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Thymic Deletion and Regulatory T Cells Prevent Antimyeloperoxidase GN

† ‡ Diana S.Y. Tan,* Poh Y. Gan,* Kim M. O’Sullivan,* Maree V. Hammett, Shaun A. Summers,* † ‡ Joshua D. Ooi,* Brita A. Lundgren,§ Richard L. Boyd, Hamish S. Scott,§ A. Richard Kitching,* † ‡ Ann P. Chidgey, and Stephen R. Holdsworth*

*Centre for Inflammatory Diseases, Monash University Department of Medicine, Clayton, Victoria, Australia; †Immune Regeneration Laboratory, Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Australia; ‡Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia; and §Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia

ABSTRACT Loss of tolerance to neutrophil myeloperoxidase (MPO) underlies the development of ANCA-associated vasculitis and GN, but the mechanisms underlying this loss of tolerance are poorly understood. Here, we assessed the role of the in deletion of autoreactive anti-MPO T cells and the importance of pe- ripheral regulatory T cells in maintaining tolerance to MPO and protecting from GN. Thymic expression of MPO mRNA predominantly localized to medullary thymic epithelial cells. To assess the role of MPO in forming the repertoire and the role of the Aire in thymic MPO expression, we 2/2 2/2 compared the effects of immunizing Mpo mice, Aire mice, and control littermates with MPO. 2/2 2/2 Immunized Mpo and Aire mice developed significantly more proinflammatory -producing anti-MPO T cells and higher ANCA titers than control mice. When we triggered GN with a subnephrito- 2/2 genic dose of anti-glomerular basement membrane antibody, Aire mice had more severe renal disease +/+ than Aire mice, consistent with a role for Aire-dependent central deletion in establishing tolerance to MPO. Furthermore, depleting peripheral regulatory T cells in wild-type mice also led to more anti-MPO T cells, higher ANCA titers, and more severe GN after immunization with MPO. Taken together, these results suggest that Aire-dependent central deletion and –mediated both play major roles in establishing and maintaining tolerance to MPO, thereby protecting against the development of anti-MPO GN.

J Am Soc Nephrol 24: 573–585, 2013. doi: 10.1681/ASN.2012090898

Systemic autoimmunity to myeloperoxidase system to discriminate between self and non-self (MPO) is directly involved in causing the glomeru- antigens. involves thymic deletion lar and vascular inflammation of ANCA-associated of with high-affinity interactions be- pauci-immune necrotizing autoimmune anti-MPO tween the T cell receptor and self-peptide MHC GN (AIMPOGN).1–3 ANCA induces neutrophil ac- complexes, preventing many potentially autoreac- tivation and endothelial cell adhesion, with the re- tive T cells from entering the periphery.9 The role of lease of neutrophil extracellular traps containing 4, MPO and proteases triggering endothelial injury. Received September 10, 2012. Accepted December 21, 2012. 5 Experimental studies demonstrate that autoim- Published online ahead of print. Publication date available at mune anti-MPO CD4+ T cells respond to glomer- www.jasn.org. ular MPO deposited by degranulating neutrophils, Correspondence: Prof. Stephen R. Holdsworth, Centre for In- directing injurious delayed type hypersensitivity flammatory Diseases, Monash University Department of Medi- (DTH)–mediated injury.6–8 cine, Monash Medical Centre, 246 Clayton Road, Clayton, VIC Immunologic tolerance is maintained by central 3168, Australia. Email: [email protected] and peripheral mechanisms, allowing the immune Copyright © 2013 by the American Society of Nephrology

J Am Soc Nephrol 24: 573–585, 2013 ISSN : 1046-6673/2404-573 573 BASIC RESEARCH www.jasn.org central tolerance in the maintenance of tolerance to the po- low-expressing mTECs (mTEC-lo) (Figure 1B). Expression tential kidney autoantigen, MPO, is largely unknown. The in mTEC-hi is consistent with the known critical involvement autoimmune regulator (Aire) is impor- of these cells in the development of the T cell repertoire.31 tant for the induction and regulation of tolerance.10–12 Aire Given that Aire is expressed only on the CD452 nonhemato- is primarily found in lymphoid organs, particularly in the poietic population, and predominantly by the mature mTECs thymus where it is predominantly found in the nuclei of (mTECs-hi) (Figure 1, C and D), we determined whether 2 2 mature, highly MHC II–expressing13–15 medullary thymic MPO expression would be altered in Aire / mice. We found epithelial cells (mTECs).16,17 Aire promotes the promiscu- that MPO expression was almost absent from the mTEC-hi 2 2 ous expression of tissue-restricted antigens (TRAs) in and mTEC-lo cells in Aire / mice (Figure 1B), suggesting a mTECs.13,16–18 However, the mechanisms by which Aire strong association with Aire-dependent peripheral antigen ex- controls the presentation of TRA expression in mTECs and pression. its effect on tolerance and autoimmunity remain to be fully defined. MPO Protein Expression Confined to Neutrophils and Despite central tolerance, some autoreactive cells escape Macrophages the selection process, entering the periphery where they may The presence of MPO protein was determined by immunohis- cause autoimmunity if activated.19,20 Naturally arising CD4 tology staining. We examined the thymi of 5-week-old Mpo+/+, 2 2 2 2 +CD25+Foxp3+ regulatory T cells (Tregs), mainly produced Mpo / , and Aire / mice (Figure 2, A–C, respectively). MPO 2 2 by the thymus by high-affinity interactions with thymic ep- protein is present in Mpo+/+ and Aire / mice, but as expected, 2 2 ithelial cells,21 are a distinct T cell population that plays a absent from Mpo / thymi (Figure 2, A–C). The MPO distri- pivotal role in the maintenance of self-tolerance. Several bution was along the corticomedullary junction. Colocaliza- studies demonstrate the importance of Tregs in the preven- tion studies demonstrated MPO localization to neutrophils tion of organ-specific autoimmunity by potently sup- (Figure 2, D–F) and macrophages (Figure 2, G–I) in the thymic pressing autoreactive T cells in a contact-dependent and compartment of all five thymus samples. Confocal immuno- cytokine-independent manner.22–26 Depletion of Tregs leads fluorescence microscopy revealed that all neutrophils and some to the spontaneous development of some autoimmune dis- macrophages are MPO positive. It was also evident that most of eases.27–29 the MPO positive cells are neutrophils. To assess the role of central and peripheral tolerance in regulating the development of autoimmunity to MPO, we Thymic Aire-Dependent MPO Expression Deletes used a validated model of MPO-induced autoimmunity.6,7,30 Potentially Autoreactive Anti-MPO T Cells and Protects Establishment of anti-MPO autoimmunity directs the devel- against AIMPOGN opment of focal necrotizing GN similar to that seen in human We assessed MPO-specific cell-mediated immune responses ANCA-associated GN. Our studies demonstrate the impor- with regard to the Th1 and Th17 subset differentiations in tance of both central and peripheral mechanisms in maintain- lymph node cells (LNs) draining the sites of MPO immuni- ing tolerance to MPO. Aire promotes thymic MPO expression zation and serum levels of MPO-ANCAtiters in groups of wild- 2 2 2 2 2 2 and enhances central deletion of autoreactive anti-MPO T type (WT), Mpo / ,andAire / mice. In both Mpo / and 2 2 cells, whereas peripheral Tregs suppress potentially autoreac- Aire / mice, the frequencies of anti-MPO autoreactive CD4+ tive MPO-specific CD4+ T cells. Both mechanisms limit anti- T cells by enzyme-linked immunosorbent spot (ELISPOT) MPO GN. assay and the titers of circulating MPO-ANCA were increased compared with Mpo+/+ (Figure3,A,B,F–H) and Aire+/+ lit- termate WT controls (Figure 3, C and I). The dermal DTH 2 2 RESULTS response to MPO was also increased in Aire / mice (Figure 3D). A control protein, ovalbumin (OVA) in Freund’scom- MPO mRNA Is Predominantly Expressed by MHC II– plete adjuvant (FCA), did not induce anti-MPO autoimmu- Expressing Medullary Thymic Epithelial Cells in an Aire- nity in Mpo+/+ mice. Circulating anti-MPO lgG, lgG1, and Dependent Manner lgG2c levels (Figure 3, E–G) were detected in normal Mpo+/+ After enzymatic digestion of thymic tissue and flow cytometric mice after MPO immunization (not in OVA-immunized mice), 2 2 2 2 sorting of thymic stromal cell (TSC) subsets, transcripts for but were significantly higher in Mpo / and Aire / mice (Fig- 2 2 2 2 MPO were detected in the Mpo+/+ mice, but not in Mpo / ure 3H). There was no difference between Mpo+/+ and Mpo / mice, which served as a negative control. Within the mouse mice in the frequency of Foxp3+ expression of the CD4+ T cells thymus, MPO mRNA is highly expressed in the CD452 TSC isolated from LNs draining the sites of injection (Figure 3, I and subpopulation, but was only minimally detected in the CD45+ J).TheabsenceofthymicMPOandAireaffectedtheinducible thymic hematopoietic subpopulation (Figure 1A). Of the repertoire of autoreactive anti-MPO CD4+ T cells and the levels CD452 population, the major cell subpopulation expressing of autoantibody in a manner consistent with a role for thymic MPO mRNA was the mTECs. MPO was expressed in both the expression of MPO in deleting potentially autoreactive anti-MPO MHC II high-expressing mTECs (mTECs-hi) and MHC II CD4+ T cells.

574 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 573–585, 2013 www.jasn.org BASIC RESEARCH

Figure 1. Expression of MPO and Aire mRNA by thymic cell populations, including hematopoietic TECs (CD45+), nonhematopoietic TECs (CD452), medullary TECs (mTECs), cortical TECs (cTECs), and non-TECs (nTECs) with either high (hi) or low (lo) coexpression of MHC II, and dendritic cells (DCs). (A) MPO is predominantly expressed on nonhematopoietic populations in the Mpo+/+ thymus. (B) 2 2 MPO is predominantly seen in MHC II coexpressing mTECs in the WT mice compared with the Aire / mice. (C) Aire is expressed on the nonhematopoietic populations in the Aire+/+ thymus. (D) Within the CD452 population, Aire is predominantly expressed by the mTECs-hi cells. The asterisks denote the statistical differences between mTECs-hi with mTECs-lo, cTECs, nTECs, or DCs (***P,0.001).

To determine the functional significance of thymic Tregs Protect against the Induction of MPO MPO expression in protecting against induced anti-MPO Autoimmunity and AIMPOGN autoimmune focal necrotizing GN, we studied the de- Treatment of naïve mice with the anti-CD25 mAb, PC61, de- 2 2 velopment of glomerular injury occurring in Aire / ver- creased the Treg population significantly in MPO- and OVA- sus littermate WT mice, using our previously published immunized mice over a 16-day period compared with mice 2 2 model.7,32 Aire / mice developed augmented anti-MPO that were not treated with the depleting antibody (Figure 5A). GN quantified by histology (Figure 4A). Glomerular ab- The CD4+CD25+Foxp32 activated T cell population was not normalities quantified include necrosis, segmental prolif- affected by the depletion and therefore remained elevated after eration, hypercellularity, and capillary wall thickening immunization (Figure 5A). The timing of administration of (Figure 4B). Exacerbation of renal injury was also demon- PC61 was carefully planned to allow the predominant effects strated by elevated proteinuria (Figure 4C) as with to be on Tregs with minimum depletion of effector T cells after glomerular accumulation of injurious CD4+ T cells and MPO immunization. It is likely that because CD25 expression macrophages (Figure 4D). is much more prominent on Tregs before immunization, these

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IL-17A–producing lymphocytes from the draining LNs was significantly enhanced in MPO-immunized mice compared with OVA-immunized PC61-treated animals, suggesting that Tregs modulate Th1- and Th17-mediated autoimmune responses to MPO (Figure 5, C and D). The MPO-immunized mice treated with PC61 made enhanced autoantibody re- sponses to MPO compared with the MPO-immunized rat IgG-treated group (Figure 5E). C57BL/6 OVA-immunized mice injected with anti-CD25 antibody do not develop significant anti-MPO auto- antibody titers, showing that Treg depletion enhances autoimmune B cell re- sponses to MPO.

Immunodepletion of Tregs Increases the Severity of Glomerular Injury To test the importance of Tregs in the protection against ANCA-associated GN, we modified the protocol of experimental autoimmune anti-MPO GN6,7,32 to induce very modest disease in C57BL/6 mice using only a single MPO immunization. The slightly shorter 16-day experimental time course also ensured that the effects of Figure 2. MPO immunostaining of murine thymii demonstrated that thymic MPO PC61 on Tregs lasted for the full experi- expression was predominantly in neutrophils. Immunohistochemical staining for MPO 2 2 ment. PC61-treated mice developed protein on murine thymic tissue sections from Mpo+/+ mice (A), Mpo / mice (B), and 2 2 enhanced functional renal injury as dem- Aire / mice (C). The immunohistochemical staining is visualized by light microscopy. onstrated by an increased urinary protein/ The higher cell density region constitutes the cortex, whereas the lower cell density region constitutes the medulla. The MPO protein distribution is in the cortico- creatinine ratio compared with both con- 2 2 medullary junction in the normal Mpo+/+ and Aire / thymi. MPO protein is not de- trol rat IgG-treated and OVA-immunized 2 2 tected in Mpo / mice. Colocalization of thymic neutrophils (D–F) and macrophages PC61-treated mice (Figure 6, A and B). (G–I) with MPO in Mpo+/+ mice with MPO. The immunofluorescence is visualized by laser The development of GN as a result of ex- scanning confocal microscopy. (D) Neutrophils (Gr-1 green) are almost all positive for acerbated autoimmunity to MPO resulting MPO (dark blue) (E), as observed by the merged image MPO and Gr-1 (light blue) (F). from Treg depletion was also demonstrated Fewer macrophages are present as demonstrated by F4/80 (green) (G), but most are MPO histologically. Anti-CD25 pretreatment positive (dark blue) (H) as observed by the merged image MPO and F4/80 (light blue) (I). caused the development of increased seg- fi 3 – 3 – Original magni cation, 400 in A C; 800 in D I. mental glomerular necrosis (Figure 6C) in MPO-immunized mice, compared with OVA-immunized and control rat IgG- cells are more susceptible to PC61 depletion. PC61 depletion treated mice. Glomeruli of PC61-treated MPO-immunized resulted in an increase in the frequency of CD4+CD25 mice showed severe segmental necrosis, protein cast for- +Foxp32 activated T cell population compared with animals mation, and glomerular cellular proliferation compared with that were not treated with the depleting antibody (6.760.73106 non-Treg-depleted MPO-immunized mice and PC61-treated versus 4.560.73106; P,0.05, t test). OVA-immunized mice, which showed only relatively mild glo- The reduction in the Treg population after anti-CD25 merular cellular proliferation to no detectable injury treatment was associated with enhanced T cell responsiveness (Figure 6C). These outcomes also confirmed that the glomer- to MPO ex vivo. LN cells from MPO-immunized PC61-treated ular injury in this model results from MPO autoimmunity with C57BL/6 mice proliferated more vigorously in response to no significant contribution from the subnephritogenic dose of MPOthanmicethatweregivencontrolratIgGandthose anti-glomerular basement membrane (GBM) antibody, which that were treated with PC61 and immunized with OVA nonetheless plays a critical role of triggering disease by induc- (Figure 5B). The frequency of MPO-specificIFN-g– and ing glomerular deposition of the autoantigen, MPO.7

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Mice with AIMPOGN augmented by anti-CD25 pretreat- immature mTEC populations may still reflect an association ment developed significant glomerular influx of cellular with Aire. Indeed, this is supported by the loss of MPO mRNA 2 2 mediators, CD4+ T cells, macrophages, and neutrophils expression in both mTEC-hi and mTEC-lo subsets in Aire / compared with control rat IgG-treated C57BL/6 mice (Fig- mice. The observation that MPO (at the mRNA level) is ex- ure 7). Accumulation of CD4+ T cells, macrophages, and neu- pressed to the same extent in mTECs-hi and mTECs-lo (WT) trophils was minimal in OVA-immunized PC61-treated mice, cells suggests that downregulation of Aire precedes downregula- demonstrating that the influx of effectors was a result of MPO tion of MPO. immunization and augmentation of autoimmunity from Treg Thymic expression of autoantigens are often measured by depletion, and not the anti-GBM antibody alone. mRNA levels.13,16,34 However, the degree to which mRNA transcripts expressed by the mTECs are translated into whole, functional protein is unknown. We colocalized MPO protein DISCUSSION expression within neutrophils and macrophages, but not mTECs. During the positive and negative selection MPO is a major autoantigen in patients with microscopic processes, a large number of developing thymocytes die polyangiitis.33 The current studies demonstrate that in mice, through .40 Thus, the presence of neutrophils and MPO is expressed in the thymus in an Aire-dependent man- macrophages in the thymus is likely to be associated with their 2 2 ner. MPO deletion from the thymus as seen in both Mpo / involvement with clearance of apoptotic cells during thymo- 2 2 and Aire / mice increases the frequency of autoreactive cyte development. In the context of thymic antigen presenta- anti-MPO CD4+ T cells produced in response to MPO im- tion, MPO peptides are likely to be expressed by mTECs as a munization. The increased intensity of immune glomerular complex with MHC II at concentrations orders of magnitude 2 2 injury in MPO-immunized Aire / mice observed after the below that seen in neutrophils and macrophages and thus too deposition of MPO in glomeruli confirms the functional im- low for the protein to be detected by immunohistology. portance of this increased level of induced anti-MPO autoim- The importance of thymic MPO expression in generating 2 2 munity and suggests that Aire-dependent thymic expression tolerance to MPO was demonstrated using Mpo / and 2 2 of MPO functionally modifies the T cell repertoire in a manner Aire / mice. Both developed significantly higher frequen- consistent with central tolerance. cies of anti-MPO CD4+ T cells after MPO immunization To assess whether the intrathymic production of MPO than was observed in WT and littermate mice. Triggering 2 2 transcripts is involved in the central tolerance of autoreactive GN in Mpo / mice was not attempted, because the target anti-MPO cells, we determined the thymic cell populations in for disease-inducing CD4+ Teffectors, which is neutrophil- which MPO was expressed. We initially validated microarray derived MPO, is absent, rendering these mice disease resis- 2 2 results13,16,34 by using quantitative PCR (qPCR) to determine tant. However, in Aire / mice, MPO expression is normal MPO mRNA expression in the broad hematopoietic (CD45+) in hematopoietic cells, making these mice susceptible to and CD452 WTmice stromal cell populations as well as in the anti-MPO-associated GN. The enhanced GN observed in thymic epithelial cell subsets. MPO mRNA was found to be these mice occurred as a result of the increased frequency predominantly in the CD452 population and the predomi- of anti-MPO CD4+ Teffectors, which developed after MPO nant cell population expressing MPO transcripts was the immunization as a consequence of the lack of thymic MPO 2 2 mTECs. mTECs are found in the medullary region of the thy- expression in Aire / mice. This increase in anti-MPO au- mus and have an important role in self-tolerance by presenting toreactive CD4+ T cells was associated with the develop- peripheral autoantigens to developing T cells. Gene expression ment of significantly higher titers of anti-MPO ANCA in 2 2 2 2 profiling patterns in mTECs revealed that many TRAs, includ- Mpo / and Aire / mice. This was likely to be in part ing autoantigens in experimental and human autoimmune due to increased T cell help for B cells, but tolerance also diseases, are expressed by mTECs.31,35 The mTEC population involves the deletion of autoreactive B cells via mechanisms of is categorized into two subpopulations (mature and imma- B cell tolerance in the bone marrow. It is also likely that de- ture) according to their levels of MHC II and costimulatory letion of autoreactive anti-MPO B cells was similarly reduced. molecule CD80 expression,36 reflecting enhanced functional ItisimportanttonotethattheTregpopulationsinboth capacities of mature mTECs (mTEC-hi) to fully participate in groups of mice are comparable and that the elevated auto- 2 2 selection and deletion of autoreactive CD4+ T cells.16,37,38 In- reactivity observed in Mpo / mice is likely to be the result of terestingly, in this study, MPO mRNA levels were similar reduced negative selection rather than decreased Tregs or that between the mature and immature mTEC populations. MPO-specific Tregs were not produced in the thymi of these Although Aire expression has been associated with the mice. mTEC-hi population of epithelial cells, more recent evidence Low levels of autoreactive CD4+ T cells not deleted by demonstrate a biphasic expression before end stage terminal central tolerance normally persist in the periphery and are differentiation, where they downregulate CD80 and MHC II silenced by tolerogenic mechanismssuchasanergy, deletion,or to intermediate levels before their cell death.39 Thus, our find- ignorance. However, immunologic tolerance mechanisms may ings of MPO mRNA expression in both the mature and pose a threat in the maintenance of tolerance because these

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2 2 2 2 Figure 3. Assessment of autoimmunity in Mpo+/+, Mpo / ,andAire / mice immunized with MPO. The frequency of IFN- g–producing LN cells (A) and IL-17A–producing LN cells (B) is enhanced in Mpo+/+ mice compared with OVA-immunized controls, but 2 2 is significantly greater in Mpo / mice. (C and D) IFN-g–producing LN cells as well as T cell responsiveness to MPO by DTH mea- 2 2 surement are also enhanced in Aire / mice compared with Aire+/+ mice. Anti-MPO ANCA IgG titers (E), IgG1 (1:40) (F), and IgG2c 2 2 (1:80) (G) are higher in MPO-immunized Mpo+/+ mice versus OVA-immunized mice, but are significantly higher in Mpo / mice.

578 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 573–585, 2013 www.jasn.org BASIC RESEARCH

autoimmunity.42–45 Recent interest has fo- cused on the role of Tregs in peripheral tol- erance in inactivating autoreactive CD4+ T cells that escaped central deletion. Admin- istration of PC61 mAb to WT mice was as- sociated with a significant increase in the frequency of anti-MPO CD4+ T cells after MPO immunization. This is consistent with a role for Tregs in the maintenance of tolerance to MPO in the periphery. The functional importance of peripheral tolerance mechanisms in protecting against autoimmune anti-MPO-mediated GN was demonstrated by the develop- ment of significantly enhanced necrotiz- ing GN after triggering of disease by depositing MPO in the glomeruli of control and PC61-treated mice using anti-GBM antibody. Administration of a subnephritogenic dose of anti-GBM anti- bodies to immunized mice induces glo- merular neutrophil recruitment and degranulation, resulting in endogenous glomerular MPO deposition. Circulating autoreactive anti-MPO CD4+ T cells di- rect the development of focal necrotizing GN with appearances similar to those seen in human ANCA-associated GN.6 It is im- portant to note that the experimental model of OVA-immunized PC61-treated WT mice did not develop renal injury despite the fact that these mice could theoretically have had potentially PC61- induced augmented anti-GBM antibody- mediated injury. This control demonstrates that the subnephritogenic dose of anti-GBM antibody does not contribute to renal in- 2 2 jury, which results from anti-MPO-specific Figure 4. Histological injury and proteinuria were significantly increased in Aire / mice developing GN. Induction of GN results in enhanced focal necrotizing GN as- autoimmunity found in intact mice when sessed by periodic acid–Schiff staining of the kidneys (A) and quantified as the per- immunomodulated by Tregs. centage of glomerular segmental necrosis (B). (C) Functional renal injury quantified by These studies demonstrate that MPO is urinalysis is also augmented after GN induction in the absence of Aire as well as expressed in an Aire-dependent manner in glomerular CD4+ T cell and macrophage infiltrates (D). *P,0.05; **P,0.01. Original the thymus and, by this mechanism, is magnification, 3400. involved in the central deletion of poten- tially autoreactive anti-MPO CD4+ T cells. hyporesponsive autoreactive cells can be activated upon Peripheral tolerance through natural CD4+CD25+Foxp3+ antigen encounter under suitable conditions, such as inflam- Tregs also protects from the development of induced MPO mation from sepsis41 or as a result of “molecular mimicry” that autoimmunity capable of inducing focal necrotizing GN when might be able to mediate immune pathology and trigger MPO is planted in the glomeruli.

2 2 Dotted lines represent naive Mpo+/+ mice. (H) Anti-MPO ANCA IgG titers are also enhanced in Aire / mice compared with Aire+/+ control littermates. (I and J) The percentage of Tregs (CD4+CD25+Foxp3+) cells within the draining LNs in both studies (Mpo+/+ versus 2 2 2 2 Mpo / and Aire+/+ versus Aire / ) were the same. *P,0.05; **P,0.01; ***P,0.001. Imm, immunization.

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MedicalCentreAnimalFacility(Melbourne, 2 2 Australia). Mpo / mice46 were bred at the 2 2 MonashMedicalCentreAnimalFacility.Aire / mice were bred at the Department of Molecular Pathology, Centre for Cancer Biology, University of Adelaide Animal Facility. The Mpo+/+ and 2 2 Mpo / mice were used at 7–10 weeks of age. 2 2 The Aire+/+ and Aire / were used at 18–20 weeks of age. All experiments were approved by the Mon- ash Medical Centre Animal Ethics Committee.

Thymus Harvesting, Single-Cell Preparation by Enzymatic Digestion, and Sorting of TSC Subsets Thymi from adult male and female Mpo+/+, 2 2 2 2 Mpo / , Aire+/+,andAire / mice aged be- tween 1 and 4 months were harvested. To obtain sufficient numbers of TSC for qPCR analysis, the thymi were pooled containing five mice per group. The thymi were thoroughly cleaned of fat and connective tissue, the lobes separated, and the capsule nicked with fine scissors to re- lease thymocytes before digestion. The thymic lobes were placed in 50 ml of cold RPMI 1640 (Gibco, Grand Island, NY) in a 100-ml Schott bottle with a magnetic flea and stirred gently for 20–30 minutes at 4°C. The supernatant was then collected and the thymi transferred into fresh RPMI 1640. Any further thymocytes were re- moved by gentle mixing with a wide bore glass pipette until the supernatant was mostly clear of thymocytes. The washes were discarded. For the thymus digestion, 5 ml of Liberase (0.2% w/v; Roche, Penzberg, Ger- many)/DNAse (0.1%; Roche) enzyme mix per thymus digest tube was added and mixed gently Figure 5. Immunodepletion of Tregs (CD4+CD25+Foxp3+) is induced by mAb PC61 with a wide bore glass pipette and placed into a treatment (in control mice given rat IgG or immunized with control antigen [OVA]). 37°C waterbath for digestion. The digesting thy- Significant reduction of Tregs in the LN draining immunization sites is confirmed at the mic tissue was agitated gently every 5–10 mi- end of experiments. (A) Immunodepletion of Tregs (CD4+CD25+Foxp3+) by mAb nutes with a glass pipette. Every 15–20 minutes, fi PC61 (in control mice given isotype rat IgG1) signi cantly reduces the proportion of Tregs the supernatant was collected and kept on ice in the draining LNs of mice immunized with MPO or control antigen (OVA) compared with every 15–20 minutes in 15-ml tubes containing control-treated mice immunized with MPO. Conversely, deletion of Tregs (by PC61) 2 ml of FACS buffer (0.1% BSA/PBS with 0.5 mM results in a statistically increased percentage of effector T cells (CD4+CD25+Foxp32) EDTA) to neutralize enzymes. Fresh enzyme mix induced by MPO immunization. The proliferation of LN cells is enhanced after Treg depletion and MPO immunization (B), as is the frequency of IFN-g–producing cells (C) was then added to the digest tube. This was re- – and IL-17A–producing cells (D) compared with control OVA-immunized and non-Treg- peated 3 4 times until the thymi were completely depleted MPO-immunized mice. (E) Elevated levels of anti-MPO IgG titers are also digested. After digestion, all fractions were pooled observed. *P,0.05; **P,0.01; ***P,0.001. Imm, immunization. and filtered using 100-mm mesh and centrifuged at 4803g for 5 minutes at 4°C to collect cells. The single-cell suspensions of adult thymus prepared CONCISE METHODS by enzymatic digestionwere then subjected to CD45+ bead depletion for the isolation of TSCs. Mice After enzymatic digestion of the thymi, cells were resuspended in Mice were on a C57BL/6 background. C57BL/6 (Mpo+/+)micewere FACS buffer at a concentration of 107 cells per 95 ml buffer. CD45 purchased from Monash Animal Services and housed at the Monash microbeads were added at a concentration of 5 ml/107 cells. Samples

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Figure 6. GN is induced in mice treated with Treg-deleting mAb PC61 or control rat IgG. The protein/creatinine ratio (A) and glomerular segmental necrosis (B) are significantly enhanced in mice with Treg deletion. (C) Histologic evaluation confirms the increased incidence of severe segmental necrosis, protein cast formation, and glomerular cellular proliferation in periodic acid–Schiff-stained sections. Original magnification, 3400. ***P,0.001. Imm, immunization. were incubated on a rotating mixer at 4°C for 20 minutes. After the remaining 20 ml and the pellet was resuspended by flicking the Ep- incubations, any unbound beads were diluted by adding up to 103 pendorf tube gently. Lysis buffer was then added to the sample from volume of FACS buffer. Cells were centrifuged for 10 minutes at the RNEasy Mini Kit (Qiagen, Hilden, Germany), vortexed thor- 3003g, the supernatant discarded, and pellets resuspended at a con- oughly to lyse the cells, and snap-frozen in liquid nitrogen and stored centration of 0.53108 cells/ml before placing on the AutoMACS at 280°C for later RNA extraction. RNA was extracted per the Separator (Miltenyi Biotech, Bergisch Gladbach, Germany) for sep- RNEasy Mini kit instructions. RNA was reverse transcribed using aration using the “Depletes” program. Fractions were eluted into Superscript III (Invitrogen Life Technologies, Carlsbad, CA) and separate tubes. The negative fraction contained the CD452 TSCs. oligo-dT oligonucleotides (Invitrogen Life Technologies) according The CD45+ fraction was run through a second time to obtain any to the manufacturer’s protocol of the Superscript III first-strand syn- remaining CD452 TSCs. Once all CD452 cells were eluted, the neg- thesis system. ative fractions were pooled and centrifuged and the cell pellets were qPCR analysis of TSC subsets was performed on a Rotor-Gene collected for further analysis. 3000 (Qiagen) in 10-ml reactions using a Platinum SYBR Green After CD45+ hematopoietic cell fraction had been depleted, the qPCR SuperMix-UDG kit (Invitrogen Life Technologies) with CD452 TSC fraction was stained and sorted into the following sub- prevalidated qPCR primers from Qiagen. After initial holds for sets for qPCR analysis: mTECs-hi (CD452 EpCAM+ UEA-1+ MHC 2 minutes at 50°C (uracil-DNA glycosylase incubation) and then IIhi); mTECs-lo (CD452 EpCAM+ UEA-1+ MHC IIlo); cortical 2 minutes at 94°C (enzyme activation), PCR was performed with 40 TECs, or cTECs (CD452 EpCAM+ Ly51+ MHC II+); and non- cycles of 94°C for 15 seconds (denaturation), 60°C for 30 seconds TECs (EpCAM2 MHC II2). The dendritic cell population was (annealing and extension), and 1 cycle of melt analysis at 65°C–-95°C sorted based on CD45+ CD11c+ MHC II+. Cells were sorted (0.5°C increments per step, 5 seconds per step). The DD threshold using a BD Influx (BD Biosciences, Franklin Lakes, NJ). (CT) method was used to calculate relative levels of target mRNA. Glyceraldehyde-3-phosphate dehydrogenase was used as +/+ RNA Isolation and qPCR for Murine MPO the housekeeper gene and the Mpo CD452 population was After cell sorting, the cells were washed twice in nuclease-free PBS and used as the calibrator sample. Data are presented as mean 6 SEM cells were pelleted by centrifugation at 5003g for 5 minutes at 4°C. determined from two to three groups of five thymi pooled for each After the second wash, the supernatant was discarded down to the population.

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atthebaseofthetailatday0.Atday7,themice were immunized again with 20 mgofnmMPOin Freund’s incomplete adjuvant subcutaneously at theneck.Ninedaysafterimmunization,GNwas induced in all mice planting endogenous MPO in their glomeruli using an established tech- nique32 of administering 1.5 mg of sheep anti- mouse GBM globulin intravenously on 2 consecutive days to assess the development of renal injury. Administration of a subnephritogenic dose of anti-GBM antibody recruits and degranulates neutrophils, depositing MPO in the target organ in the same manner likely to occur in the human disease through neutrophil degranulation. Mice were humanly culled at day 20. The time course of this model was previously used and published by our group.7

Induction of MPO Autoimmunity and Immunodepletion by PC61 in the Model of AIMPOGN Systemic autoimmunity to recombinant mouse MPO (rmMPO), which induces autoreactivity to nmMPO,47 was assessed by immunizing 2 2 Mpo+/+ or Mpo / mice with 10 mg of rmMPO in FCA subcutaneously at the base of the tail. Control Mpo+/+ mice were immunized with 10 mg of OVA in FCA. Mice were humanely culled 10 days postimmunization when T cell autoreactiv- ity to MPO was established.8 The assessment of the role of Tregs in the model of experimental autoimmune anti-MPO Figure 7. Glomerular leukocytic infiltration in mice developing autoimmune anti-MPO GN was performed at day 22 by CD4+CD25 GN. Infiltration of glomerular CD4+ T cells (A), macrophages (B), and neutrophils (C) is +Foxp3+ Tregs depletion in naïve Mpo+/+ significantly enhanced in mice immunized with MPO (versus OVA control). However, Treg depletion with mAb PC61 is associated with significantly further augmented leukocyte mice (n=8, each group) by a single intraperito- infiltration of all leukocyte subpopulations. **P,0.01; ***P,0.001. Imm, immunization. neal injection of 1 mg of a well characterized anti-IL-2 receptor–specificTreg-deleting Expression of MPO in Mouse Thymi by monoclonal rat anti-mouse CD25 mAb, PC61,48 whereas control Immunoperoxidase Stainings and Immunofluorescence mice were administered 1 mg of isotype rat IgG1. At day 0, experi- For the detection of MPO protein, mouse thymi were fixed in Bouin’s mental autoimmune anti-MPO GN was induced by immunizing 8- fixative and embedded in paraffinwax,and3-mm-thick sections were week-old male mice (n=8, each group) with 40 mg of nmMPO in FCA stained using periodic acid–Schiff reagent. (450 ml subcutaneously at the base of the tail and the neck; Sigma, St. For colocalization studies, mouse thymi were embedded in OCT Louis, MO) to assess the role of Tregs in maintaining tolerance to and snap-frozen. Frozen tissues were cut at 6 mm and stained using MPO. PC61-treated control mice (n=8) were immunized with OVA rabbit anti-cow Cytokeratin (Dako, Glostrup, Denmark), mouse (Sigma) in FCA. Nine days after immunization, GN was induced in anti-mouse MPO FITC (Hycult Biotech, Uden, The Netherlands), all mice by planting endogenous MPO in their glomeruli using a well rat anti-mouse Gr-1 647 (BD Biosciences), or rat anti-mouse F4/80 established technique6,7,30 of administering 1.5 mg of sheep anti- 647 (BD Biosciences). Slides were examined on the same day by mouse GBM globulin intravenously on 2 consecutive days to assess confocal microscopy using a Ti-E invert microscope with a C1 con- the functional capacity of Tregs to immunomodulate induced ANCA- focal laser point scan head attached (Nikon, Tokyo, Japan). associated vasculitis. Mice were humanely culled 4 days (day 14) after the second intravenous injection. The single immunization with 2/2 Induction of MPO Autoimmunity in AIMPOGN in Aire nmMPO and the shorter overall time course of this experiment +/+ and Aire Mice were to induce very mild disease in control antibody-treated mice 2 2 Aire / (n=7) and control Aire+/+ littermates (n=4) were immunized and allow PC61 to have its effects on Tregs throughout the with 20 mg of native mouse MPO (nmMPO)47 in FCA subcutaneously experiment.

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Assessment of Systemic Autoimmune Responses to protein/creatinine ratios were determined. The urine samples from 2 2 MPO Aire+/+ and Aire / mice were tested by dipstick (Combur 10 Test; IFN-g and IL-17A production was assessed by ELISPOT (BD Bio- Roche) for proteinuria and scored on a 0–3+ scale. sciences and eBiosciences, respectively) with harvested draining LN To assess histologic renal injury, kidney tissue was fixed in for- cells. Both IFN-g and IL-17A ELISPOT assays were performed ac- malin and embedded in paraffin, and 4-mm tissue sections were cut 5 cording to the manufacturer’s instructions. Briefly, 5310 cells/well and stained with periodic acid–Schiff using coded slides. Because were incubated with or without 5 mg/ml of antigen (OVA or focal/segmental necrosis of glomeruli is a major characteristic of human rmMPO) for 18 hours at 37°C in 5% CO2 on ELISPOT plates pre- anti-MPO GN, this abnormality was used to quantify glomerular injury coated with purified anti-mouse IFN-g the previous night. The in mice in these studies. Segmental necrosis on glomerulus was diag- next day, IFN-g–secreting cells were detected using biotinylated nosed when .25% of the glomerular area was necrotized. The percent- anti-mouse IFN-g, streptavidin-horseradish peroxidase, and 3- age of glomeruli with segmental necrosis was used as a quantitative amino-9-ethyl-carbazole. The number of IL-17A–secreting cells indicator of the severity of histologic glomerular injury. The proportion 5 was determined by first incubating 5310 cells/well with or without of glomerular necrosis was determined by evaluating a minimum of 50 5 mg/ml of antigen for 18 hours at 37°C in 5% CO2 on ELISPOT glomerular cross-sections per mouse. plates precoated with purified anti-mouse IL-17A the night before. For assessment of glomerular macrophages, neutrophils, and CD4+ T The next day, IL-17A–secreting cells were detected using biotinylated cell infiltrations, frozen periodate lysine paraformaldehyde- anti-mouse IL-17A, avidin-horseradish peroxidase, and 3-amino-9- fixed kidneys were cut at 6 mm and stained using a three-layer ethyl-carbazole. Spots were enumerated with an automated ELISPOT immunoperoxidase technique, including FA/11 (anti-CD68) for mac- reader system. rophages, RB6–8C5 (anti-Gr-1; DNAX, Palo Alto, CA) for neutro- For the T cell proliferation assay, a single-cell suspension from phils, and GK1.5 (anti-CD4+; American Type Culture Collection, 5 theLNswaspreparedaseptically.Weincubated5310 cells/well Manassas, VA) for CD4+ T cells. A minimum of 40 glomeruli per in triplicate in the presence or absence of 5 mg/ml of antigen (OVA mouse were assessed using coded slides, and results are expressed as or rmMPO) at 37°C in 5% CO2 for 72 hours. Cells were pulsed cells per glomerular cross-section (c/gcs). with 0.5 mCi 3H-thymidine/well for the final 18 hours, and were then harvested onto glass fiber filters using an automated Statistical Analyses cell harvester. All data are presented as the mean 6 SEM. An unpaired t test was used Circulating mouse anti-MPO antibody titers in sera were mea- for statistical analysis, and one-way ANOVA and Tukey’s post hoc test sured by ELISA. The sera were prepared from cardiac puncture blood were used for multiple group comparisons. after mice were sacrificed. One hundred microliters of rmMPO or 5 mg/ml of nmMPO in 0.05 M carbonate/bicarbonate buffer (pH 9.6) was coated onto 96-well polystyrene microtiter plates (Nunc; ACKNOWLEDGMENTS Thermo Fisher Scientific, Waltham, MA) for 18 hours at 4°C. The assay was performed at room temperature. The absorbance values This study is supported by grants from the National Health and were read at 450 nm using a Bio-Rad 550 microplate reader (Bio-Rad, Medical Research Council of Australia (606684 to S.R.H and 1023059 Hercules, CA) and analyzed using Microplate Manager software (ver- to H.S.S.), the Swedish Society of Medicine (SLS-96661 to B.A.L), and sion 5.2; Bio-Rad). the Swedish Research Council (524-2010-6723 to B.A.L.).

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