indicated. B, Human GPS patient serum inhibition of Ag binding by Tg mouse serum. The dilution of Tg mouse serum that gave 50% max- decreased (not shown). imal binding to ␣3(IV) NC1 collagen was incubated with varying di- Characterization of mAb derived from HϩL Tg mice lutions of inhibitor (GPS patient serum, shown as reciprocal dilution on x-axis). Shown are results using serum from two Tg mice, Tg1 and Tg2, Autoreactive Tg Ig were also recovered as monoclonal Ig by fu- and human serum from two GPS patients, GPS1 and GPS2, and a sion of both unmanipulated and in vitro LPS-stimulated Tg mouse healthy control (normal serum). The Journal of Immunology 6097

Ig that binds epitopes recognized by human patient serum IgG. When the H and L chain transgenes are coexpressed in vivo, transgene-expressing B cells are readily recovered from the spleen. However, peripheral total B cell numbers are significantly decreased in Tg mice despite L chain editing. Genetic introduction of Rag enzyme deficiency to preclude Ig editing and remove influences from endogenous lymphocyte populations results in elimination of Tg B cells. Thus development of Tg anti-␣3(IV)NC1 collagen B cells is halted in the bone marrow, where cells are deleted unless rescued by a Rag-dependent process. The central tolerance phenotype indicates that tolerizing self-Ag is expressed in the bone marrow. The nature of this putative tolerogen is unclear. The ␣3(IV) collagen chain targeted by GPS patients’ autoantibodies has a restricted tissue distribution, being syn- thesized primarily in specialized basement membranes of glomerulus, renal tubules, alveoli, cochlea, eye, and testis (4). Moreover, pathogenic epitopes are conformational and normally buried in the native ␣3(IV) NC1 hexamer, partially hidden from Downloaded from circulating Ab and B cells (5–8). Epitope exposure, possibly facilitated by environmental factors and dynamic interactions of au- FIGURE 6. Transgene expression in Rag-deficient anti-␣3(IV)NC1 collagen HϩL Ig Tg mice. A, Serum total IgM concentration in ␮g/ml as toantibodies and collagen protein (9), is presumed key to disease determined by ELISA. Median in ␮g/ml, 287.5 non-Tg Rag-sufficient, 0.0 onset. Thus it has been predicted that anti-␣3(IV)NC1 collagen B non-Tg Rag-deficient, 227.6 Tg Rag-sufficient, and 0.31 Tg Rag-deficient, cells are not tolerized in vivo under normal circumstances, but p Ͻ 0.001. B, Number of B220ϩ cells in spleen as by determined by flow

rather exist ignorant of Ag. Our finding of deletion and editing http://www.jimmunol.org/ cytometry. Median in ␮g/ml, 39.2 non-Tg Rag-sufficient, 1.0 non-Tg Rag- challenges this view, and rather suggests the presence of a potent Ͻ ϭ deficient, 19.4 Tg Rag-sufficient, and 0.7 Tg Rag-deficient, p 0.001. n tolerogen in the bone marrow. One candidate is tumstatin, an 4–7 mice/group. ϳ232 amino acid, 30-kDa circulating form of ␣3(IV)NC1 cleaved from the carboxyl terminus of the ␣3(IV) collagen chain by the action of metalloproteinases (41). Fragments of collagen IV are Anti-NC1 Ig HϩL chain Tg with superimposed Rag deficiency reported in sera of healthy individuals, and Kalluri and colleagues To further dissect the contributions of editing to the survival of Tg detected circulating tumstatin in plasma of normal mice at a mean B cells, we crossed HϩL Tg mice with mice deficient in the Rag concentration of 336 ng/ml using a direct ELISA with anti- enzyme. Rag-1 and Rag-2 are required for variable region gene tumstatin Ab (4, 42). Alternatively, a cross-reactive epitope on an by guest on September 29, 2021 rearrangements that generate productive Ig and T cell receptors as yet unidentified endogenous protein may tolerize anti- (27). Loss of either enzyme thus eliminates influences from en- ␣3(IV)NC1 collagen B cells. Our results raise the possibility of a dogenous (non-Tg) Ig, including receptor editing or revision, al- therapeutic immunoregulatory role for tumstatin or related lelic inclusion, and follicular competition, as well as influences of soluble Ag. immunoregulatory T cells (28–37). Ig Tgs are expressed on the Despite a default tolerance phenotype of profound central dele- Rag-deficient background because the prerearranged VDJ and VJ tion, Tg B cells are present in substantial numbers in the periphery genes bypass the requirement for Rag (38, 39). of Tg Rag-sufficient mice. This Rag-dependent escape is due in Resultant triple-mutant HϩL Tg Rag-deficient mice had small part to L chain editing, in which persistent activation of Rag en- spleens with few lymphocytes. Flow cytometric analysis detected zymes catalyzes secondary rearrangements to replace existing pro- no IgM-expressing B220ϩ B lymphocytes in either the spleen or ductive ones (28–30, 40, 43, 44). Editing to an innocuous speci- bone marrow of Tg Rag-deficient mice (Fig. 4, B and D and Fig. ficity can be an effective mechanism to abrogate autoreactivity. 6B), indicating complete central deletion. Spleen size and flow Direct evidence of editing in the anti-␣3(IV)NC1 collagen B cells cytometric plots of splenocytes from Tg Rag-deficient and non-Tg includes expression of ␭ L chains in cells that already carry the Rag-deficient littermates were essentially identical. The spleens of ␬ both Tg and non-Tg Rag-deficient mice contain a small population rearranged Tg and PCR-based recovery from Tg spleens of tran- ␬ of B220lowIgMϪC19Ϫ cells, consistent with previous reports in scripts from productively rearranged endogenous -chains. It is which negative CD19 staining suggests that these are not B lineage notable, however, that splenic B cell numbers in Rag-sufficient ␣ cells (38–40). Consistent with these findings, ELISA analysis re- anti- 3(IV)NC1 collagen Tg mice are only one-fourth to one-fifth vealed no detectable Ig in serum of wild-type Rag-deficient mice the number in wild-type littermates. Although editing is a common and only trace quantities of Tg IgM/␬ Ig in serum of young HϩL phenomenon, accounting for ϳ25% of splenic B cells in healthy Tg Rag-deficient mice (Fig. 6). Residual serum IgM recovered individuals (45), it is also highly efficient. Editing typically mini- from Tg Rag-deficient mice reacted with ␣3(IV)NC1 collagen in mizes deletional cell loss such that normal or near normal numbers ELISA (not shown). of splenic B cells are maintained within a diverse polyclonal B cell population (40). Discussion Persistent B cell depletion in the anti-␣3(IV)NC1 collagen Tg To better understand the immunological events that precipitate or- model may be attributed in part to accelerated B cell development, gan destruction in patients with Goodpasture syndrome, we ex- imparted by the transgene, that limits the window for serial editing plored mechanisms regulating autoreactive B cells that bind the (46). The duration of the editing-susceptible period may be crucial Goodpasture Ag. For this purpose we developed a novel HϩL to its effectiveness as a salvage mechanism. Its prolongation pro- chain Ig Tg mouse model expressing an anti-␣3(IV)NC1 collagen vides additional opportunity for secondary Ig gene rearrangements. 6098 REGULATION OF ANTIGOODPASTURE AUTOREACTIVITY

In this regard, tolerance-induced slowing of immature B cell turn- Our proposed model for regulation of Tg anti-␣3(IV)NC1 col- over and enforced bcl2 expression are credited with rescue of wild- lagen B cells, dependent on central selection and editing, must also type B cells reactive with a ubiquitous membrane Ag (47). In the explain the autoreactivity recovered from Tg mice. Most animals anti-␣3(IV)NC1 collagen Ig Tg model, the capacity of editing to have low levels of serum anti-␣3(IV)NC1 collagen autoantibodies, restore normal B cell numbers may also be thwarted by Tg H chain and autoreactivity is recovered from hybridomas derived from Tg dominance. If a single H chain generates autoreactivity with a mouse spleens. This autoreactivity may depend on the same edit- variety of different L chains, many secondary rearrangements will ing process that permits the cells to escape deletion. Editing that fail to abolish autoreactivity. This possibility is supported by re- occurs in trans on a second L chain allele, as occurs both physi- sults of additional transfection experiments (not shown) in which ologically and in H and L chain Ig Tg mice, permits expression of introduction of the Tg H chain construct into a Vk8Jk5 L chain- both L chain proteins in the same B cell (40). Dual L chain ex- only hybridoma cell line (gifted by Dr. M. Radic; Ref. 48) pro- pression is reported in wild-type mice and healthy humans (64, duced a novel IgM,␬ Ig that bound ␣3(IV)NC1 collagen in ELISA. 65). In mouse spleens, an estimated 10% of mature B cells express Regardless of mechanism, in the anti-␣3(IV)NC1 collagen HϩL both ␬ alleles (66) and 20% of ␭-producing cells coexpress a Tg model, neither editing nor proliferation and expansion of pe- ␬-chain (64). Dual L chain expression is reported in a significant ripheral B cell pools is sufficient to replenish splenic populations to fraction of peripheral B cells in mice bearing the 3H9H-56R anti- normal levels. DNA or the 3–83 anti-MHC class I Ig Tg (67, 68). It is notable that Rag-dependent survival of a subset of Tg anti-␣3(IV)NC1 col- young 3H9H/56R anti-DNA mice do not secrete anti-DNA Ab in ␣ lagen B cells may depend on secondary rearrangements at the en- vivo (67). Nonetheless, analogous to the anti- 3(IV)NC1 collagen dogenous Ig H chain loci. Although endogenous IgMb H chain is Ig Tg reported here, 3H9H/56R anti-DNA Tg B cells captured as Downloaded from ␭ rarely detected on the surface of peripheral B cells in neonatal and hybridomas secrete both -encoded autoantibody and nonautore- ␬ young anti-␣3(IV)NC1 collagen Ig Tg mice (Fig. 4), dual positive active editor L chain (67). Similarly, Ig Tg mice engineered to IgMaϩIgMbϩ and isolated IgMbϩ B cell populations emerge in a coexpress two L chains spontaneously produce the autoreactive Tg subset of older Tg mice (not shown). This coexpression of an Ig; lack of or escape from tolerance in this model is attributed to endogenous H chain may promote survival of autoreactive cells in receptor dilution (32). In this scenario the innocuous (nonautore- a manner analogous to L chain editing, as is proposed for high active, editor) receptor presumably promotes cell survival, activa- http://www.jimmunol.org/ tion, and differentiation, ultimately supporting secretion of both affinity anti-dsDNA B cells in the R4A-␥2b H chain Ig Tg model editor and autoantibody (44). The extent to which similar mecha- (49). In nonautoimmune mice productive rearrangements at both H nisms permit autoreactivity in the anti-␣3(IV)NC1 collagen Ig Tg chain alleles are detected in ϳ2–4% of B cells (50), although mice may be dissected by genetic exclusion of endogenous L chain recovery of dual H chain-producing B cells in this setting is rare loci. Nonetheless, the presence of spontaneous anti-␣3(IV)NC1 (51). H chain inclusion may function physiologically as a salvage collagen autoreactivity in the Tg mice, and at low levels in healthy mechanism to ensure formation of a functional pre-BCR (50). The humans (12), invites speculation as to whether this specificity is extent of dual H chain expression is generally higher, although preserved because it confers some immunological advantage to the

variable, in IgM Tg or Ig gene-targeted models (52–57), and may by guest on September 29, 2021 host, as proposed for anti-phosphocholine autoantibodies (32). be detected at the level of the cell population or individual cells The presence of trace quantities of Tg Ig in a few Tg Rag- (32, 46, 58). The contribution of H chain inclusion to autoreactive ϩ deficient mice in the absence of detectable IgM B cells in the cell survival and autoimmunity appears to be minimal for at least periphery further suggests that rare anti-␣3(IV)NC1 collagen B some specificities. In anti-phosphocholine Ig Tg mice, endogenous cells, possibly anergic cells with below-threshold levels of surface H chain is coexpressed by 20% of Tg B cells; however, back-cross Tg expression, do escape censoring and can be activated in a T- with mice lacking the IgM transmembrane exons (muMT mice), independent manner. Our studies do not indicate significant B1 which eliminates endogenous H chains, fails to alter splenic B cell lineage development in the peritoneum, although the significance numbers or block autoantibody production (32). of the rare peritoneal IgMϩ B220ϩ cells in a few Tg Rag-deficient ␣ Rag deficiency may influence anti- 3(IV)NC1 collagen Tg B mice (data not shown) is unclear. cell fates by additional mechanisms. However, none seems likely It is notable that the majority of serum IgM in Rag-sufficient Tg to account for profound central deletion. Loss of endogenous B mice derives from the relatively few endogenous (non-Tg IgMb) B cells removes competitor populations that would otherwise restrict cells, despite domination of the peripheral B cell repertoire by Tg autoreactive cell entry into follicles and germinal centers in sec- IgMa-bearing B cells. The role of homeostatic mechanisms in sup- ondary lymphoid organs (33, 34). The absence of T cells can in- porting normal serum IgM levels in the setting of B cell lymphope- fluence autoreactive B cell fate and survival in the periphery by a nia or in Ig Tg models expressing a tolerance phenotype is unclear. variety of mechanisms (34, 59, 60), including a permissive role in Adult mouse serum IgM normally derives approximately equally B cell anergy that appears to be Ag or model specific (39, 61). from infrequent B1 lineage cells and from Ag-recruited B2 lineage However, a significant role has not been defined for T cells or cells (69). The low serum Tg IgMa levels suggest that many Tg B circulating T cell-derived cytokines in the bone marrow-special- cells either have not been activated in vivo or are anergic. ized microenvironments that support early B cell development, Rag-sufficient Tg mice also demonstrate an unexplained signif- survival, selection, and emigration (62). Congenitally athymic icant T cell depletion. It is possible, and perhaps likely, that the (nude) mice are not autoimmune, despite subtle abnormalities of B restricted diversity and size of the endogenous B cell population cell development and primary V gene repertoire (63), and intro- influences the T cell repertoire. It is now well appreciated that B duction of the nude mutation does not discernibly alter outcomes cells have important Ab-independent regulatory roles that include in anti-phosphocholine Ig Tg mice (32). Similar to our finding in Ag presentation for T cell activation or tolerance induction, coor- the Rag-deficient anti-␣3(IV)NC1 collagen Ig Tg, central deletion dination of T cell migration and differentiation, regulation of T cell is efficient in mice lacking Rag and bearing an Ig Tg reactive with polarization and effector function, regulation of dendritic cell func- the ubiquitous cell membrane self-Ag MHC H-2k, whereas abun- tions, and production of immunomodulatory cytokines and che- dant B cells are present in Rag-deficient Ig Tg mice lacking this mokines (70). Depletion of regulatory T cells could promote ac- deleting self-Ag (38). tivation of Tg-expressing B cells and contribute to serum The Journal of Immunology 6099 autoantibody levels. Conversely, naive Tg anti-␣3(IV)NC1 colla- T-cells in Goodpasture’s syndrome recognize the N-terminal NCI domain on a3 gen B cells may engage collagen-reactive T cells to promote their type 1V collagen. Kidney Int. 49: 1127–1133. 15. Salama, A. D., A. N. Chaudhry, J. J. Ryan, E. Eren, J. B. Levy, C. D. Pusey, deletion, although it seems unlikely that such interactions would L. Lightstone, and R. I. Lechler. 2001. In Goodpasture’s disease, CD4ϩ T cells significantly deplete a diverse T cell repertoire. escape thymic deletion and are reactive with the autoantigen alpha3(IV)NC1. J. Am. Soc. Nephrol. 12: 1908–1915. In conclusion, our findings invite speculation about the origin of 16. Wong, D., R. G. Phelps, and A. N. Turner. 2001. The Goodpasture antigen is pathogenic autoantibodies in GPS in humans. Results from the Tg expressed in the human thymus. Kidney Int. 60: 1777–1783. model suggest that anti-␣3(IV)NC1 collagen B cells must either 17. Kalluri, R., T. M. Danoff, H. Okada, and E. G. Neilson. 1997. Susceptibility to anti-glomerular basement membrane disease and Goodpasture syndrome is linked escape central deletion, possibly through editing with dual receptor to MHC class II genes and the emergence of T cell-mediated immunity in mice. expression, or they must arise de novo from somatic events in J. Clin. Invest. 100: 2263–2275. mature B cells in the periphery, such as hypermutation and sec- 18. Sackey, F. N., K. L. Congdon, G. F. Brady, H. Hopfer, Y. Zhang, K. M. Mackin, and M. H. Foster. 2008. Shared variable domain elements among anti-collagen ondary Ig rearrangements (71) and escape regulation at these more antibodies reactive with Goodpasture epitopes. In Autoimmunity: Role, Regula- distal points. Tight regulation at multiple stages may explain the tion, and Disorder. F. L. Vogel and L. F. Zimmermann, eds. Nova Science Pub- relative rarity of this deadly autoimmune disease. lishers, Hauppauge, NY. In press. 19. Fitzsimons, M. M., H. Chen, and M. H. Foster. 2000. Diverse endogenous light chains contribute to basement membrane reactivity in nonautoimmune mice Acknowledgments transgenic for an anti-laminin Ig heavy chain. Immunogenetics 51: 20–29. Dr. Raghu Kalluri (Harvard University) is gratefully acknowledged for 20. Foster, M., Q. Liu, H. Chen, D. Nemazee, and B. Cooperstone. 1997. Anti- ␣ laminin reactivity and glomerular immune deposition by in vitro recombinant provision of recombinant human 3(IV)NC1 and Dr. David Howell antibodies. Autiommunity 26: 231–243. (Duke University Medical Center and Durham Veterans Affairs Medical 21. Cooperstone, B. G., M. M. Rahman, E. H. Rudolph, and M. H. Foster. 2001. In Center) for providing patient Goodpasture serum. We thank the Duke vitro and in vivo expression of a nephritogenic Ig heavy chain determinant: Downloaded from University Comprehensive Cancer Center Hybridoma, Cell Culture, and pathogenic autoreactivity requires permissive light chains. Immunol. Cell Biol. 79: 222–230. DNA sequencing facilities and Dr. Carol Wikstrand for expertise. We 22. Pelanda, R., S. Schaal, R. M. Torres, and K. Rajewsky. 1996. A prematurely thank Erica Ungerwitter, Joanna Bradley, Jacquie Anderson, and Rus- expressed Ig␬ transgene, but not V␬J␬ gene segment targeted into the Ig␬ locus, sell Williams for technical assistance. can rescue B cell development in ␭5-deficient mice. Immunity 5: 229–239. 23. Rudolph, E. H., K. L. Congdon, F. N. Sackey, M. M. Fitzsimons, and Disclosures M. H. Foster. 2002. Humoral autoimmunity to basement membrane antigens is regulated in C57BL/6 and MRL/MpJ mice transgenic for anti-laminin Ig recep-

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