The autoimmune targets in IPEX are dominated by gut epithelial proteins
Authors: Daniel Eriksson, Rosa Bacchetta, Hörður Ingi Gunnarsson, Alice Chan, Federica Barzaghi, Stephan Ehl, Åsa Hallgren, Frederic van Gool, Fabian Sardh, Christina Lundqvist, Saila M. Laakso, Anders Rönnblom, Olov Ekwall, Outi Mäkitie, Sophie Bensing, Eystein S. Husebye, Mark Anderson, Olle Kämpe, Nils Landegren
Journal: Journal of Allergy and Clinical Immunology (2019)

Regulatory T (Treg) cells play a key role in immune system homeostasis by suppressing detrimental immune responses against self-proteins, in particular in the intestine.1Tanoue T. Atarashi K. Honda K. Development and maintenance of intestinal regulatory T cells.Nat Rev Immunol. 2016; 16: 295-309Crossref PubMed Scopus (277) Google Scholar Patients with mutations in the gene FOXP3 display a severe defect of Treg-cell function that results in IPEX syndrome (OMIM 304790) characterized by impaired immune tolerance and multiorgan autoimmunity. Dermatitis, type 1 diabetes, and in particular autoimmune enteropathy are hallmarks of the syndrome. Intestinal Treg cells normally suppress immune responses against harmless dietary antigens and commensal microorganisms, but the intestinal self-proteins that are guarded by Treg cells have not yet been fully identified.2Russler-Germain E.V. Rengarajan S. Hsieh C.S. Antigen-specific regulatory T-cell responses to intestinal microbiota.Mucosal Immunol. 2017; 10: 1375-1386Crossref PubMed Scopus (50) Google Scholar Patients with IPEX and enteritis typically harbor autoantibodies against harmonin (USH1C) and villin, which are structural proteins predominantly expressed in brush border epithelia of the intestine and renal tubules.3Kobayashi I. Imamura K. Kubota M. Ishikawa S. Yamada M. Tonoki H. et al.Identification of an autoimmune enteropathy-related 75-kilodalton antigen.Gastroenterology. 1999; 117: 823-830Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Kobayashi I. Kubota M. Yamada M. Tanaka H. Itoh S. Sasahara Y. et al.Autoantibodies to villin occur frequently in IPEX, a severe immune dysregulation, syndrome caused by mutation of FOXP3.Clin Immunol. 2011; 141: 83-89Crossref PubMed Scopus (43) Google Scholar Autoantibodies against glutamate decarboxylase-65 (GAD-65, GAD2),5Baekkeskov S. Aanstoot H.J. Christgau S. Reetz A. Solimena M. Cascalho M. et al.Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase.Nature. 1990; 347: 151-156Crossref PubMed Scopus (1466) Google Scholar which is a marker for islet cell autoimmunity in type 1 diabetes, have been detected in patients with IPEX already in their first week of life. These studies suggest that the autoimmune pathogenesis of IPEX involves a humoral response that can be exploited for identifying the immune targets.6Kinnunen T. Chamberlain N. Morbach H. Choi J. Kim S. Craft J. et al.Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells.Blood. 2013; 121: 1595-1603Crossref PubMed Scopus (112) Google Scholar Hitherto, no studies have been undertaken to characterize autoantigens in IPEX syndrome in a comprehensive way. Here, we sought to identify the autoantibody targets in patients with IPEX on a broad scale to gain better understanding of the autoimmune manifestations of IPEX and insights to peripheral tolerance mechanisms mediated by Treg cells. We used panels of more than 9000 human proteins to characterize autoantibody targets in 14 patients with IPEX and 24 healthy controls (detailed methodology and additional tables/figures are available in this article's Online Repository at www.jacionline.org). In total 20 proteins clearly stood out as patient-specific autoantigens (Fig 1; see Table E1 in this article's Online Repository at www.jacionline.org). Among them, the 3 previously known IPEX autoantigens included in the panels were all present: harmonin,3Kobayashi I. Imamura K. Kubota M. Ishikawa S. Yamada M. Tonoki H. et al.Identification of an autoimmune enteropathy-related 75-kilodalton antigen.Gastroenterology. 1999; 117: 823-830Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar α-IFN,7Rosenberg J.M. Maccari M.E. Barzaghi F. Allenspach E.J. Pignata C. Weber G. et al.Neutralizing anti-cytokine autoantibodies against interferon-α in immunodysregulation polyendocrinopathy enteropathy X-linked.Front Immunol. 2018; 9: 544Crossref PubMed Scopus (11) Google Scholar and GAD-65,5Baekkeskov S. Aanstoot H.J. Christgau S. Reetz A. Solimena M. Cascalho M. et al.Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase.Nature. 1990; 347: 151-156Crossref PubMed Scopus (1466) Google Scholar verifying a reliable detection of autoantibodies (Fig 1, C, and Fig E1). Interestingly, the new autoantigens showed predominant expression in tissues typically affected in patients with IPEX, in particular in the intestinal epithelium. In the brush border of enterocytes, ANKS4B and harmonin form a complex that anchors transmembrane links reaching between the tips of microvilli to the enterocyte cytoskeleton (Fig 2, A). Competition assays revealed no autoantibody cross-reactivity, suggesting that harmonin and ANKS4B were independent autoantigens (see Fig E2 in this article's Online Repository at www.jacionline.org). Another novel autoantigen, ACSL5, is primarily expressed in the small intestine, where it regulates the enterocyte proliferation along the crypt-villus axis. The protein with the strongest autoantibody signal in the protein array data, HNF4A, is a nuclear receptor with an expression pattern that closely follows that of harmonin and ANKS4B. HNF4A has important functions in differentiation of intestinal and renal epithelia. To validate the proteome-wide autoantibody data with an independent method, we developed radio-ligand binding assays for a subset of the new candidate autoantigens ANKS4B, HNF4A, PPARG, and RXRA, as well as for 3 established autoantigens: GAD-65, harmonin, and villin. We extended the group of patients with IPEX with 3 additional patients in the validation experiments (n = 17). To better evaluate the clinical specificity, we also included a larger group of healthy subjects (n = 75) and sera from other autoimmune and inflammatory diseases: inflammatory bowel disease (n = 20), autoimmune polyendocrine syndrome (APS) type 1 (n = 20), and APS-2 (n = 40). The novel autoantigens ANKS4B, HNF4A, PPARG, and RXRA were all confirmed in the radio-ligand binding assay and showed high specificity for IPEX in the investigated clinical material (see Figs E3 and E4 in this article's Online Repository at www.jacionline.org). Among the 16 patients with IPEX and enteropathy, 11 of 16 (69%) were positive for autoantibodies against harmonin, 9 of 16 (56%) against ANKS4B, 7 of 16 (44%) against HFN4A, and 8 of 14 (57%) against ACSL5 (ACSL5 autoantibody data were available only for the discovery case group) (Fig 2, B). Hence, 11 of 16 patients (69%) displayed autoantibodies against 1 or more autoantigens in the intestinal epithelium. Although the number of patients without enteropathy was too small to assess any statistical associations with specific autoantibodies, we noted that the only patient without enteropathy was negative for all the intestinal autoantibodies. Two patients were negative for all investigated autoantibodies. Both these patients displayed clinical manifestations and mutations in FOXP3 typical for IPEX (Table E1). Besides HNF4A, our list of top 20 targets from the protein array contained 10 additional nuclear receptors, all with highly correlated results. Because nuclear receptors share an evolutionary template featuring a highly conserved DNA-binding domain and a moderately conserved ligand-binding domain, we performed competition assays to test for cross-reactivity. Focusing on a subset of the nuclear receptor autoantigens, HNF4A, RXRA, and PPARG, we could demonstrate a cross-reactivity between these autoantigens (see Fig E5 in this article's Online Repository at www.jacionline.org). Hence, the nuclear receptor autoantibodies were specific for a subset of nuclear receptors, but cross-reactive within this subset. Investigations of truncated nuclear receptor protein in the array indicated that the nuclear receptor autoantibodies targeted a shared epitope in the ligand-binding domain. IPEX can rapidly become fatal if not aggressively treated. Although immunosuppressive therapy can give transient or partial improvement of disease symptoms, allogenic hematopoietic stem cell transplantation (HSCT) offers the only possibility of cure. By investigating consecutive samples from patients with IPEX taken before and after treatment, we found that autoantibodies remained detectable following immunosuppressive treatment with rapamycin but completely disappeared 1.5 years following successful HSCT (see Fig E6 in this article's Online Repository at www.jacionline.org). The replenishment of stem cells with normal functioning FOXP3 might explain disease suppression and disappearance of autoantibodies following HSCT not observed with immunosuppressive treatments. Although this is by far the most comprehensive study of autoantibody targets in IPEX, less than half of the protein coding genes were covered in our screen. For instance, the previously identified skin autoantigen in IPEX, keratin 14,8Huter E.N. Natarajan K. Torgerson T.R. Glass D.D. Shevach E.M. Autoantibodies in scurfy mice and IPEX patients recognize keratin 14.J Investig Dermatol. 2010; 130: 1391-1399Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar was not present in the investigated panel. In summary, we have identified intestinal self-antigens that are implicated in the enteropathy of IPEX, which may advance the understanding of FOXP3-dependent Treg cells in controlling immune tolerance toward self-antigens in the gut. Patients with IPEX were included from Italy, the Unites States, and Germany. All patients fulfilled clinical diagnostic criteria for IPEX, requiring a disruptive FOXP3 mutation and manifestation of enteropathy and/or endocrinopathies. Blood donors not known to suffer from autoimmune disease were included as healthy controls, both in the discovery (n = 24) and in the validation control (n = 75) groups. The validation control groups also included additional subjects with autoimmune diseases: 20 subjects with autoimmune polyendocrine syndrome type 1 (APS1); 20 subjects with APS2 with type 1 diabetes; 20 subjects with APS2 without type 1 diabetes; 10 subjects with ulcerative colitis; and 10 subjects with Crohn disease. Protein arrays containing more than 9000 full-length human proteins (ProtoArray v5.1, PAH05251020, ThermoFisher, Waltham, Mass) were used to study autoantibodies in serum samples diluted 1:2000. The arrays were probed according to the manufacturer's protocol using the recommended reagents: (PA055, ThermoFisher), Alexa Fluor 647 goat antihuman IgG (A21445, ThermoFisher), Dylight 550 goat anti-GST (#DY550011-13-001, Cayman Chemicals, Ann Arbor, Mich), and the LuxScan HT24 (BioCapital) micro-array scanner. Autoantibodies were measured by immunoprecipitation of antigens radiolabeled with 35S-methionine. Complementary DNA clones were purchased from Origene and subcloned into pTNT or Sp64 expression vectors (L5610, Promega, Madison, Wis), which were used for transcription and translation in vitro (Promega TNT Systems): USH1C SC336653 (NM_001297764), ANKS4B SC306281 (NM_145865), HNF4A SC307232 (NM_178849), PPARG SC322218 (NM_138712), RXRA SC118299 (NM_002957), VIL1 SC123608 (BC_017303). Immunoprecipitation was conducted in 96-well filtration plates (Millipore, Burlington, Mass). Serum samples (2.5 μL) were incubated overnight at 4°C with 30'000 cpm of radiolabeled protein. Immune complexes were captured with protein A Sepharose (Protein A Sepharose 4 Fast Flow, GE Healthcare, Chicago, Ill) during a 45-minute incubation. After 10 washing steps, radioactive decay was measured in a beta counter (1450 Microbeta Triplex, Wallac). All serum samples were tested in duplicate in separate wells. BSA (4%) was included in each plate to serve as a negative standard. For each antigen, a selected IPEX or APS1 patient serum was included in all 96-well plates to serve as a positive standard for index calculation. Autoantibody indices were calculated according to the following: (sample value − negative standard value) ÷ (positive standard value − negative standard value) × 100. The upper limits of the normal range were defined as an index value of 5 SDs above the mean of the healthy controls. Quantile normalization was applied to reduce the effects of technical variation between different protein array experiments.E1Bolstad B.M. Irizarry R.A. Astrand M. Speed T.P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6185) Google Scholar Protein targets affected by printing contaminations were identified using a previously described approach and removed (n = 4).E2Landegren N. Sharon D. Freyhult E. Hallgren A. Eriksson D. Edqvist P.H. et al.Proteome-wide survey of the autoimmune target repertoire in autoimmune polyendocrine syndrome type 1.Sci Rep. 2016; 6: 20104Crossref PubMed Scopus (38) Google Scholar Protein targets were sorted by their difference between cases and controls. For each protein, the strongest signal intensity in the control group was subtracted from the second to strongest patient signal intensity. Thereby, targets with elevated signal in 2 or more patients could be selected. Clusters of autoantigens were identified by correlation. All study subjects gave their informed consent for participation in accordance with their local institutional review boards (IRBs). The study was performed in accordance with the Declaration of Helsinki and approved by the local ethics committees (2008/296-31/2 and 2016/2553-31/2 by Stockholm, UP 00-052 by Uppsala, IRB protocol TIGET02 and TIGET06 by Ospedale San Raffaele, and IRB protocol 34131 by Stanford University). The STRING database was used for functional enrichment analysis of PFAM protein domains, and GO-terms describing biological processes.E3Szklarczyk D. Morris J.H. Cook H. Kuhn M. Wyder S. Simonovic M. et al.The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible.Nucleic Acids Res. 2017; 45: D362-D368Crossref PubMed Scopus (3584) Google Scholar Protein primary structures retrieved from the array manufacturer were aligned with Clustal omega.E4Goujon M. McWilliam H. Li W. Valentin F. Squizzato S. Paern J. et al.A new bioinformatics analysis tools framework at EMBL-EBI.Nucleic Acids Res. 2010; 38: W695-W699Crossref PubMed Scopus (1233) Google Scholar, E5Sievers F. Wilm A. Dineen D. Gibson T.J. Karplus K. Li W. et al.Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.Mol Syst Biol. 2011; 7: 539Crossref PubMed Scopus (7957) Google Scholar Mutations were annotated using the gene transcript hg19_knownGene_uc004dnf.4, chrX:49106897-49121288. RNA-Seq data from the GTEx database were used to investigate protein expression.E6Carithers L.J. Ardlie K. Barcus M. Branton P.A. Britton A. Buia S.A. et al.A novel approach to high-quality postmortem tissue procurement: The GTEx Project.Biopreserv Biobank. 2015; 13: 311-319Crossref PubMed Scopus (366) Google Scholar, E7Lonsdale J. Thomas J. Salvatore M. Phillips R. Lo E. Shad S. et al.The Genotype-Tissue Expression (GTEx) project.Nat Genet. 2013; 45: 580Crossref PubMed Scopus (3530) Google Scholar, E8Available at: www.gtexportal.org. Accessed August 22, 2017.Google Scholar To detail the tissue distribution of novel autoantigens, we investigated immunohistochemical stainings retrieved from the Human Protein Atlas.E9Available at: www.proteinatlas.org. Accessed February 15, 2018.Google Scholar, E10Uhlen M. Fagerberg L. Hallstrom B.M. Lindskog C. Oksvold P. Mardinoglu A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (5734) Google ScholarFig E2Competition assays for USH1C and ANKS4B. USH1C and ANKS4B autoantibodies were tested for cross-reactivity. Radio-ligand binding assays were performed in presence of increasing amounts of unlabeled protein. Radiolabeled antigens (*) were competed against unlabeled protein. The x-axes denote the ratios of labeled to unlabeled protein. No competition was observed, suggesting that USH1C and ANKS4B were independent antigens.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Verification of novel autoantigens with independent methods in extended case and control groups. The previously identified autoantigens harmonin (USH1C) and glutamate decarboxylase-65 (GAD2), and the novel candidate autoantigens harmonin-interacting protein (ANKS4B), HNF4A, RXRA, and PPARG, showed elevated signals in the protein array screening (A), and were validated using radio-ligand binding assays (B). All 14 patients with IPEX from the discovery screen and 3 new patients with IPEX were assessed. Additional control subjects were included: 75 healthy blood donors; 20 subjects with APS1; 40 subjects with APS2 (10 with and 10 without type 1 diabetes mellitus); and 20 subjects with inflammatory bowel disease (10 with ulcerative colitis and 10 with Crohn disease).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Comparison of protein array and RLBA results. Patients with IPEX who had weakly elevated signals in the protein array screen came out positive in the validation experiments using the sensitive radio-ligand binding assays. The x-axes display the fluorescence signal intensities detected in the protein array experiment on a logarithmic scale. The y-axes display autoantibody index from the RLBAs. Each data point represents a patient with IPEX (n = 14). Dotted lines mark the cutoff levels for the RLBAs. RLBA, Radio-ligand binding assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Nuclear receptor autoantibodies target a shared epitope in the ligand-binding domain. A, The RLBA results for PPARG and HNF4A autoantibodies were similar. Results from single patients with IPEX are connected with lines (n = 14). B-D, Competition assays were used to study autoantibody cross-reactivity. Radiolabeled nuclear receptor autoantigens (*) were competed against unlabeled protein of the same autoantigen species, other nuclear receptor autoantigens, and ANKS4B as a negative control. The x-axes denote ratios of labeled to unlabeled protein. E, The heatmap displays signal intensities for the nuclear receptors in the protein array ordered by sequence similarity. The 4 patients with IPEX with the strongest signals for nuclear receptor autoantigens are included. The strongest signal among healthy controls has been summarized in 1 column, to illustrate that all healthy controls had weak signals for all nuclear receptors. RLBA, Radio-ligand binding assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Autoantibodies are undetectable following allogenic HSCT. A, The autoantibody profiles detected in consecutive samples from 4 patients with IPEX undergoing immunosuppressive treatment and/or HSCT. Each subpanel includes data from a single patient. Complete normalization of autoantibody levels was seen 1.5 years and 5 years after HSCT. B, A principle component (PC) space calculated from RLBA results. The investigated patient and control samples are indicated with dots. Patients sampled before (diamonds) and after (squares) HSCT moved (connecting lines) away from the scattered cluster of patients with IPEX (red) to the cluster of healthy controls (black), as autoantibodies became undetectable. RLBA, Radio-ligand binding assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Clinical and genetic characteristics of patients with IPEX syndromePatientAge at samplingEnteropathyT1DMDermatitisAdditional diagnosesFOXP3 mutation10 yYes, 1 moNoYes, 1 moAutoimmune hepatitis; nephrotic range proteinuria, hematuriac.1270_1272delinsC (p.C424L-fs*34)228 yYes, 5 moNoYes, 5 moArthritis; ITP; renal cell carcinoma; lymphomac.816+2 T del (splice donor variant)310 yGastritis, 8 yNoNoLow-molecular-weight proteinuriac.210+1 G>C (splice donor variant)410 yYes, 4 moYes, birthYes, birthAIHA; thrombocytopeniac. 1040 G>A (p.R347H) (FKH dom)53 yYes, 1 moNoNoNonec.750-752del (p.E250del)65 moYes, 1 moYes, birthNoNonec.736-1 G>A (splice acceptor site)717 yYes, 2 moNoNoNonec.1222 G>A (p.V408M)81 yYes, 6 moYes, 10 dYes, 3 moNonec.1117 TT>GC (p.F373A) (FKH dom)918 yYes, 1 moYes, 2 yYes, 1 moAIHA; alopecia; autoimmune thyroiditis; arthritisc.1150 G>A (p.A384T) (FKH dom)106 yYes, 40 dNoYes, 40 dAIHA; lymphadenopathy; CMV hepatitisc.1078 C>T (p.L360F)113 yYes, 2 yNoNoNonec.1190 G>A (p.R397Q)1228 yYes, 7 moYes, 18 yYes, 18 yHepatitisc.1040 G>A (p.R347H)131 yYes, 3 moYes, 2 wYes, 1 moAutoimmune thyroiditis; neurodevelopmental delay; hepatitisc.210+2 T>G (splice donor variant)141 yNoYes, 10 moNoNonec.210+2 T>G (splice donor variant)1527 yYes, 3 moYesYesNonec.1150 G>A (p.A384T) (FKH dom)161 yYes, 4 moNoYes, 2 moNonec.816+5C>T (splice donor variant)173 yYes, 2 moNoYes, 3 moAIHAc.751-753delGAG (p.Glu251del)AIHA, Autoimmune hemolytic anemia; CMV, cytomegalovirus; FKH dom, forkhead domain; ITP, immune thrombocytopenic purpura; T1DM, type 1 diabetes mellitus. Open table in a new tab AIHA, Autoimmune hemolytic anemia; CMV, cytomegalovirus; FKH dom, forkhead domain; ITP, immune thrombocytopenic purpura; T1DM, type 1 diabetes mellitus. We thank the Autoimmunity Profiling National Facility in Stockholm, Science for Life Laboratory, for excellent technical assistance.