Basic Immunology and Its Clinical Application (Advances in Experimental Medicine and Biology, 1444) [2024 ed.] 9819997801, 9789819997800

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Advances in Experimental Medicine and Biology  1444

Mitsuru Matsumoto   Editor

Basic Immunology and Its Clinical Application

Advances in Experimental Medicine and Biology Volume 1444

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2022 CiteScore: 6.2

Mitsuru Matsumoto Editor

Basic Immunology and Its Clinical Application

Editor Mitsuru Matsumoto Professor Emeritus Tokushima University Tokushima, Japan

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-981-99-9780-0    ISBN 978-981-99-9781-7 (eBook) https://doi.org/10.1007/978-981-99-9781-7 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore Paper in this product is recyclable.

Preface

Recent advances in our knowledge of immunology coupled with the development of new technologies are remarkable. However, I would think that knowledge of basic immunology has not been fully utilized at the bedside yet. Many people would admit that the vaccination initiated by Edward Jenner is one of the most successful examples of the so-called clinical application of basic immunology although this modern concept was not discernable at that age. Even after the discoveries of the development of mRNA vaccination against SARS-CoV-2 and effective immune checkpoint inhibition for cancer immunotherapy, there is still good room for expanding the clinical application of our knowledge of basic immunology. This book summarizes the recent achievements in this area and highlights what can be further explored to make immunology a more practical science. This book’s exploration of ongoing and upcoming applications of basic immunology may, in turn, deepen our understanding of basic immunology. In this way, we can say that basic immunology and its clinical applications are two wheels of the same cart. Our goal of this book is not intended to cover all the fields of the basic immunology-clinical application axis. Obviously, this is not an immunology textbook that covers all the basic immunological themes. Instead, we would like to focus on several good examples in the fields so that immunologists working in other fields would become interested in the application of their knowledge to clinical work. We will highlight the immunological fields that have been already applied as well as those that can be applied before too long. I am pleased to say that we have successfully achieved this mission in this book as follows. First of all, all the authors are the top runners in the corresponding areas. In this regard, I paid special attention to gathering authors who are well motivated to apply their knowledge of basic immunology to the bedside. As a result, we have covered a unique range of topics, from Toxoplasmosis to CAR T cells. I have also included several chapters regarding how the discoveries of the genes responsible for human immune-related diseases were successfully studied by animal models. Although this may look like the reverse of the application of basic immunology to clinical application, this will demonstrate how the knowledge obtained by the animal models is useful to achieve our task. I strongly believe that this book will attract many clinicians with a strong mind for research, as well as young and promising immunologists who have started their research careers. Tokushima, Japan

Mitsuru Matsumoto v

Contents

Part I From the Bench to the Bedside 1 Novel  Insights into the Autoimmunity from the Genetic Approach of the Human Disease����������������������������������������������������   3 Pärt Peterson 2 Learning  the Autoimmune Pathogenesis Through the Study of Aire ������������������������������������������������������������������������������������  19 Mitsuru Matsumoto and Minoru Matsumoto 3 E  xtrathymic AIRE-Expressing Cells: A Historical Perspective����������������������������������������������������������������������������������������  33 Dominik Filipp, Jasper Manning, and Jana Petrusová 4 Neoself  Antigens Presented on MHC Class II Molecules in Autoimmune Diseases������������������������������������������������������������������  51 Hui Jin and Hisashi Arase 5 Regulatory  T Cells for Control of Autoimmunity ������������������������  67 Ryoji Kawakami and Shimon Sakaguchi 6 Autoinflammatory  Diseases Due to Defects in Degradation or Transport of Intracellular Proteins ������������������������������������������  83 Izumi Sasaki, Takashi Kato, Nobuo Kanazawa, and Tsuneyasu Kaisho 7 Endosomal  Toll-Like Receptors as Therapeutic Targets for Autoimmune Diseases����������������������������������������������������������������  97 Kensuke Miyake, Takuma Shibata, Ryutaro Fukui, Yusuke Murakami, Ryota Sato, and Ryosuke Hiranuma Part II Manipulating the Immune System 8 Control  of the Development, Distribution, and Function of Innate-Like Lymphocytes and Innate Lymphoid Cells by the Tissue Microenvironment���������������������������������������������������� 111 Koichi Ikuta, Takuma Asahi, Guangwei Cui, Shinya Abe, and Daichi Takami

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9 Necroptosis  and Its Involvement in Various Diseases ������������������ 129 Hiroyasu Nakano 10 RNA  Metabolism Governs Immune Function and Response�������������������������������������������������������������������������������������������� 145 Masanori Yoshinaga and Osamu Takeuchi Part III Coopting with Microorganisms 11 Development  of Orally Ingestible IgA Antibody Drugs to Maintain Symbiosis Between Humans and Microorganisms�������������������������������������������������������������������������������� 165 Reiko Shinkura 12 TCR  Signals Controlling Adaptive Immunity against Toxoplasma and Cancer������������������������������������������������������ 177 Masaaki Okamoto and Masahiro Yamamoto Part IV Novel Methodologies for the New Era of Immunology 13 Molecular  Imaging of PD-1 Unveils Unknown Characteristics of PD-1 Itself by Visualizing “PD-1 Microclusters”���������������������������������������������������������������������� 197 Wataru Nishi, Ei Wakamatsu, Hiroaki Machiyama, Ryohei Matsushima, Yosuke Yoshida, Tetsushi Nishikawa, Hiroko Toyota, Masae Furuhata, Hitoshi Nishijima, Arata Takeuchi, Makoto Suzuki, and Tadashi Yokosuka 14 Development  of Immune Cell Therapy Using T Cells Generated from Pluripotent Stem Cells �������������������������� 207 Hiroshi Kawamoto, Kyoko Masuda, and Seiji Nagano 15 Dissecting  the Immune System through Gene Regulation ���������� 219 Hideyuki Yoshida 16 HLA  Genetics for the Human Diseases������������������������������������������ 237 Takashi Shiina and Jerzy K. Kulski

Contents

Part I From the Bench to the Bedside

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Novel Insights into the Autoimmunity from the Genetic Approach of the Human Disease Pärt Peterson

Abstract

Keywords

A u t o i m m u n e - p o l y e n d o c r i n o p a t h y -­ candidiasis-ectodermal dystrophy (APECED) is a monogenic inborn error of autoimmunity that is caused by damaging germline variants in the AIRE gene and clinically manifests with multiple autoimmune diseases in patients. Studies on the function of the AIRE gene, discovered in 1997, have contributed to fundamental aspects of human immunology as they have been important in understanding the basic mechanism of immune balance between self and non-self. This chapter looks back to the discovery of the AIRE gene, reviews its main properties, and discusses the key findings of its function in the thymus. However, more recent autoantibody profilings in APECED patients have highlighted a gap in our knowledge of the disease pathology and point to the need to revisit the current paradigm of AIRE function. The chapter reviews these new findings in APECED patients, which potentially trigger new thoughts on the mechanism of immune tolerance.

APECED · AIRE gene · Thymus · Immune tolerance · Autoimmunity

P. Peterson (*) Institute of Biomedical and Translational Medicine, University of Tartu, Tartu, Estonia e-mail: [email protected]

1.1 Introduction The immune system protects us from harmful agents while keeping body cells and tissues intact and functional. To maintain this delicate balance, it needs to be controlled and regulated. During the last decades, studies of human monogenic diseases and their parallel animal models have revealed an important understanding of the pathogenesis of these diseases. In addition, studies on certain so-called experiments of nature, have provided broader insight into the basic functioning of the human immune system and have underscored the fundamental roles of individual genes in keeping an immune balance between self and non-self. Their associated functional and regulatory pathways are central to the development and maintenance of immune cells, contributing to immune tolerance and homeostasis. Consequently, monogenic autoimmune diseases are highly informative for our understanding of immune balance as they link defined monogenic defects with clinical phenotypes.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 M. Matsumoto (ed.), Basic Immunology and Its Clinical Application, Advances in Experimental Medicine and Biology 1444, https://doi.org/10.1007/978-981-99-9781-7_1

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Monogenic inborn errors of autoimmunity typically manifest at an early age and have a more severe disease course than multifactorial autoimmune diseases. Another characteristic feature is that they often involve multiple organ systems with variable severity. While the cases of monogenic autoimmunity are individually rare, the prevalence of individual symptoms may vary considerably. This chapter discusses autoimmune-­ polyendocrinopathy-­candidiasis-ectodermal dystrophy (APECED) or autoimmune polyendocrine syndrome (APS1) as one of the most well-known examples of monogenic autoimmune disease. I will give a retrospective on the AIRE gene identification by our team in the 1990s and then an overview of the AIRE gene that is mutated in APECED, and discuss the clinical and immunological features of this disease.

1.2 The Identification of the AIRE Gene The monogenic basis of APECED disease was known already for a long time. In the 1960s, reports pointed to the clinical and genetic heterogeneity among patients with Addison’s disease and that Addison’s disease could be part of two separate and distinct clinical syndromes, depending on the type of associated disorders [1, 2]. This led later to the distinction between APS1 and APS2 [3]. Finnish physician Jaakko Perheentupa gave APS1 the alternative name APECED to highlight its clinical picture and monogenic etiology. My first encounter with APECED disease was when I started working at the University of Tampere, Finland, in the early 1990s. My supervisor was Professor Kai Krohn, who had already worked with APECED patient-derived autoantibodies in the 1970s [4]. These autoantibodies reacted with protein fractions extracted from the adrenal cortex. The topic of my PhD thesis became the cloning and characterization of these autoantigens, using lambda phage cDNA library screening. As a result of this, we were able to identify three target proteins belonging to the P450 cytochrome family—steroid 17 hydroxy-

P. Peterson

lase (P450c17), steroid 21-hydroxylase (P450c21), and side chain cleavage enzyme (P450scc) [5–7], associated with the Addison’s disease in APECED. Along with the screening of autoantibodies, we had several contacts with the patients and their family members, which enabled us to collect peripheral blood material for cells and serum, and other tissue samples from the Finnish patients. Therefore, quite soon after completing my thesis in 1996, we became interested in identifying the gene mutated in APECED. From the beginning, it was obvious that the gene should somehow function in the regulation of the immune system and impact the mechanism of immune tolerance, differentiating between self and non-self responses. At the same time, Professor Leena Peltonen’s research group at the University of Helsinki was working on gene identification, linking the disease mutations to 21q22.3, the terminal end of the longer arm of chromosome 21 [8]. The chromosomal region 21q22.3 was problematic as markers on this region showed an exceptionally high recombination rate, but their linkage disequilibrium analysis narrowed the critical genomic region to around 500 K base pairs. In the mid-1990s, the sequence of the human genome was not yet identified, but there was an ongoing global effort, the Human Genome Project, to sequence all the genes of the human genome. We soon understood that we needed to cooperate with experts in human genome analysis and gene cloning. Kai contacted Prof Stylianos Antonarakis from the University of Geneva and Dr. Hamish Scott, who did his postdoc in Geneva. They were a perfect team to cooperate on the identification and characterization of novel, human disease-associated genes, which were at that time mapped to fragments of the human genome sequence. They also had been collecting DNA samples from Swiss and North Italian APECED patients, which, along with Finnish samples, made up a cohort of patients from different ethnic origins. At that time, only a handful of genes were known in the 21q22.3 genomic region. One known gene localized to the APECED region was

1  Novel Insights into the Autoimmunity from the Genetic Approach of the Human Disease

encoding liver-type phosphofructokinase (PFKL), which later turned out as a neighboring gene to AIRE.  PFKL, however, was soon excluded as an APECED gene as it had a very distinct function and was expressed outside of immune tissues. Another candidate gene that was located at the end of chromosome 21 and sharing amino acid sequence similarity with P450 steroid hydroxylase autoantigens, lanosterol synthase (LSS), proved to be quickly wrong too. The sequences of the large fragments of genomic DNA from chromosomal regions became available in GenBank as bacterial artificial chromosomes (BACs). The genome was mapped by markers, usually specific nucleotide repeats, whose relative positions or links to each other were known. However, one of the challenges in the search for the genes was that their exact position and order on BAC and other large segments of the genome were unknown. It was also not clear how to map the correct exons and introns to the genomic sequence, and the main key to this was to use a large collection of expressed sequence tags (ESTs) and exon prediction programs. ESTs were fragments of mRNA sequences derived through sequencing reactions performed on randomly selected clones from cDNA libraries of human tissues. They were usually short, less than a few hundred nucleotides, but were produced and made available in GenBank in large batches. Nevertheless, finding a gene was more like seeking a needle in a haystack. Most of the work was using a Blast program to pull together the EST fragments and align them to the genomic BAC regions. The hope was to get an intact structure of the new gene with proper exons and introns, and their junction regions using Grail and Genscan programs, which looked for open reading frames and exon-intron boundaries in the genomic sequence. The next task was to align the translated sequence to known proteins to see whether it has meaningful similarity to protein domains. Then we sequenced the exon regions in the patients and healthy controls to identify possible mutations that disrupt the reading frame, repeating this procedure over again. We went through a number of exons of candidate genes, including CFPA410

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then known as C21ORF2, which is 30  K base pairs away from the AIRE gene and causes inherited retinopathies [9]. Finding novel genes from the human genome was a tedious task. Stylianos Antonarakis suggested that we should team up with Professor Nobuyoshi Shimizu from Keio University, Tokyo, whose research group worked on sequencing the terminal region of chromosome 21 as part of the Human Genome Project. Human chromosome 21 was considered a model chromosome for the Human Genome Project because of its small size and its association with Down syndrome. Prof Shimizu had a large lab that worked on sequencing human chromosomal fragments and on the identification of novel disease genes, which also included APECED.  They had constructed a human genomic bacterial artificial chromosome (BAC) library consisting of tens of thousands of clones, each containing an average DNA insert size of 100 K base pairs, and covering the human genome three times. Dr. Jun Kudoh, who was the key investigator on the APECED project from Shimizu’s lab, and his colleague Dr. Kentaro Nagamine, studied the BAC contigs from the 21q22.3 region consisting of the sequences of DNA segments that overlapped in a way that provided a contiguous representation of this region [10]. One of these contigs included a 450 K base pair region covering the critical region identified by Peltonen’s team. They were immediately eager to work together on this joint effort. The collaboration between the three research groups consisted of frequent emails and phone calls, and rapid sharing of the data when one or another team discovered a small additional piece of this larger mosaic. The entire collaboration was highly inspiring and stimulating, and at times, it felt like an obsession as thinking about the APECED gene took up most of my awake brain capacity. Meanwhile, we continued working to characterize the expression of the putative transcripts in immune tissues. We used PCR-amplified cDNA probes from predicted exons to perform northern blotting with RNA from immune organs. Most of the probes gave no signal, but one probe, with the name GR1, gave strong positive bands identify-

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ing several transcripts in the thymus tissue and lower expression in lymph nodes. However, the spleen, peripheral blood leukocytes, bone marrow, and appendix were all negative, as well as the autoimmune target tissues of APECED (adrenal gland, liver, and pancreas). Considering our main hypothesis that APECED is caused by a defect in immune tolerance, the finding of strong expression in the thymus was very promising. In the summer of 1997, the sequencing at Shimizu’s laboratory revealed APECED mutations in the gene, which overlapped with the GR1 probe that hybridized to the thymic RNA in the northern blot. This was the breakthrough in the hunt for the gene. The novel gene was located just proximal to the earlier known PFKL gene, which we have excluded as the APECED gene. The gene had 14 exons, and the mutation in exon 6, causing a stop codon at position 257 (R257X), was present in several patients. Two Swiss patients from one family and four Finnish patients were homozygous for the R257X allele, while other Finnish patients were heterozygous. The sequencing at Shimizu’s lab, Keio University, soon identified other mutations in the same gene. Further rapid amplification of cDNA ends (RACE) showed a new transcript that consisted of 1635 base pairs and also identified minor splice variants. The initial working name was the APECED gene. However, using the disease name for a gene was considered misleading as there were examples where defects in several genes caused a disease or vice versa; various mutations in a gene could cause different forms of diseases like muscular dystrophy. Therefore, we initially proposed to name AIR for Autoimmune Regulator. When the paper by Nagamine et  al. was submitted to Nature Genetics, the editors notified us that another transcript is called AIR and we should change the name. Indeed, the noncoding antisense transcript from the IGF2R gene, involved in genetic imprinting, was called AIR in the literature (now AIRN). To make the distinction, we renamed the gene AIRE. From its protein sequence, it was obvious that AIRE was related to transcriptional control as it had a nuclear localization signal and two PHD-­

P. Peterson

type zinc finger motifs. Further cloning and transfection of the cDNA in fusion with GFP showed its location in nuclear dot-like bodies [11]. The mRNA in situ hybridization and antibodies to AIRE revealed its expression in rare epithelial cells in the thymic medulla and in vivo locations in similar nuclear bodies as after the transfection. The finding that medullary epithelial cells in the thymus express AIRE was compatible with the defect in immune tolerance seen in APECED, and this was something that we expected as we knew that the negative selection of potentially self-reacting lymphocytes is taking place in the thymic medulla [12]. In parallel with our group, the team led by Prof Leena Peltonen identified the gene and came to very similar conclusions. Their gene mapping approach was also based on studying BAC and cosmids clones, and their fluorescence in situ hybridization confirmed the chromosomal localization. By large-scale sequencing, they ended up with the APECED gene and its exon-intron structure in the vicinity of PFKL. Because R257X was present in most of the Finnish patients, they named it as a Finnish major mutation and calculated its carrier frequency as 1:250  in Finland. Both papers were published in Nature Genetics in the December 1997 issue back-to-back [13, 14]. After the gene identification, we had several meetings at Keio University hosted by Prof Shimizu. At that time, it was fascinating to see the huge sequencing capacity of their facilities, having a row of multi-capillary ABI3700 machines, the main DNA sequencer of that time, whereas we had only one. Shimizu had many projects ongoing in parallel including the identification of Parkin (PRKN) gene causing juvenile Parkinson’s disease and the sequencing of medaka, a small freshwater fish living in rice paddies in Japan. Through this international collaboration, we later cloned and characterized another AIRE-neighboring gene, DNMT3L, which interacts with DNMT3A and DNMT3B in de novo DNA methylation [15, 16]. Over the subsequent 2 years, our team spent considerable time identifying APECED-causing mutations in the AIRE gene [17–19].

1  Novel Insights into the Autoimmunity from the Genetic Approach of the Human Disease

The discovery of AIRE contributed to a new period of understanding thymic immune tolerance. At the same period with AIRE cloning, the seminal studies on the expression of tissue-­ specific antigens in the medullary thymic epithelial cells (mTECs), called “ectopic” or “promiscuous” gene expression, were made by Bruno Kyewski and Ludger Klein, and others [12, 20, 21]. Their experiments demonstrated that quantitative as well as qualitative differences in transcriptional regulation of intrathymic gene expression can set the threshold for tolerization and lead to the exclusion of self-antigens from central tolerance and thus predispose to autoimmunity. A critical proof demonstrating Aire role in immune tolerance came from studies on Aire-­ deficient mice by Mark Anderson, Christophe Benoist, and Diane Mathis and colleagues at Harvard University in 2002 [22].

1.3 AIRE Has Domains Characteristic of the Transcriptional Regulator AIRE protein sequence and domain structure are well conserved among mammalians [23]. Most of the placental mammalian species have substantial similarity not only in protein sequence but also in upstream regulatory elements of the gene. Expectedly, the primates share the highest sequence similarity throughout the chromosomal region of the gene. The structure of Aire is also conserved in two other mammalian groups; marsupials (opossum) and monotremes (platypus), which both are evolutionally conserved with placental mammals in their immunoglobulin and T cell receptor loci [24]. Birds, amphibians, and fish seem to have a gene that has similar domains with Aire in the N-terminal region but not in C-terminus, and its role in immune mechanisms in lower animal classes remains unknown. AIRE domains highlight its role in transcriptional regulation: a conserved nuclear localization signal, the HSR and SAND domains in its N-terminus, and two PHD-type zinc fingers. Close to the HSR domain AIRE has a bipartite

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nuclear localization signal for the transport into the nucleus, where it forms dot-like structures, which resemble PML bodies [25–27]. The SAND is a conserved domain found in a variety of proteins involved in transcriptional regulation and chromatin remodeling [28]. SAND was originally suggested to function as a DNA binding domain; however, instead of the critical KDWK amino acid motif needed for DNA binding, AIRE has a KNKA sequence. Within the N-terminal HSR and SAND regions, it has a similarity with the Sp100 protein, an autoantigen in primary biliary cirrhosis. The Sp100 protein family contains other members, Sp110 and Sp140 that, interestingly, also localize subcellularly to the nuclear bodies and have one PHD finger but have a bromodomain instead of the second PHD finger [29–31]. The structural similarity of Sp100 protein family members with AIRE provokes interesting parallels as they too have a role in immune regulation. AIRE contains two PHD-type zinc fingers, which are the structurally conserved chromatinbinding domain of approximately 65 amino acids and are found in about 150 human proteins [32]. Many PHD fingers act as nucleosome interaction determinants and selectively bind unmodified or methylated at lysine 4 histone H3 tails. Indeed, the first PHD finger of AIRE interacts with unmethylated histone H3 at lysine 4 (H3K4me3) and mediates AIRE binding to chromatin [33– 37]. AIRE-PHD fingers are needed for its transcriptional activity, and pathological missense or truncation mutations in the patients disrupt their structures. The second AIRE PHD finger, however, does not interact directly with histone H3 even though it is important for the transactivation capacity. The two AIRE-PHD fingers have independent non-interacting folds connected by an unstructured proline-rich linker, and the second PHD finger likely acts in the interaction of other proteins via so far unknown chromatin-associated nuclear partners [38]. Through binding to critical chromosomal factors, AIRE enhances transcriptional elongation and promotes the upregulation of hundreds of genes in thymic epithelial cells [39–41]. The top interaction partners of AIRE are DNA-dependent

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protein kinase (DNA-PK) and topoisomerase 2-alpha (TOP2A), which work together in relaxing torsional tensions of the DNA double helix [42–45]. Abramson et al. described in detail the AIRE interacting complex that contains DNA-PK, TOP2 but also poly ADP ribose polymerase 1 (PARP-1), and Ku proteins [42]. The interaction of AIRE with DNA-PK and TOP2A appears to be a key event in AIRE target gene expression. AIRE is an atypical transcriptional regulator with an unusual capacity to enhance gene expression without promoter and RNA processing elements [46]. It has been shown to interact with chromatin at super-enhancer regions where it regulates the expression of multiple genes, often located in clusters or larger chromosomal domains [47, 48].

1.4 AIRE Has a Unique Role in Guarding Self-Tolerance AIRE has a central and unique role in the immune system as it promotes the expression of a large number of self-antigens in the thymus [49, 50]. The expression in mTECs matches with the high expression of MHC class II and CD80 to ensure efficient antigen presentation and is tightly controlled. AIRE is regulated by TNF family member RANK-RANKL and CD40-CD40L signaling, both activating the NF-κB pathway [51–53]. This upstream enhancer region is shared across mammals and is indispensable for RANK-induced Aire expression as a defect in these NF-κB sites completely abolishes Aire expression in the thymus [54, 55]. In the more proximal promoter region, the gene has a CpG island and its demethylation is necessary for the AIRE expression [56]. Also, a gene locus-insulating chromatin modifier CTCF and several transcriptional regulators such as Irf4 and Irf8 [57] and Ets factors, common for epithelial-specific expression [58], add to the tight regulation of the gene. In addition to AIRE expression in mTECs, multiple studies have shown extrathymic expression of AIRE in secondary lymphoid organs, where it may support peripheral immune tolerance with various functions [59–61]. However, the peripheral

P. Peterson

expression level is substantially lower compared to the thymus, and its contribution to the peripheral immune tolerance beyond its role in the thymus would need more studies. Aire-deficient mouse studies have provided insight into understanding the mechanism of Aire. Studies on Aire-deficient mouse models of APECED revealed that Aire plays an important role in the generation of T cell tolerance in the thymus, mainly by inducing the expression of a large repertoire of transcripts, many of which encode proteins normally restricted to organs residing in the periphery [49]. The lack of AIRE expression in the thymus results in a defect in the clonal deletion of autoreactive T cells and ultimately to autoimmunity [22, 62, 63]. Airedeficient mice exhibit a defective negative selection of thymic CD4+ T cells, leading to the presence of autoreactive T cells in the periphery. Aire deficiency is closely associated with decreased production of thymic regulatory T cells (Tregs), which play a key role in suppressing autoreactive responses in the periphery. Airedeficient mice show decreased numbers of Tregs already in the perinatal period with a specific subtype of Tregs lacking very early in life [64]. Most of the Treg specificities are independent of Aire, whereas some Treg-specific clones are Aire-dependent and likely drive the autoimmunity [65, 66]. Several studies have confirmed lower numbers of Tregs and/or impaired suppressive capacity in APECED patients [67–70] and fewer Treg clones with common TCRβ sequences, which instead were found among conventional T cells [71]. Single-cell studies have revealed remarkable heterogeneity in mTECs [47, 72, 73]. A recent study by Michelson et al. divided mTECs into various subpopulations based on their resemblance to peripheral tissues, which were called “mimetic cells” and suggested that Aire is partially and variably required for the development of these subpopulations [74]. They showed that Aire engages lineage-defining transcription factors to instigate peripheral cell types, in the context of earlier studies proposing the thymic medulla to function as a mixture of epithelial cells [75, 76]. Mouse studies have shown that

1  Novel Insights into the Autoimmunity from the Genetic Approach of the Human Disease

Aire is also necessary for the maturation program of mTECs and that the mTEC compartment does not mature normally in the absence of Aire [77, 78]. As its deficiency in mice leads to impaired differentiation of the mTEC-high population in the thymus, it may affect the self-antigen expression via a scarcity of these various epithelial cell populations by developmental block [79].

1.5 APECED Starts Early in Childhood and Is Heterogenic APECED is a rare disease but more frequent in some populations that have been isolated in the past, such as Iranian Jews (1:9000), Sardinians (1:14,000), and Finns (1:25,000) [80]. Although it can be also underdiagnosed, the prevalence in other countries is very low and varies from 1:43,000 in Slovenia to 1:500,000 in France [81, 82]. APECED usually starts in early childhood and is known for its classical triad of chronic mucocutaneous candidiasis (CMC), hypoparathyroidism, and Addison’s disease, the onset following the aforementioned order. However, multiple other diseases, and the majority of these are autoimmune, can manifest in the patients [80, 81, 83]. CMC usually affects the skin and mucosal tissues. Hypoparathyroidism, which is relatively rare alone, is the most frequent and sometimes the only endocrine component in APECED.  Sporadic Addison’s disease is also rare, but common in APECED patients. Overall, the number and severity of clinical symptoms vary greatly between patients, even among those with identical mutations. The symptoms tend to increase with age with the median age of onset for the classical triad of the disease (CMC, hypoparathyroidism, and Addison’s disease) being 11 years [84]. The incidence at a later age is quite rare. The reasons why the parathyroid gland and adrenal cortex are the main autoimmune targets in APECED remain unknown, and would deserve investigation, but are challenging to study as animal models do not follow the same disease pattern. However, the selective destruc-

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tion of the adrenal cortex should prompt future studies addressing the pathological etiology of Addison’s disease in APECED. Other endocrine and nonendocrine autoimmune disorders may occur in APECED patients. The most common are type 1 diabetes (T1D), autoimmune thyroid disease, and hypophysitis. APECED (also known as APS1) is similar to APS2 in that patients with both autoimmune polyendocrinopathies develop Addison’s disease and have an increased risk to have T1D and/or autoimmune thyroid disease. However, APS2 is multifactorial and is associated with HLA, while APECED is not. Gonadal insufficiency is more common in females. Patients often suffer from gastrointestinal autoimmunity, enteropathy with malabsorption, hepatitis, autoimmune gastritis with or without vitamin B12 and iron deficiencies, and exocrine pancreatitis. The patients have other lesions affecting ectodermal structures namely enamel dysplasia, vitiligo, alopecia, keratoconjunctivitis, and nail dystrophy. In addition, urticaria-like erythema and hyposplenism or splenic atrophy have been reported [84, 85], which increase the susceptibility to bacterial infections. Interestingly, APECED appears to exhibit substantial clinical heterogeneity between ethnic populations, although systematic analyses are rare due to the low prevalence. For example, American patients do not seem to develop the full classical triad of candidiasis, hypoparathyroidism, and Addison’s disease as the first manifestations. A systematic analysis of over 150 APECED patients from the US revealed enrichment of nonendocrine autoimmune manifestations relative to other APECED cohorts [83]. They developed a hexad of nonendocrine disease manifestations, including urticarial eruption, autoimmune gastritis, intestinal malabsorption, autoimmune pneumonitis, autoimmune hepatitis, and Sjögren’s-like syndrome, with much higher frequency (40–80%) compared to European APECED patients (