The Immunodominant Myeloperoxidase T-Cell Epitope Induces Local Cell-Mediated Injury in Antimyeloperoxidase Glomerulonephritis

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The immunodominant myeloperoxidase T-cell PNAS PLUS epitope induces local cell-mediated injury in antimyeloperoxidase glomerulonephritis

Joshua D. Ooia, Janet Changa, Michael J. Hickeya, Dorin-Bogdan Borzab, Lars Fuggerc, Stephen R. Holdswortha,d, and A. Richard Kitchinga,d,e,1

aCenter for Inﬂammatory Diseases, Department of Medicine, Monash University, and Departments of dNephrology and ePediatric Nephrology, Monash Medical Centre, Clayton 3168, Victoria, Australia; bDepartments of Medicine and Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232; and cMedical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom

Edited by Emil R. Unanue, Washington University School of Medicine, St. Louis, MO, and approved August 9, 2012 (received for review June 19, 2012) Microscopic polyangiitis is an autoimmune small-vessel vasculitis MPO Abs can induce neutrophil- and complement-mediated that often manifests as focal and necrotizing glomerulonephritis FNGN (14–18), enhanced by infection-related signals like LPS and renal failure. Antineutrophil cytoplasmic Abs (ANCAs) specific (14, 19, 20). for myeloperoxidase (MPO) play a role in this disease, but the role Although there is a rationale for autoreactive CD4+ cells of autoreactive MPO-specific CD4+ T cells is uncertain. By screening contributing to the development of disease in microscopic pol- overlapping peptides of 20 amino acids spanning the MPO mole- yangiitis, their role is less clear. There is evidence that MPO- cule, we identified an immunodominant MPO CD4+ T-cell epitope ANCA production requires antigen-specific CD4+ T cells (21, + (MPO409–428). Immunizing C57BL/6 mice with MPO409–428 induced 22). Furthermore, autoreactive MPO-specific CD4 T cells can focal necrotizing glomerulonephritis similar to that seen after be induced experimentally in animals (23), MPO-specific T cells whole MPO immunization, when MPO was deposited in glomeruli. that produce IFN-γ are present in the peripheral blood of fi + −/− Transfer of an MPO409–428-speci c CD4 T-cell clone to Rag1 humans with acute ANCA-associated vasculitis (24–26), and mice induced focal necrotizing glomerulonephritis when glomeru- urinary CD4+ effector/memory cells reflect disease activity (27). lar MPO deposition was induced either by passive transfer of MPO- Effector/memory CD4+ T cells, together with macrophages, ANCA and LPS or by planting MPO409–428 conjugated to a murine tissue factor, and fibrin, are present in glomeruli of patients with antiglomerular basement membrane mAb. MPO409–428 also in- ANCA-associated glomerulonephritis (28, 29). Finally, in a mu- duced biologically active anti-MPO Abs in mice. The MPO409–428 rine model of anti-MPO FNGN, where autoimmunity to MPO is epitope has a minimum immunogenic core region of 11 amino induced and glomerulonephritis is triggered by injection of sheep acids, MPO415–426, with several critical residues. ANCA-activated anti-mouse glomerular basement membrane (GBM) Ab, CD4+ neutrophils not only induce injury but lodged the autoantigen + T-cell depletion during the effector phase attenuated disease MPO in glomeruli, allowing autoreactive anti-MPO CD4 cells to (23). Based on this evidence, we hypothesize that MPO-specific induce delayed type hypersensitivity-like necrotizing glomerular effector CD4+ cells are important in disease by localizing to lesions. These studies identify an immunodominant MPO T-cell glomeruli and inducing a delayed type hypersensitivity (DTH)- fi epitope and rede ne how effector responses can induce injury like lesion. Anti-MPO CD4+ cells may localize to glomeruli by – in MPO-ANCA associated microscopic polyangiitis. recognizing MPO within glomeruli, acting as a planted glomerular autoantigen deposited by ANCA-activated neutrophils that autoimmunity | lymphocytes | T helper 1 cells | macrophages have degranulated and/or formed neutrophil extracellular traps

(NETs) (30). IMMUNOLOGY mall-vessel vasculitis is often induced by autoimmunity to Although MPO’s B-cell epitopes have been the subject of Sneutrophil granule proteins, predominantly myeloperoxidase studies mapping them to areas within the heavy chain (31, 32), (MPO) and proteinase 3 (Pr3) (1), as well as lysosomal mem- the T-cell epitopes of MPO are undefined. Identifying MPO’sT- brane protein-2 (2). Although there is some overlap, autoim- cell epitopes is important in understanding the pathogenesis of munity to MPO is strongly associated with microscopic poly- anti-MPO disease and would represent progress toward less toxic angiitis and reactivity to Pr3 results in granulomatosis with therapies focused on the autoimmune response. In the current polyangiitis (GPA). The kidneys are frequently affected by focal studies, we have defined an immunodominant CD4+ T-cell MPO and segmental necrotizing glomerulonephritis (FNGN), leading epitope and then used this epitope to test the hypothesis that to rapidly progressive glomerulonephritis and end-stage renal antigen-specific CD4+ T cells recognize both this epitope and failure. Due to the presence of auto-Abs to MPO and Pr3, this MPO itself in glomeruli and induce FNGN. This immunodo- disease is also known as antineutrophil cytoplasmic Ab (ANCA)- minant T-cell epitope exists across at least three different MHC associated vasculitis (3). Morbidity and mortality rates remain II alleles and also can induce MPO-ANCA. Our studies redefine high, with a 5-y survival rate of 46–85% in microscopic poly- our understanding of anti-MPO disease to now include a distinct angiitis (4), and most treatments have limited effectiveness and significant toxicities (5).

Evidence for a pathogenic role for ANCA in microscopic Author contributions: J.D.O., S.R.H., and A.R.K. designed research; J.D.O. and J.C. polyangiitis includes the use of plasma exchange as therapy, performed research; M.J.H., D.-B.B., and L.F. contributed new reagents/analytic tools; a case report of lung hemorrhage in a neonate following pla- J.D.O., J.C., S.R.H., and A.R.K. analyzed data; and J.D.O. and A.R.K. wrote the paper. cental transfer of MPO-ANCA, and other observations in The authors declare no conﬂict of interest. humans (6, 7). Moreover, ANCA can activate neutrophils and This article is a PNAS Direct Submission. promote their adhesion in vitro (8) and in vivo (9–12). The ad- 1To whom correspondence should be addressed. E-mail: [email protected]. hesion of neutrophils in target tissues, particularly the kidney, See Author Summary on page 15547 (volume 109, number 39). induces injury by means of the release of injurious oxidants and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. enzymes, including MPO itself (13). In addition, transferred anti- 1073/pnas.1210147109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1210147109 PNAS | Published online September 5, 2012 | E2615–E2624 Downloaded by guest on September 27, 2021 role for effector CD4+ T cells that recognize MPO, which are planted in glomerular capillaries by MPO-ANCA–activated neutrophils. The pathogenesis of effector responses in microscopic polyangiitis should now be considered to include a sequential mix of types II (Ab-mediated) and IV (DTH-like) hypersensitivity. Results

MPO409–428 Is the Immunodominant T-Cell Epitope of MPO. To define CD4+ T-cell epitopes within MPO, we immunized groups of C57BL/6 mice with pools of MPO 20-mers (from the mouse MPO sequence, with each peptide overlapping by 12 aa; Table S1). Separate groups of mice were injected with peptide pools from the proregion of MPO (peptides 1–17), the light chain (peptides 18–31), or one of four quarters (H1–H4) of the heavy chain (peptides 32–46, 47–61, 62–76, or 77–89). Splenocytes were restimulated with each third of the relevant immunizing pool (e.g., splenocytes from the group immunized with peptides 1–17 were restimulated with peptides 1–6, 7–12, or 13–17), and recall responses were measured by IFN-γ enzyme-linked immu- nospot (ELISPOT) assay. The five peptide groups inducing the strongest responses were peptide pools 7–12, 23–27, 52–56, 57–61, and 62–66 (Fig. 1A). Additional groups of mice were then immunized with one of these groups of peptides, and their draining lymph node (LN) cells were restimulatedwitheachoftheindividual peptides from within the immunizing pool. The five individual peptides that induced the strongest recall responses were peptides 10, 24, 52, 57, and 61 (Fig. 1B). To determine the immunodominant MPO T-cell epitope, we then immunized separate groups of mice with each of these peptides and restimulated their draining LN cells ex vivo with either the immunizing peptide or recombinant mouse MPO using proliferation assays and ELISPOT assays for IFN-γ and IL-17A. Comparing recall responses, MPO peptide 52 (MPO409–428, PRWNGEKLYQEARKIVGAMV) induced the strongest responses both to itself and to whole recombinant mouse MPO (Table 1). Proliferative responses were significantly higher than those induced by all other peptides. Although proinflammatory cytokine production after peptide 52 immunization was numer- Fig. 1. MPO T-cell epitopes in C57BL/6 mice. (A) Overlapping peptides (89 ically greater than after all other peptides, peptide 61 also in- 20-mers) spanning the entire mouse MPO sequence were used to immunize duced moderately strong IL-17A and IFN-γ production. groups of mice. Pro, proregion peptides (1–17); Light, light chain peptides Mice immunized with native mouse MPO (n = 5) developed (18–31). H1 (32–46), H2 (47–61), H3 (62–76), and H4 (77–89) are heavy chain recall responses to peptide 52 but not to a control peptide, ov- peptides. Splenocytes were restimulated ex vivo with each third of the immunizing pool (x axis), and responses were measured by IFN-γ ELISPOT assay albumin (OVA)323–339 (proliferation assay stimulation index: 2.1 ± 0.2 vs. 1.0 ± 0.1, IFN-γ ELISPOT assay: 8.0 ± 1.8 spots, and (mean number of spots minus baseline). Each dot represents the mean re- ± sponse from an individual mouse, and results are representative of two in- IL-17A ELISPOT assay: 10.3 1.7 spots; no samples restimu- dependent experiments with a minimum of four mice per group. (B) Groups lated with OVA323–339 showed any spots above media alone). of mice were immunized with one of the five strongest responding pools of Immunization with peptide 53 or 54, both of which overlap with peptides (peptides 7–12, 23–27, 52–56, 57–61, or 62–66) and draining LN cells peptide 52 and induce recall responses by IFN-γ ELISPOT assay were restimulated ex vivo with individual peptides (x axis). Each dot represents in mice immunized in peptide pool 52–56 (Fig. 1 A and B), did the mean response from an individual mouse, and results are representative not induce responses to peptide 52, 53, or 54 or to recombinant of two independent experiments with a minimum of four mice per group. MPO (Fig. S1).

– T-Cell Autoreactivity to MPO409–428 (MPO Peptide 52) Is Not MHC II-, I- immunizing C57BL/6 mice with the complete 20-aa MPO409 428 Ab-, Restricted. Because there are no clear MHC class II associ- (PRWNGEKLYQEARKIVGAMV) and measuring proliferative ations with anti-MPO glomerulonephritis, we sought to de- recall responses to shortened 16-mers and 12-mers of MPO409–428 termine if MPO peptide 52 (MPO409–428) is immunoreactive in (Fig. 3A). There were no detectable recall responses when mice expressing different MHC II molecules. We immunized MPO409–428 was shortened from the C terminus to either a 16- BALB/c mice (expressing I-Ad) and humanized HLA- mer, amino acids 409–424, or to a 12-mer, amino acids 409–420 DRB1*15:01 transgenic (Tg) mice (without mouse MHC class (Fig. 3A). However, recall responses with amino acids 413–428, II) (33) with the pool of MPO peptides 52–56. In both strains, containing the 16 C-terminal amino acids, were comparable to peptide 52 was the immunogenic peptide (Fig. 2 A and B). recall responses induced by full-length amino acids 409–428. The BALB/c and HLA-DRB1*15:01 Tg mice immunized with mouse recall response to the 12 C-terminal amino acids 417–428, al- MPO peptide 52 developed autoreactive responses to both MPO though detectable, was reduced compared with MPO409–428.To peptide 52 and to whole recombinant MPO (Fig. 2 C and D). delineate the core immunoreactive residues of MPO409–428 further, mice were immunized with the 16-mer amino acids 413–428

Deﬁning the Core and Critical Residues of the MPO409–428 Epitope. We and recall responses to sequential 12-mers overlapping by 11 aa then further delineated the key areas of this T-cell epitope. We were measured. Restimulation of mice immunized with amino identiﬁed the critical peptide length for immunoreactivity by acids 413–428 with the 12-mers containing amino acids 414–425

E2616 | www.pnas.org/cgi/doi/10.1073/pnas.1210147109 Ooi et al. Downloaded by guest on September 27, 2021 Table 1. Immune responses induced by the ﬁve most immunogenic MPO peptides PNAS PLUS Responses to immunizing peptide Responses to recombinant MPO

Peptide Proliferation, SI IFN-γ, spots IL-17A, spots Proliferation, SI IFN-γ, spots IL-17A, spots

10 2.3 ± 0.5 18 ± 413± 1 2.2 ± 0.2 21 ± 17± 1 24 3.8 ± 1.1 14 ± 212± 3 3.0 ± 0.3 19 ± 312± 1 52 9.2 ± 0.8* 64 ± 10** 67 ± 8** 4.3 ± 0.2* 39 ± 5*** 45 ± 5** 57 2.4 ± 0.4 32 ± 533± 4 2.5 ± 0.2 22 ± 320± 4 61 5.6 ± 1.0 55 ± 10 64 ± 13 3.0 ± 0.3 32 ± 531± 3

Groups of mice were immunized with individual peptides, and immune responses to the immunizing peptide or to whole recombinant mouse MPO were measured ex vivo using an [3H]-thymidine proliferation assay and IFN-γ and IL-17A ELISPOT assays. SI, stimulation index; spots, mean number of spots minus baseline. Results are presented as the mean ± SEM and are representative of three independent experiments, each with a minimum of ﬁve mice per group. *P < 0.05 vs. peptides 10, 24, 57, and 61; **P < 0.05 vs. peptides 10, 24, and 57; ***P < 0.05 vs. peptides 10 and 24.

and 415–426 induced recall responses comparable to those substituted 11-mers [alanine itself (MPO420) was substituted by obtained by restimulation with the 16-mer immunogen amino a serine]. Each 11-mer had one residue substituted by alanine in acids 413–428 (Fig. 3B). Therefore, the core immunoreactive sequential order. An individual amino acid was deemed a critical residues of MPO409–428 are the 11 amino acids (415–425; residue if its substitution by an alanine abrogated a positive ex KLYQEARKIVG). The critical residues within this 11-mer were vivo proliferative response. The substitution of tyrosine, arginine, delineated by immunizing C57BL/6 mice with KLYQEARKIVG isoleucine, or valine deﬁned these amino acids as the critical and restimulating draining LN cells with individual alanine- residues within this epitope (Fig. 3C).

MPO409–428 Immunization Induces Nephritogenic T-Cell Responses. To determine the nephritogenicity of the anti-MPO T-cell responses generated by MPO409–428 immunization, a previously established model of anti-MPO–directed glomerulonephritis was used (23, 34). In this model, C57BL/6 mice immunized with mouse MPO lose tolerance to MPO, but this autoimmunity, in itself, is not sufficient to induce glomerulonephritis, which is triggered by injecting low-dose sheep anti-mouse GBM Ab. This Ab induces transient glomerular neutrophil recruitment with MPO deposition in glomeruli. MPO409–428-immunized mice developed renal injury with increased albuminuria and blood urea nitrogen to a similar degree as mice immunized with whole native mouse MPO (Fig. 4 A and B). MPO409–428-immunized mice developed FNGN comparable to mice immunized with whole native mouse MPO, characterized by segmental glomerular necrosis and fibrin deposition within the glomerular tuft. Mice immunized with OVA323–339 and given anti-GBM Ab exhibited only mild lesions (Fig. 4 C–J). Analysis of glomerular cellular effectors showed IMMUNOLOGY increased infiltration of CD4+ T cells and macrophages compared with control mice, similar to that seen after whole native mouse MPO immunization (Fig. 4 K–M). Concurrently, a group of mice (n = 6) immunized with the next highest responding MPO peptide from Table 1 (peptide 61, MPO481–500) and given anti-GBM Ab did not develop more proteinuria or histological glomerular injury than OVA323–339-immunized mice (6.1 ± 1.1 vs. 5.3 ± 0.7 mg over 24 h and 16.1 ± 2.4 vs. 16.2 ± 2.0% of glomeruli affected by necrosis, respectively).

+ −/− Transfer of MPO409–428-Specific CD4 T Cells into Rag1 Mice Induces FNGN. To determine definitively whether T-cell autoreactivity to this dominant MPO epitope is nephritogenic, we generated Fig. 2. Immunogenicity of MPO peptide 52 (MPO409–428) is not restricted to fi MHC II I-Ab. The immunogenicity of MPO peptide 52 was examined in mice mouse MPO409–428-speci c T-cell clones from C57BL/6 mice. d + expressing I-A (BALB/c) or human DR2 (DR2 Tg) (with the absence of murine CD4 lines from OT-II Tg mice (i.e., OVA323–339-specific) de- MHC II and Tg expression of human HLA-DRB1*15:01). When immunized rived under the same conditions were control cells. We trans- – −/− with the pool of peptides 52 56, peptide 52 is capable of inducing recall ferred T-cell clones into naive Rag1 mice (MPO409–428-specific responses in the context of I-Ad (BALB/c, n =4)(A) or HLA-DR2 (DR2 Tg, n =4) 6 or OVA323–339-specific: 5 × 10 cells per mouse) and then im- (B). When mice were immunized with peptide 52 (MPO409–428) (BALB/c, n =6; munized mice with the clones’ cognate peptide. As before, MPO DR2 Tg, n = 4), LN cells from both strains exhibited recall responses to both was deposited in glomeruli using low-dose sheep anti-mouse the peptide itself (MPO peptide 52, ●) and recombinant mouse MPO (rmMPO, ○ □ GBM Ab and mice were culled 5 or 14 d after disease induction. ) compared with control OVA323–339 ( ), as measured by proliferation (C and fi D). Each dot represents the mean response from an individual mouse. ***P < At 5 d, mice that received the MPO409–428-speci c T-cell clone 0.001. Results are representative of three independent experiments with developed a DTH response to MPO injected intradermally 24 h a minimum of four mice per experiment. SI, stimulation index. before the end of the experiment, which was not detected in

Ooi et al. PNAS | Published online September 5, 2012 | E2617 Downloaded by guest on September 27, 2021 compared with control clone recipients (Fig. 5 I–K). The degree of perivascular and tubulointerstitial inflammation was also increased, with more CD4+ T cells and macrophages but similar numbers of neutrophils in the tubulointerstitium (Fig. S2 and Table S2). Glomerular crescent formation was observed in mice receiving the MPO409–428-specific T-cell clone at day 14 (6 ± 2% of glomeruli affected) but not in mice receiving OVA323–339- specific T cells. In some mice, pulmonary histology showed signs of interstitial pneumonitis and lymphocytic bronchiolitis, but these findings were inconsistent and not present in all clone transfer studies.

+ Localizing MPO409–428 to Glomeruli Induces CD4 T-Cell Clone- Mediated FNGN. To determine whether the presence of the T-cell epitope MPO409–428 itself in glomeruli can induce T-cell– + mediated renal injury in mice with MPO409–428 CD4 T cells, we injected a biotinylated MPO409–428 peptide conjugated to a mouse anti-mouse monoclonal IgG1 (clone 8D1) specific for the mouse noncollagenous domain of the α3 chain of type IV collagen, which does not induce pathological albuminuria or major histological changes (35). Glomerular deposition of biotinylated MPO409–428 was demonstrated by direct immunofluorescence using streptavidin-FITC Ab (Fig. 6 A and B). Injection of the MPO409–428-8D1 conjugate itself did not increase glomerular neutrophil recruitment (Fig. 6C) or renal MPO activity (naive mice: 1.8 ± 0.2, 8D1 alone: 2.2 ± 0.5, 8D1-MPO409–428: −3 2.0 ± 0.4; expressed as ΔA460/min × 10 ) at a 4-h time point. −/− Compared with control Rag1 mice receiving OVA323–339- specific cells and MPO409–428-8D1, mice receiving the MPO- 6 specific clone (25 × 10 cells) followed by MPO409–428-8D1 developed glomerular infiltrates of CD4+ T cells, macrophages, and neutrophils (Fig. 6 A–C) with FNGN (Fig. 6 H–K). Glo- merular crescent formation was observed in four of six mice receiving the MPO409–428-specific clone (3%, 5%, 5%, and 10% Fig. 3. Core immunogenic residues of MPO409–428.(A) C57BL/6 mice were of glomeruli affected) but not in mice receiving OVA323–339- immunized with MPO409–428 (MPO peptide 52), and reactivity to shortened specific cells. These pathological abnormalities translated into 3 16-mers and 12-mers was assessed by [ H]-thymidine proliferation in drain- functional injury with increased albuminuria and blood urea ni- ing LN cells. Ex vivo reactivity to the shortened 16-mers and 12-mers was trogen levels (Fig. 6 L and M). Similarly, perivascular in- compared with reactivity induced by MPO peptide 52. (B) MPO413–428 was fl fi 3 ammation, tubulointerstitial injury, and in ltration of effector used to immunize mice, reactivity to individual 12-mers was assessed by [ H]- fi thymidine proliferation in draining LN cells, and ex vivo reactivity to the cells were observed in mice that received the MPO409–428-speci c clone (Table S2). These experiments show that MPO – itself individual 12-mers was compared with MPO413–428. The core 11 amino acid 409 428 residues are the 11 amino acids shared by MPO414–425 and MPO415–426 in glomeruli, without the initial presence of neutrophils or en- (underlined) because both induced comparable reactivity to the immunizing dogenous MPO, is sufficient to induce effector MPO-specific MPO413–428.(C) To determine the critical residues, mice were immunized with T-cell localization and injury. the core 11 amino acids, MPO415–425, and draining LN cells were restimulated ex vivo with alanine-substituted 11-mers. Alanine-substituted positions: +, LPS and MPO-ANCA Recruit Neutrophils and Deposit MPO, Leading to MPO415–425;K,ALYQEARKIVG; L, KAYQEARKIVG; Y, KLAQEARKIVG; Q, CD4+ T-Cell–Mediated FNGN. Relapse of ANCA-associated vascu- KLYAEARKIVG; E, KLYQAARKIVG; A, KLYQESRKIVG; R, KLYQEAAKIVG; sec- litis is associated with infections (36, 37). At least part of the ond K, KLYQEARAIVG; I, KLYQEARKAVG; V, KLYQEARKIAG; G, KLY- explanation for these clinical observations may come from ex- QEARKIVA; −, OVA323–339; MPO, whole recombinant mouse MPO. Reactivity to individual alanine-substituted 11-mers was compared with reactivity to perimental evidence showing that infection primes neutrophils

MPO415–425 and showed that tyrosine (Y), arginine (R), isoleucine (I), and va- [and potentially other cells (20)], allowing ANCA to bind to and line (V) were critical to the response. *P < 0.05; ***P < 0.001. Results are activate neutrophils (9, 19). This results in their recruitment to representative of two independent experiments, each with a minimum of target tissues, especially the kidney (9, 15). Modeling this process four mice per group. Each dot represents the mean response from an in- involves injecting LPS and transferring anti-MPO IgG (gener- − − dividual mouse. SI, stimulation index. ated in Mpo / mice) into mice. Although these Abs can induce injury (14), recruiting neutrophils into glomeruli may also result + ± ± in the deposition of MPO within glomeruli, where effector CD4 control mice (footpad swelling: 0.4 0.1 vs. 0.0 0.0 mm). cells could potentially recognize MPO as a planted glomerular Splenocytes harvested at 14 d from mice that received MPO409– antigen. Having shown that MPO-specific CD4+ cells can rec- fi – 428-speci c clones showed recall responses to both MPO409 428 ognize the immunodominant MPO T-cell epitope, MPO – , γ ± ± 409 428 and MPO (IFN- ELISPOT assay: 156 33 and 170 40 in the glomerulus, we next tested the hypothesis that MPO from spots, respectively). anti-MPO Ab-activated neutrophils acts as an autoantigen in fi + Mice receiving the MPO409–428-speci c T-cell clone developed target tissues, resulting in effector CD4 cell-mediated injury. progressive FNGN with increased albuminuria, blood urea ni- Mice were injected with LPS and anti-MPO IgG (generated by − − trogen, segmental glomerular necrosis, and fibrin deposition immunizing Mpo / mice with murine MPO). Histological compared with mice that received OVA323–339-specific T cells analyses 3 h after injection showed [as previously demonstrated and anti-GBM Ab (Fig. 5 A–F). There were increased CD4+ T (20)] glomerular neutrophil recruitment after LPS and anti- cells and macrophages, but not neutrophils, within glomeruli MPO IgG (Fig. 7 A, C, and D). There was also increased renal

E2618 | www.pnas.org/cgi/doi/10.1073/pnas.1210147109 Ooi et al. Downloaded by guest on September 27, 2021 MPO activity (Fig. 7B). Confocal microscopy using anti-CD45 PNAS PLUS and MPO-specific Abs revealed the presence of leukocyte-associated MPO as well as extracellular MPO that had been deposited in the glomerulus (Fig. 7E). Injecting LPS and anti-MPO Abs after transferring either 5 × 6 6 + 10 or 25 × 10 MPO409–428-specific CD4 T-cell clones induced significant renal disease compared with transfer of LPS and OVA323–399-specific cells. MPO-specific effector T cells resulted in increased albuminuria, blood urea nitrogen, and FNGN with glomerular necrosis and fibrin deposition (Fig. 7 F–M). Renal injury was more severe after 25 × 106 MPO-specific cells compared with 5 × 106 MPO-specific cells. Glomerular crescent formation was observed in three of six mice that received 25 × 106 MPO-specific cells (with 10%, 15%, and 45% of glomeruli affected) but not in mice receiving 5 × 106 MPO-specific cells. There were also increases in glomerular infiltration of CD4+ T cells, macrophages, and neutrophils, similar to those observed when using the MPO409–428-8D1 conjugate to plant the T-cell epitope in glomeruli (Fig. 7 N–P). Perivascular and tubulointerstitial injury followed a similar pattern (Table S2). Transfer of 6 50 × 10 MPO409–428-specific cells resulted in markedly acceler- ated renal disease specific to mice receiving MPO-specific cells, and experiments were terminated at 6 d (Fig. S3).

MPO409–428 Induces Biologically Active MPO-ANCA. Indirect immunofluorescence using purified IgG on ethanol-fixed peritoneal mouse neutrophils showed a p-ANCA pattern specificto MPO409–428-immunized mice and not seen in serum IgG from OVA323–339-immunized mice or when IgG from MPO409–428- − − immunized mice was applied to neutrophils from Mpo / mice (Fig. 8 A–C). To determine if immunization with MPO409–428 induced MPO-ANCA, we tested sera from MPO409–428-immunized C57BL/6 mice for MPO-specific IgG by ELISA. Sera from OVA323–339- or whole native mouse MPO-immunized mice served as negative and positive controls. MPO409–428-immunized mice developed MPO-ANCA IgG, albeit at lower titers compared with whole native mouse MPO immunization (Fig. 8D). To determine whether MPO-ANCA induced by MPO409–428 had functional activity, we transferred purified MPO-ANCA into LPS-primed C57BL/6 recipient mice. Compared with IgG from OVA323–339- immunized mice, serum IgG from MPO409–428-immunized mice resulted in increased glomerular neutrophil recruitment at 3 h, (Fig.

8E), showing that these auto-Abs are biologically active. IMMUNOLOGY Discussion The glomerulus is a common target in MPO-ANCA–associated microscopic polyangiitis, with the severity of renal disease often defining the outcome. Although ANCA- and neutrophil-induced injury is important in microscopic polyangiitis, the presence of CD4+ T cells (26) and the MPO itself (28, 30) within lesions implies that MPO-specific autoreactive T cells may localize to glomeruli and cause injury. The current studies demonstrate a role for MPO-specific CD4+ cells in experimental ANCA-associated FNGN and define an immunodominant CD4+-cell epitope of MPO (MPO409–428). Our findings support a model whereby both ANCA and autoreactive effector CD4+ cells are important in microscopic polyangiitis. After tolerance is lost to MPO (with MPO409–428 as a potential key epitope), antigen-

Fig. 4. MPO409–428 immunization induces nephritogenic autoimmunity. fi C57BL/6 mice were immunized with either OVA323–339 [control (n =5),□], increased after MPO-speci c immunization. Some glomeruli from MPO409– – MPO409–428 (n =6,●), or native mouse MPO (n =4,○), and disease was 428- and whole MPO immunized mice exhibited segmental necrosis (H&E) (C triggered by recruiting neutrophils to glomeruli with low-dose sheep anti- and E–G) with glomerular fibrin deposition (fibrin as the brown reaction – fi mouse GBM Ab. Mice immunized with an irrelevant antigen (OVA323–339) product) (D and H J). Photomicrographs of glomeruli and brin deposition are and injected with low-dose sheep anti-mouse GBM Ab developed minimal taken at a magnification of 400×. (Scale bars: E–J,10μm.) Glomerular infiltrates + injury, whereas mice immunized with MPO409–428 developed FNGN similar of CD4 T cells (K) and macrophages (L) but not neutrophils (M) were increased < < to that induced by immunization with whole-mouse MPO. Functional indices after MPO409–428 immunization. gcs, glomerular cross section. *P 0.05; **P of glomerular injury, albuminuria (A) and blood urea nitrogen (B), were 0.01; ***P < 0.001.

Ooi et al. PNAS | Published online September 5, 2012 | E2619 Downloaded by guest on September 27, 2021 specific CD4+ T cells provide T-cell help to autoreactive B cells in producing MPO-ANCA. In many people who develop MPO- ANCA, these auto-Abs interact with and activate neutrophils, causing the neutrophils to be recruited to glomerular capillaries. Neutrophils mediate injury but, critically, also release the autoantigen MPO, which then is present in glomeruli and can be recognized by autoreactive MPO-specific CD4+ cells. Glomer- ular localization of these damaging MPO-specific effector T cells results in the recruitment of effector leukocytes, the development of FNGN, and renal impairment. In contrast to other autoimmune diseases, there have been no consistent MHC class II associations in microscopic polyangiitis. HLA typing studies in patients with ANCA-associated vasculitis have focused largely on GPA (most frequently marked by autoimmunity to Pr3). In this disease, a variety of MHC class II associations have been found (reviewed in 38), with DPB1*04:01 being the most consistent. There are, however no clear positive associations with microscopic polyangiitis, which usually fea- tures autoimmunity to MPO with MPO-ANCA. We therefore d tested the immunogenicity of MPO409–428 in mice with I-A and mice with HLA-DRB1*15:01, finding that this peptide could induce immune responses in more than one MHC II haplotype. Further studies in C57BL/6 mice identified a minimum immunogenic peptide, MPO415–425, and several critical amino acids within this peptide. When glomerular disease was triggered with low-dose anti- GBM Ab, MPO409–428 peptide-immunized mice (but not OVA323–339-immunized mice) developed significant FNGN, showing that immunity to this peptide induced FNGN. MPO is a major constituent of neutrophils and is released when neutrophils induce inflammation, contributing to tissue injury by generating damaging oxidants (13, 39). However, in microscopic polyangiitis following ANCA-induced glomerular neutrophil localization, neutrophils release MPO that is deposited in glomeruli (28, 30). Immunologically, the glomerulus becomes a site where MPO, the autoantigen, is present and available for antigen recognition by effector T cells. Therefore, after generating T helper (Th) 1 IFN-γ–secreting MPO409–428-specific clones that induced dermal DTH in vivo, we performed a series of transfer experiments demonstrating that MPO-specific CD4+ cells induce FNGN after MPO has been deposited in the glomerular microvasculature. This antigen- specific disease (not found in recipients of OVA-specific CD4+ T cells) was characterized by segmental glomerular necrosis; glomerular fibrin deposition; CD4+ T-cell, macrophage, and neutrophil recruitment; and functional renal injury (albuminuria and raised blood urea nitrogen). Tubulointerstitial infiltrates and injury, in the form of tubular dilation, necrosis, and protein cast formation, as well as perivascular inflammation were present in mice receiving MPO-specific CD4+ cells. Because injection of anti-GBM Ab induces neutrophil recruitment, increased renal MPO activity, and free MPO within the glomerulus, we initially injected these Abs to trigger disease (34, 39–41). However, this strategy of inducing glomerular MPO deposition is confounded by anti-GBM Ab-induced neutrophil Fig. 5. Transfer of T-cell clones specific for MPO409–428 induces progressive 6 recruitment and the enzymatic effects of MPO, which, together, FNGN. T-cell clones specific for MPO409–428 (5 × 10 ) were transferred into Rag1−/− mice (●), disease was triggered 7 d later with low-dose sheep anti- complicate the study of MPO as an autoantigen. By defining an mouse GBM Ab, and experiments were ended after a further 5 d (n =6)or14 immunodominant MPO peptide, we have defined a role for − − d(n = 4). Control Rag1 / mice that received sheep anti-mouse GBM Ab with MPO as a planted autoantigen distinct from its effects as an + fi ○ a CD4 T-cell line speci c for OVA323–339 ( )(n =6,5d;n = 4, 14 d) developed injurious effector molecule. To determine whether MPO409–428 fi minimal injury, but mice given MPO409–428-speci c cells developed pro- itself could be recognized as a nephritogenic peptide within gressive functional injury with pathological albuminuria (A), increased blood glomeruli, we used a modification of our previously published urea nitrogen levels (B), and FNGN (C) with glomerular fibrin deposition (D). delivery system (35). By conjugating MPO – to a non- Histologically, glomeruli from control mice at 14 d were near normal (E) 409 428 immunogenic mouse anti-mouse GBM monoclonal IgG1 Ab, we compared with those of mice receiving MPO409–428-specific cells (F). (Scale bars: E and F,20μm.) Progressive increases in glomerular CD4+ T cells (G) and could plant MPO409–428 in glomeruli. Compared with some other macrophages (H) but not neutrophils (I) over time were seen in recipients of murine IgG subclasses, the murine IgG1 subclass has limited MPO-specific T cells. gcs, glomerular cross section. *P < 0.05; **P < 0.01; capacity to cause injury itself, because it fixes complement poorly ***P < 0.001. and has a relatively low affinity for leukocyte-activating Fcγ

E2620 | www.pnas.org/cgi/doi/10.1073/pnas.1210147109 Ooi et al. Downloaded by guest on September 27, 2021 receptors (42). At the dose that we used, the Ab itself induces PNAS PLUS minimal leukocyte recruitment and no albuminuria (35). Therefore, compared with polyclonal sheep IgG or anti-MPO Abs, it served as a relatively inactive carrier that could localize + MPO409–428 to glomeruli. When effector CD4 cells were transferred into mice, only MPO-specific cells localized to glomeruli and induced significant FNGN with renal impairment. Studies in patients with ANCA-associated vasculitis show associations between infection and disease relapse and activity, suggesting a role for infection in the pathogenesis of these diseases (36, 37). Having shown that MPO-specific T cells could recognize MPO409–428 within glomeruli to induce injury, we triggered disease with transferred anti-MPO Abs and LPS, a prototypic infection-related signal. This is likely to be more analogous to the “clinical” situation, where enzymatically active and autoantigenic MPO lodges in glomeruli when it is released by MPO-ANCA–activated neutrophils. LPS has activating effects on both neutrophils and intrinsic glomerular cells, including induction of the production of neutrophil chemo- attractants by glomerular endothelial cells (20). Although, as expected, there was some relatively mild injury induced by the initial LPS and anti-MPO Ab-induced glomerular neutrophil recruitment, MPO-specific T cells caused significant and dose-dependent FNGN, with the highest dose of cells resulting in rapidly progressive renal failure in an antigen-specific manner and pre- mature termination of experiments. These experiments show that the humoral and cellular arms of the autoimmune response to MPO collaborate to induce ANCA-associated vasculitis. There are several ways by which MPO, an abundant protein in neutrophils, can become lodged in glomeruli in ANCA-associated glomerulonephritis. Although ANCA-induced neutrophil recruitment results in neutrophil degranulation, ANCA also promotes NET formation (30). These NETs are extracellular, include both MPO and Pr3, and are present in glomeruli in ANCA-associated glomerulonephritis. In addition, neutrophil microparticles, which are present in ANCA-associated vasculitis, contain MPO and can localize to endothelial cells (43). It remains to be determined which cell type presents MPO409–428 to effector CD4+ cells in glomeruli, allowing antigen-specific local MPO recognition. Leukocytes traditionally present antigens to effector CD4+ cells. Dendritic cells (rare in glomeruli) or monocyte/macrophages may be important in ingesting MPO

present within glomeruli to allow glomerular T-cell recognition IMMUNOLOGY of antigen, whereas neutrophils themselves can also express MHC II and present antigens under some circumstances (44). Alternatively, intrinsic glomerular cells could be important, because they can both internalize MPO (45) and be in contact with intracapillary MPO-speciﬁc CD4+ T cells. Studies in murine glomerulonephritis induced by a planted foreign antigen support a role for glomerular cells in both antigen recognition and activation of effector CD4+ cells (46–48). Chimeric mice lacking MHC II expression (46) or the costimulatory molecule CD40 (47) in renal tissue cells did not develop severe T cell-mediated glomerulonephritis, and in the same model, glomerular CD80 and CD86 contributed to injury (48).

+ Fig. 6. Planting MPO409–428 in glomeruli induces CD4 cell-mediated FNGN. Injecting a mouse anti-mouse α3 chain of type IV collagen [α3(IV)NC1] IgG1 −/− mAb (clone 8D1) conjugated to biotinylated MPO409–428 into naive Rag1 later by targeting MPO409–428 to glomeruli via MPO409–428 conjugated to mice results in deposition of biotinylated MPO409–428 peptide in glomeruli. clone 8D1. Mice were culled 14 d (n = 6) after triggering disease. Control −/− + After 4 h, a streptavidin-FITC Ab showed no biotin in glomeruli of mice Rag1 mice were injected with the OVA323–339-specific CD4 T-cell line injected with unconjugated 8D1 (n =6)(A), but clear biotin signal was followed by the 8D1-MPO409–428 conjugate (○)(n = 6). The presence of observed within glomeruli of mice injected with the 8D1-biotinylated MPO409–428 in glomeruli resulted in glomerular localization of MPO-specific MPO409–428 conjugate (n =6)(B). The tubular fluorescence in both panels is but not OVA-specific T cells (D), with macrophage and neutrophils in glo- endogenous biotin within renal tubules. (C) Injection of 8D1-biotinylated meruli (E and F). Recruitment of these leukocytes resulted in FNGN with

MPO409–428 conjugate (●) did not result in an increase in glomerular neu- glomerular fibrin deposition (G–J). The 8D1-MPO409–428 conjugate with OVA- + trophils compared with naive (□) or unconjugated 8D1 mAb alone in re- specificCD4 cells did not induce functional renal injury, but MPO409–428- −/− + + cipient (○) Rag1 mice at 4 h. MPO409–428-specific CD4 T-cell clones (25 × specific CD4 cells resulted in both albuminuria (K) and renal impairment (L). − − 106) were transferred into Rag1 / mice (●), and disease was triggered 7 d **P < 0.01; ***P < 0.001. (Scale bars: A, B, I, and J,20μm.)

Ooi et al. PNAS | Published online September 5, 2012 | E2621 Downloaded by guest on September 27, 2021 Fig. 7. Coinjection of LPS and anti-MPO Abs deposits MPO in glomeruli and triggers cell-mediated glomerular injury and FNGN. Injecting LPS and anti-MPO − − Abs into naive Rag1 / mice (n = 4 in each group) results in neutrophil recruitment (A, C, and D) and increased renal MPO activity after 4 h (B). (Scale bars: C and D,20μm.) (E) When assessed by confocal microscopy, free [nonleukocyte (CD45)-associated] MPO could be detected in glomeruli (CD45, red; MPO, green; + nonleukocyte-associated MPO in green). (Scale bar: 5 μm.) The dotted line shows the outline of the glomerulus. MPO409–428-specific CD4 T-cell clones were transferred into Rag1−/− mice (5 × 106, n =6;25× 106, n =6;●), and disease was triggered 7 d later by injecting LPS and anti-MPO Abs. Mice were culled after −/− + 6 a further 14 d. Control groups were Rag1 mice injected with LPS and anti-MPO Abs, followed by an OVA323–339-specific CD4 T-cell line (5 × 10 , n =4;25× 106, n =5;○). Mice with OVA-specific T cells injected with LPS and anti-MPO Abs showed only mild injury with modest albuminuria (F), no renal impairment

(G), and low proportions of glomeruli affected by segmental necrosis (H) and fibrin deposition (I). Renal injury was markedly increased by MPO409–428-specific CD4+ T cells in a dose-dependent manner. Representative photomicrographs of glomeruli show only mild glomeruli hypercellularity in control mice (J) but FNGN in mice receiving MPO-specific CD4+ cells (K). A similar pattern was seen with glomerular fibrin deposition [control cells (L) and MPO-specific CD4+ cells (M)]. (Scale bars: J–M, 25 μm.) Glomerular leukocyte recruitment showed a dose-dependent increase in CD4+ cells (N) and antigen-specific increases in macrophages (O) and neutrophils (P). gcs, glomerular cross section. *P < 0.05; **P < 0.01; ***P < 0.001.

Both effector Th1 cells and Th17 cells are important in ex- and evidence exists for the involvement of both Th1 and Th17 perimental glomerulonephritis induced by a planted foreign cells in human immune renal injury (55, 56). Although some antigen (35, 49–51), whereas in autoimmune diseases, cell-me- human observational studies and our recent experimental data diated injury can be mediated by either Th1 cells, Th17 cells, or support a role for Th17 cells and IL-17A in ANCA-associated both Th1 and Th17 cells (52–54). We have shown that either Th1 glomerulonephritis (34, 57, 58), other studies implicate Th1 cells cells or Th17 cells can induce injury in glomerulonephritis (35), (59). The current studies, in showing that injury can be mediated

E2622 | www.pnas.org/cgi/doi/10.1073/pnas.1210147109 Ooi et al. Downloaded by guest on September 27, 2021 ANCA–activated neutrophils and autoreactive effector CD4+ T PNAS PLUS cells that recognize MPO within glomeruli. Materials and Methods MPO Peptides, Proteins, and Mice. Peptide libraries for MPO T-cell epitope screening assays were synthesized as PepSets (Mimotopes). Peptides were 20 aa long and overlapped by 12 aa. Peptide sequences are based on National Center for Biotechnology Information reference NP_034954 (mouse MPO; Table S1). Individual peptide immunization and restimulation assays were performed with >90% pure peptides by HPLC (Mimotopes or AusPep).

OVA323–339 was purchased from Auspep. Native mouse MPO was puriﬁed from a mouse cell line, 32Dcl3, and recombinant mouse MPO was generated using a baculovirus system, as previously described (60). C57BL/6 and BALB/c mice were obtained from Monash Animal Services, Monash University. − − − − − − Rag1 / , humanized MHC class II / HLA-DRB1*15:01 Tg (33), Mpo / ,and OT-II mice were bred at Monash Medical Centre Animal Facilities. Male mice, aged 6–8 wk, were used for experiments and kept in speciﬁc pathogen-free conditions at Monash Medical Centre Animal Facilities.

Identiﬁcation of the MPO T-Cell Epitopes. To identify the T-cell immunogenic regions of MPO, groups of C57BL/6 mice were initially immunized s.c. with peptide pools that correspond to the proregion of the molecule (peptides 1– 17), the light chain (peptides 18–31), or one of four quarters (H1–H4) of the heavy chain (peptides 32–46, 47–61, 62–76, and 77–89; 10 μg of peptide per mouse) in Freund’s incomplete adjuvant (Sigma). Spleens were harvested 6 d postimmunization. Splenocytes were stimulated ex vivo with each third of the immunizing pool, and responses were measured by IFN-γ ELISPOT assay.

Fig. 8. MPO409–428-immunized mice develop biologically active MPO-ANCA. Groups of mice were then immunized s.c. in the base of the tail with one of (A–C) Representative micrographs (400×) of ethanol-fixed peritoneal neu- the peptide pools 7–12, 23–27, 52–56, 57–61, or 62–66 (10 μg of peptide per trophils stained by indirect immunofluorescence with purified serum IgG. mouse) in Freund’s complete adjuvant. Draining LN cells were harvested 10 (Scale bars: 1 μm.) The result is representative of two individual experiments d postimmunization. Finally, C57BL/6 mice were immunized s.c. in the base with IgG purified from pooled sera. (D) Serum from C57BL/6 mice immunized of tail with one of the five peptides that induced the strongest responses

with MPO409–428 develops detectable levels of MPO-specific IgG, as measured (peptides 10, 24, 52, 57, and 61; 10 μg per mouse) in Freund’s complete by serum IgG ELISA (n = 6 per group). nmMPO, native mouse myeloperox- adjuvant. Draining LN cells were harvested 10 d postimmunization. Re- idase. (E)Purified serum IgG was transferred into LPS-primed syngeneic activity to individual peptides and to whole recombinant MPO was com- recipients, and glomerular recruitment of neutrophils was assessed 3 h later pared by [3H]-thymidine proliferation and IFN-γ and IL-17A ELISPOT assays. (n = 4 per group). □, serum from OVA323-339 immunized C57BL/6 mice; ●, ○ serum from MPO409-428 immunized C57BL/6 mice; , serum from native Murine Model of anti-MPO–Directed Glomerulonephritis. Mice were immu- mouse myeloperoxidase immunized mice. ***P < 0.001. nized s.c. in the base of tail with 100 μg of MPO409–428,OVA323–339,or40μgof native mouse MPO in Freund’s complete adjuvant, followed by a similar s.c. ’ γ– injection 7 d later in the back of the neck in Freund s incomplete adjuvant. by IFN- secreting Th1 cells, support a model wherein both Th1 Seven days later, mice received an i.v. injection of 1 mg of sheep anti-mouse and Th17 cells mediate injury in anti-MPO disease. GBM Ab on 2 d consecutively. Injury was assessed 4 d after the last i.v. The MPO409–428 epitope can also induce MPO-ANCA with injection. some biological activity, because transfer of pooled IgG from −/− 6 – × IMMUNOLOGY MPO409–428-immunized mice with LPS could induce modest T-Cell Clone Transfer Models. Each Rag1 mouse received 5 50 10 fi glomerular neutrophil recruitment. The target areas for human (MPO409–428- or OVA323–339-speci c cells) T cells, followed by s.c. injection of μ ’ MPO-ANCA have been identified in regions within the MPO 100 g of the cognate peptide in Freund s complete adjuvant. MPO was deposited in glomeruli 7 d posttransfer by (i) i.v. injection of 1 mg of sheep heavy chain (MPO – , corresponding to mouse MPO – ) 387 745 360 718 anti-mouse GBM Ab, (ii) i.v. injection of 150 μg of 8D1 mAb conjugated to (31), which include our T-cell epitope, although it is not neces- MPO409–428,or(iii) i.p. injection of LPS (0.5 μg/g) and i.v. injection of protein sary for autoreactive T-cell and B-cell epitopes to be from similar G-purified anti-MPO IgG Ab (50 μg/g) derived from immunizing Mpo−/− mice parts of the autoantigen. Targeted antigen-specific therapy is (20). Injury was assessed at day 5 or at day 14 posttransfer. To conjugate

a long-term aim in treating autoimmune disease. The identifi- MPO409–428 to 8D1 mAb, 5 mg/mL (grown in-house) was reacted with 0.1 mg/ cation of an important epitope within MPO provides a platform mL N-succinimidyl-6-maleimido-caproate (Sigma) for 2 h. MPO409–428 (10 mg/ for further work aimed at developing antigen-specific therapies, mL) was combined with 8D1 mAb at 10-fold molar excess for 3 h, the re- given the apparently relatively restricted range of autoantigens in action was stopped by adding 2 mM cysteine, and unconjugated peptide was removed by dialysis in PBS. Conjugation was confirmed by Western blot microscopic polyangiitis with MPO-ANCA. using streptavidin-HRP Abs (BD Biosciences). When considered with published data on the role of ANCA in Additional methods are detailed in SI Materials and Methods. disease, our studies demonstrate that tissue injury in microscopic polyangiitis is mediated by the following series of events. Fol- Statistics. Where there were three or more groups (the majority of experi- lowing neutrophil priming and activation by the coordinate ac- ments), including experiments across different time points, one-way ANOVA tion of infection-related signals and MPO-ANCA, neutrophils followed by Tukey’s posttest was used to assess differences. Where there are recruited to glomeruli. Here, they initiate injury not only by were only two groups, a Student t test was used. Means and SEMs are shown < < < releasing injurious mediators but by depositing MPO, the auto- (*P 0.05, **P 0.01, and ***P 0.001). antigen, in glomerular capillaries. The presence of MPO within fi + Study Approval. These studies were conducted in strict accordance with the these small vessels allows effector MPO-speci c CD4 T cells to Australian code of practice for the care and use of animals for scientific localize to glomeruli and induce a DTH-like necrotizing glo- purposes by the National Health and Medical Research Council of Australia. merulonephritis. Therefore, the pathogenesis of microscopic Animal studies were approved by the Monash University Animal Ethics polyangiitis includes distinct and important roles for both MPO- Committee.

Ooi et al. PNAS | Published online September 5, 2012 | E2623 Downloaded by guest on September 27, 2021 ACKNOWLEDGMENTS. We thank Prof. J. Dowling for advice on pulmonary Australia) for technical advice on 8D1 conjugation. These studies were funded histology; Prof. F. Carbone for the anti-Vα Ab; Dr. C. Lo, A. Li, and C. Lo for by National Health and Medical Research Council of Australia Program Grant technical assistance; and Dr. H. Braley (Commonwealth Serum Laboratories, 334067 and Project Grant 1008849.

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