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AUTOIMMUNITY AND

Topic Editors Luigi D. Notarangelo and Rosa Bacchetta

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Frontiers in August 2013 | and Immunodeficiency | 1 AUTOIMMUNITY AND IMMUNODEFICIENCY

Topic Editors: Luigi D. Notarangelo, Harvard , USA Rosa Bacchetta, Fondazione Centro San Raffaele Del Monte Tabor, Italy

Immune regulation results from a finely tuned network of distinct mechanisms operating throughout life and balancing the need to clear and prevent self-aggression. Primary (PIDs) are “experiments of nature” where the ability to fight against is deeply impaired. The study of patients with PIDs has been instrumental to identify and characterize key components and mechanisms Image created by Aisha Sauer and Luigi D. Notarangelo using that govern development Servier Medical Art. and function of the . Recently, it has become clear that in congenital monogenic the ability of the immune system to build and maintain active tolerance to self can be specifically altered, so that autoimmune symptoms may easily prevail over infections in these . In addition, increasing observations have brought the attention to the fact that hypomorphic in genes that control T and/or development are often associated with clinical and laboratory features of , thus expanding the spectrum of PID phenotypes. For example, mutations in genes driving development can lead to defective lymphostromal cross-talk in the and impinge of negative selection of self-reactive T cells and/or Treg function. Similarly, disorders of B cell development may associate with defects of editing and/ or with abnormalities of peripheral B cell homeostasis. On the other hand, can provoke defective immune responses by targeting and/or immune cells.

Frontiers in Immunology August 2013 | Autoimmunity and Immunodeficiency | 2 This Research Topic will focus on i) summarizing updated clinical and immunological features of diseases characterized by immune dysregulation of known and still undefined origin and ii) gathering new insights into the mechanisms of T and B cell development, function and interaction, in order to broader the comprehension of the pathogenesis of autoimmunity and to ultimately advance the definition of novel therapeutic strategies.

Frontiers in Immunology August 2013 | Autoimmunity and Immunodeficiency | 3 Table of Contents

05 Immunodeficiency with Autoimmunity: Beyond the Paradox R. Bacchetta and L. D. Notarangelo 06 Immune Dysregulation, Polyendocrinopathy, , X-Linked Syndrome: A Paradigm of Immunodeficiency with Autoimmunity Federica Barzaghi, Laura Passerini and Rosa Bacchetta 31 Autoimmunity in Wiskott–Aldrich Syndrome: An Unsolved Enigma Marco Catucci, Maria Carmina Castiello, Francesca Pala, Marita Bosticardo and Anna Villa 45 Autoimmune Dysregulation and Purine Metabolism in Adenosine Deaminase Deficiency Aisha Vanessa Sauer, Immacolata Brigida, Nicola Carriglio and Alessandro Aiuti 64 The STAT5b Pathway Defect and Autoimmunity Takahiro Kanai, Jennifer Jenks and Kari Christine Nadeau 72 APECED: Is This a Model for Failure of T Cell and B Cell Tolerance? Nicolas Kluger, Annamari Ranki and Kai Krohn 84 Pathogenesis of Autoimmunity in Common Variable Immunodeficiency Klaus Warnatz and Reinhard E. Voll 90 Autoimmune Cytopenias In Common Variable Immunodeficiency Jenna C. Podjasek and Roshini S. Abraham 97 TH17 Cells in Autoimmunity and Immunodeficiency: Protective or Pathogenic? Ashish K. Marwaha, Nicole J. Leung, Alicia N. McMurchy and Megan K. Levings 105 Dendritic Cells a Double-Edge Sword in Autoimmune Responses Giada Amodio and Silvia Gregori

Frontiers in Immunology August 2013 | Autoimmunity and Immunodeficiency | 4 Editorial published: 12 April 2013 doi: 10.3389/fimmu.2013.00077 Immunodeficiency with autoimmunity: beyond the paradox

R. Bacchetta 1* and L. D. Notarangelo 2*

1 San Raffaele Telethon Institute for Gene (HSR-TIGET), Division of Regenerative Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy 2 Division of Immunology and the Manton Center for Orphan Research, Children’s Boston, Harvard Medical School, Boston, MA, USA *Correspondence: [email protected]; [email protected] Edited by: Eric Meffre, Yale University School of Medicine, USA Reviewed by: Eric Meffre, Yale University School of Medicine, USA

The association of immunodeficiency and autoimmunity may Autoimmune polyendocrinopathy--ectodermal dystro- ­represent a paradox, yet it has been described in an increasing num- phy (APECED) is an autosomal recessive condition due to ber of conditions. Use of unbiased genomic approach to identify of the (AIRE) gene. Patients with APECED novel forms of primary immunodeficiencies (PIDs), along with in- present with predominant organ specificaut­ oimmunity and autoanti- depth functional studies in biological samples from affected indi- bodies with multiple specificities. AIRE has been shown to play a criti- viduals continue to unravel novel mechanisms underlying immune cal role in allowing expression of self- in the thymus, thereby dysregulation in patients with altered ability of fighting pathogens. permitting deletion of self-reactive T or their diversion In particular, it has been clearly established that genetic defects that to Treg cells. Thus APECED stands as the prototypic monogenic dis- affect T and B cell development compromise not just the ability order of central T cell tolerance. While it is still questionable whether to generate a diversified repertoire of lymphocytes capable of rec- deficiency of AIRE also affects , recent data indicate ognizing multiple pathogens, but also impinge on mechanisms of that the autoimmune-associated tissue damage may not be primarily central and peripheral tolerance, hence favoring autoimmune and due to autoantibodies, but rather to autoreactive CD8+ T cells. inflammatory manifestations. Moreover, recent studies in patients affected with Common Yet, the diagnosis of autoimmune symptoms in the context of Variable Immunodeficiency, a condition in which proper specific PIDs is troublesome, the prognosis unclear, and the treatment chal- production is deficient in favor of pathogenic autoanti- lenging. In the present collection of manuscripts, several experts body secretion, have highlighted the importance of mechanisms in the field provide an overview of the spectrum of different forms that control B cell development and receptor editing in maintaining of monogenic defects of the immune system manifesting also with immune homeostasis. autoimmunity, and discuss established and novel mechanisms Finally, two manuscripts call the attention to the dual role of cer- involved in immune dysregulation. tain cell types and their ability to acquire different immunological Studies on patients with Immunedysregulation-Polyendo- functions depending on the environment in which they differenti- crinopathy‑Enteropathy-X-linked (IPEX) Syndrome, have paved ate, as described for Th17 cells and dendritic cells, at the end of the the way to understand the phenotype arising from impaired Topic. Possibly, the future of medicine should aim to implement peripheral tolerance due to dysfunctional regulatory T cells physiological plasticity and to empower epigenetics modifications (Treg) expressing mutated FOXP3. However, this important T in order to recover from inborn errors of Nature. cell subpopulation can also be affected in other forms of PID, Received: 19 February 2013; accepted: 09 March 2013; published online: 12 April 2013. such as Wiskott–Aldrich syndrome (WAS) and adenosine deami- Citation: Bacchetta R and Notarangelo LD (2013) Immunodeficiency with autoimmunity: nase (ADA) deficiency. In these disorders, the underlying genetic beyond the paradox. Front. Immunol. 4:77. doi: 10.3389/fimmu.2013.00077 defect affects multiple cell types, resulting in impaired immune This article was submitted to Frontiers in Primary Immunodeficiencies, a specialty of defense, but also poor Treg function. Similarly, STAT5B muta- Frontiers in Immunology. Copyright © 2013 Bacchetta and Notarangelo. This is an open-access article distributed tions disrupt an essential intracellular transcriptional activa- under the terms of the Creative Commons Attribution License, which permits use, dis- tor for Treg cells, causing reduction of Treg number in affected tribution and reproduction in other forums, provided the original authors and source individuals. are credited and subject to any copyright notices concerning any third-party graphics etc.

www.frontiersin.org April 2013 | Volume 4 | Article 77 | 5 REVIEW ARTICLE published: 31 July 2012 doi: 10.3389/fimmu.2012.00211 Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: a paradigm of immunodeficiency with autoimmunity

Federica Barzaghi 1,2, Laura Passerini 1 and Rosa Bacchetta1*

1 Division of , Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy 2 Vita Salute San Raffaele University, Milan, Italy

Edited by: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare Luigi Daniele Notarangelo, Harvard monogenic primary immunodeficiency (PID) due to mutations of FOXP3, a key transcrip- Medical School, USA tion factor for naturally occurring (n) regulatoryT (Treg) cells.The dysfunction ofTreg cells is Reviewed by: Andrew Gennery, Newcastle the main pathogenic event leading to the multi-organ autoimmunity that characterizes IPEX University, UK syndrome, a paradigm of genetically determined PID with autoimmunity. IPEX has a severe Nancy Bunin, Children’s Hospital of early onset and can become rapidly fatal within the first year of life regardless of the type Philadelphia, USA and site of the mutation. The initial presenting symptoms are severe enteritis and/or type- *Correspondence: 1 mellitus, alone or in combination with eczema and elevated serum IgE. Other Rosa Bacchetta, San Raffaele Telethon Institute for Gene Therapy, autoimmune symptoms, such as hypothyroidism, cytopenia, , nephropathy, arthri- San Raffaele Scientific Insitute, Via tis, and alopecia can develop in patients who survive the initial acute phase. The current Olgettina 58, 20132 Milano, Italy. therapeutic options for IPEX patients are limited. Supportive and replacement e-mail: [email protected] combined with pharmacological are required to control symptoms at onset. However, these procedures can allow only a reduction of the clinical manifesta- tions without a permanent control of the disease. The only known effective cure for IPEX syndrome is transplantation, but it is always limited by the avail- ability of a suitable donor and the lack of specific guidelines for marrow transplant in the context of this disease. This review aims to summarize the clinical histories and genomic mutations of the IPEX patients described in the literature to date. We will focus on the clinical and immunological features that allow differential diagnosis of IPEX syn- drome and distinguish it from other PID with autoimmunity. The efficacy of the current therapies will be reviewed, and possible innovative approaches, based on the latest high- lights of the pathogenesis to treat this severe primary of childhood, will be discussed.

Keywords: IPEX, FOXP3,Treg, , neonatal diabetes, neonatal eczema, HSCT

INTRODUCTION early onset while highlighting the occurrence of unusual symp- Immune dysregulation, polyendocrinopathy, enteropathy, X- toms. The genetic analysis is always required for accurate diagnosis, linked (IPEX) syndrome is a rare monogenic primary immun- although other tests such as tissue biopsy and/or odeficiency (PID), characterized by multi-organ autoimmunity. It detection are important, as complementary tools, in the diagnostic is caused by mutations in the factor forkhead box p3 process and follow-up. (FOXP3), the master gene of T regulatory (Treg) cells. The dis- IPEX syndrome can be fatal in early infancy if not recognized, ease shows an X-linked hereditary pattern: only males are affected, therefore a timely diagnosis is essential to start appropriate treat- whereas the carrier mothers are healthy. ment. Treating IPEX patients poses a threefold challenge: autoim- Although IPEX syndrome is a rare disease, the recent increase munity, infections supported by the autoimmune damage, and the in the number of patients referred for diagnosis suggests that severity of the overall picture. Both novel and existing therapeutic the occurrence of the disease has been underestimated so far. At approaches will be discussed with an emphasis on the central role present, 63 FOXP3 mutations have been published, for an overall of Treg cell impairment in the pathogenesis of IPEX syndrome. number of 136 patients described, and of these about half have been diagnosed in the last 3 years. This also indicates that the GENETICS OF IPEX SYNDROME awareness of the disease has been growing with a better under- Immune dysregulation, polyendocrinopathy, enteropathy, X- standing of the role of FOXP3 and Treg cells in maintaining linked syndrome was described for the first time in 1982 in a large peripheral tolerance. family with 19 affected males across five generations,as an X-linked Overall, the analysis of cases reported so far (Table 1) confirms syndrome with that was lethal in most male infants by the relevance of the three main clinical manifestations and their the first months or years of life (Powell et al., 1982). Only 20 years

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 6 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy  weeks days months months Outcome Exitus Exitus Exitus Exitus 10 14 years Exitus 10 2 years Exitus 12 Exitus 19 5 weeks 1/5, alive 4/5 age na Alive Alive, A – – HSCT PD, CSA, , IgIV, na IgIV IgIV, CS Dexamethasone, CSA na TPN, plasma IV Therapy cholangitis , nodularis; ; infections, clinical findings Hypothyroidism; infections, sepsis Sepsis , hypothyroidism, , peritonitis, thrombocytopenia, peritonitis Infections, sepsis 3/5, food allergy5/5 Cachexia, hypotonia, , Autoimmune cytopenia Anemia Additional µ g/L ); 3 00 (/mm IgE (IU/mL) 3170; 9999– 40,0 230–900; 755–3492 936; 250 500; 22 na na na na 620; 30 , hyper IgE + Peeling skin + + + − − + + + + + 5/5 4/5 na; 4/5 + − + − + − + Diarrhea T1DM Eczema+ Eos + − (Vomiting) − − (Atonic gut) − (Ileo) + + + 5/5 – – – na diagnosis Post mortem Post mortem mortem na Age at onset 6 weeks 2 months 3 weeks Birth Birth 2 weeks – 3 weeks − 3 months # # # # A 1 month 9 years A A A T T BirthT Post > > > > 4A > G; Mutation Age at > c.1150G c.1290_1309 del_insTGG c.1150G c.1150G c.1189C > c.1189C c.1189C > Gc.1113T na > c.1150G c.1044 + c.750_752 delGGA f f , , f f f f f f f f , 2 , 1 f f f ◦ , F1 , F1 , F1 f f f na F1-V1 F1-IV10 F1-V5 1 3 F1-V4 2 3 3-F1; F2 − 14, 15, 27, 28 V6 V2 V7 al. al. by Pt al. | Clinical features, therapy and outcome in reported IPEX patients. Wildin Wildin et al. y-Lahad and Wildin , (2001) et al. (2001) Ferguson et al. (2000) , Bennett et al. , (2001b) McGinness et al. (2006) (2000) , Bennett et al. (2001b) (2000) , Bennett et al. (2001b) Reported Peake Ferguson et Ferguson et Lev , (1996) et al. (2001) (2001) Chatila et al. (2000) Wildin et Table 1

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 211 | 7 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy (Continued) years years years Exitus Exitus Exitus 4 months age na 19 3 years Exitus 4.5 months Exitus 2 years 7 months Exitus 14 5 years Exitus 10 Alive Alive rofecoxib, A, FK506, HSCT na FK506, betamethasone na MPD, FK506, HSCT PD, CS HSCT , rituximab, IgIV Steroids, CSA, FK506, MTX, rituximab, IgIV, HSCT na , AHA, – Hypothyroidism, thrombocytopenia, sepsis ITP , cholestatic hepatitis Infection (sepsis) , mild hepatitis, progressive renal insufficiency eczema, hypothyroidism, neutropenia, infections tubulonephropathy* tubulonephropathy*, infections, sepsis Lymphadenopathy, , ITP AHA, immune Thyroiditis, Thyroiditis, + + na na na; na na na na; na osis + − Ichthy Ichthyosis na; 1750− ITP,AHA, autoimmune − + − + + + − − + + + + − + + + + + + + + + ears – na na na na 13 y 9 years ys na na 15 da na 3 months < 1 month na 2 months # A A 459A T > > + 3T > G > G113T 1 month 4 months G c.1150G delCT c.227delT c.111 c.1 > c.1040G > c.748_750 delAAG, c.543C > Gc.1087A na c.1044 , 3, f f , 1 , 4 f na f f f f f f 1 4 1 1 1 3 F2, 5 c.1293_1294 2 2 2 al. al. ashi ennett et al. (2001) B et al. (2001, , 2011) Fuchizawa et al. (2007) , Otsubo et al. (2011) Kobay Baud et , (2001b) Kobayashi et al. (2011) (2002) , McMurchy et al. (2010) (2002) Kobayashi et al. (2001, 2011) Wildin et Wildin et al.

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 8 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy  years ears ears Outcome Exitus 6 weeks 10 11years 7 y na na 2 y 15 years 4 months 9 years Alive Alive Alive Alive age Alive age Alive Alive A A, , IgIV, Steroids, FK506, MMF,HSCT Alive na PDN, AZA, CS PD, CSA, IgIV, G-CSF CSA, PD HSCT Steroids, FK506, MPD, CS HSCT Steroids, PD, rapa AZA, rapa AZA, rapa AZA, CSA, MTX, Therapy – – clinical findings the pancreas Hepatitis ridging nails, autoimmune neutropenia, severe anemia, subclinical thyroiditis sepsis interstitial nephritis Lymphoid infiltration of Additional 0 ); 3 na (/mm IgE (IU/mL) na na; 2895–7275 na; 300 na; 763 na; 2000 – 768; 8423 – + − + + na; 17,370 CSA induced chronic − − − − − + + + + ; 33 Alopecia, longitudinal − + − + + + Diarrhea T1DM Eczema Eos + + + + + + + + diagnosis na 4 months < 1 year Age at 2 months 2 weeks – onset 3 weeks 2 months < 2 months < 4 months 7 years 10 years neonatal 3 months # A 7 months 9 years 7T > G c.227delT Mutation Age at c.227delT > c.1150G c.111 + + promoter region c.968- 20A > C c.1117- > GC 1118TT 3 f f ◦ na f na f na 1 1, 4, 1, 2 1 3 1 1, 12, 12, 12 A-1 A-2 al. by Pt al. | Continued et al. (2003) Nieves et al. (2004) (2005) Reported Owen (2005) , Fuchizawa et al. (2007) , Kobayashi et al., (2011) Otsubo et al. (2011) Mazzolari et Bindl et al. (2005) Bacchetta et al. (2006) , Gambineri et al. (2008) , McMurchy et al., (2010) Passerini et al. (2011b) Tanaka et Table 1

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 211 | 9 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy (Continued) ys ears ears ears ears Exitus 7 y 22 y Exitus 54 da < 2 years 5 months 6 years 9 y 4 y 10 years 7 years 4 years Alive Alive Alive Alive Alive Alive Alive Alive PD, , CSA, Intermittent FK506 None PD, MPD FK506, infliximab na FK506, steroids, steroids FK506 MPD, CSA, HSCT PDN, PD, AZA alive na rituximab, HSCT TPN TPN, CSA, , arthritis, Recurrent Hypothyroidism, Infections lymphadenopathy psoriasiform dermatitis, hepatomegaly hypothyroidism, MGN, food , infections lymphadenopathy, hepatosplenomegaly and , recurrent arthritis -responsive Infections Allergic asthma AHA, ITP Alopecia, AHA, Thyroiditis 2780; 374 N; 200 na na; high na; high na; high–; 1564 food allergies 552; 28,800 Hypothyroidism, N; 74 na; high Thyroiditis, AHA na + + − + + + + − − § + + - + + + − − + + + + + + + + + + + + + + + na na 22 years Post mortem na na 6 months < 1 years mortem ys neonatal 18 da 1 day na na na 4 months na A 4 days Post C T, T 14 months 7 years AC 4A > G T > A Neonatal + AG 1GG c.543C > c.970T > c.454 > c.1-7G > c.1169G c.210_210 + c.751_753 delG c.751_753 delGAG g.-6247_- 4859del c.303_304 delTT c.323C > f 5 , , f f f f , 5 , 5 f f f na na na f 6 2 1 1 1 2-p2 1, 2 3, 2, 1, 2 c.3G > 2 2 2-p1 3 ti al. al. Rao 07) , et al. (2006) (2006) De Benedet Myers et Gavin et Moudgil et al. (20 (2006) et al. (2007)

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 10 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy  years years years years Outcome Exitus 79 days 4 9 5 years 1 9 6 years 8 years Alive Alive Alive Alive Alive Alive Alive A, PD, AZA, infliximab, uximab, PD, NGT, illeostomy, mercaptopurine, steroids Imuran, CS FK506, MMF,PD, HSCT alemtuzumab, HSCT Rapa HSCT rit Rapa FK506, IgIV FK506 TPN, FK506, TPN, FK506, TPN, steroids, TPN, NGT, Rapa, Therapy allergies, reactive allergies, cheilitis, – clinical findings Food infections onychodystrophy, recurrent infections, sepsis, Hp Food infections, ITP,motor delay, hypoglycemic seizures, anemia of chronic diseases, osteopenia, hypogammaglobulinemia airways disease, AHA, infections recurrent infections, sepsis food allergies, AHA, MGN, Recurrent airway AHA Additional ); 3 > 3000 Food allergies, cheilitis, > 2000 34 (/mm IgE (IU/mL) na; 1 na na na 2400; 365– na; 6 950; + + + − + Rush na; 5320 – − na na na − § − + − + Diarrhea T1DM Eczema Eos + + + Colitis + Colitis + Colitis + + + ears na diagnosis 2 y na na na mortem Age at eeks onset < 2 months na na 3 weeks na 5 w A birth Post A na T 2 months na > G Mutation Age at > c.817-1G c.1061delC > c.210G Splice junction Intron 9 > c.1271G c.1226A g.-6247_- 4859del g.-6247_- 4859del , , f f 7 na na , , 6 f f ◦ , 1 , 2 na na na f na na na f 2 1 4 IV.2 7 1 3 3 IV.1 8 et al. Moes ed by Pt 1 | Continued et al. Report Heltzer Rao (2007) (2007) et al. (2010) et al. (2007) , Halabi-Tawil et al. (2009) , Patey-Mariaud de Serre et al. (2009) , Torgerson Table

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 211 | 11 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy (Continued) years months months years 5 years 6 years Exitus Exitus 7 years 6 10 years 1 4 months 1 2 years 6 months Exitus 3 5 years Exitus 5 9 months 9 Alive Alive Alive Alive Alive, Alive Alive A, CSA, CSA, IgIV FK506, – PD, AZA HSCT Steroids Sterois, CS FK506, AZA, Rapa, IgIV Rapa, Cx, VCR, PN MPD, FK506, IgIV MPD, CSA, FK506, AZA, IgIV, HSCT MPD, MPD, na AZA TPN, HSCT Alive yroiditis, alopecia, AHA, – – Hypogammaglobulinemia, anemia, , laryngeal papillomatosis, Norwegian MGN Insufficiency interstitial pneumonia EBV-induced Sepsis sepsis Hepatitis thrombocytopenia, sepsis Liver dysfunction, , Asthma, Adrenal Th 0 2000 1494 > 3 na na na 4900; 5400; na Thrombocytopenia, 803; 391 2187; na Hypothyroidism, hepatitis, 710; na; 517700; – na Dermatitis na − + + − − + + − + + − + − − + − + + + + + + + + + − + + + + + + + + + ears ears na na 6 y < 1 years Post mortem 4 months < 1 y days < 1 years 6 years na 19 Neonatal Neonatal Neonatal Neonatal A neonatal 1 years A A 2 months na A A T > 4A > G # 5G > 2T > G Neonatal 6 months + + + 50G > 50G > > c.1150G Exon 10 c.1271G c.11 > Cc.1099T 8 daysc.11 na exon 10 3 months c.2T > C c.543C > c.816 c.967 na f 1, 11 , 7 , 2 f na f f na f na na na 1 1 2 3 1 1, 1 1 1 3, 3 c.210 4 7 6 al. et al. (2008) (2008) (2008) , hizawa al. (2007) et al. (2007) (2007) , McLucas et al. (2007) Fuchizawa et al. (2007) , Otsubo et al. (2011) Suzuki et al. (2007) Lucas et al. (2008) Gambineri et al. (2008) , Passerini et al. (2011b) Lucas et Burroughs et Fuc Gambineri et al. Gambineri et al. Gambineri et al. (2007) , Gambineri et al. (2008) , Passerini et al. (2011b) Passerini et al. (2011b) Taddio

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 12 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy  years ears ears months months Outcome 15 23 years Exitus, 11 Exitus, 11 8 y 2 y Exitus 6 months 15 years 7 years 3 years Alive Alive Alive Alive Alive Alive HSCT rapa A PDN, AZA, PD, CS MPD, CSA Alive MPD, FK506, na IgIV,TPN, steroids, CSA, PDN, MPD, FK506, FK506 mesalazine, infliximab, AZA, 6-MP,rapa FK506, TPN, HSCT Steroids, AZA AZA, Rapa Therapy T, CMV AHA and GI , anemia, food ections Infections clinical findings failure, gastrectomy infection Hypothyroidism, AHA, inf Respiratory infections, AHA, ITP External otitis, sepsis, bacteremia, hepatosplenomegaly AIH, AIT Alopecia, AHA, AI AIH, AHA, Additional 0 ); 3 > 230 Pancreatic exocrine (/mm IgE (IU/mL) 498; 1966 na; 700 N; na na 3753; 157 na 1550; 5218 Sepsis, nephropathy MPD, PD, CSA, na; high – + Xerosis na; + + + + Dermatitis na + + − + − − − + + − − − − Severe Diarrhea T1DM Eczema Eos + chronic gastritis + + + + + + + + diagnosis 1 years < 1 year na 7 years na < 5 years Age at onset Neonatal < 1 year na Neonatal Birth 1 week 6 weeks A T T T 4 months 5 months T > > G G Mutation Age at c.725T > C 4 months 11years > Gc.1015C Neonatal 5 months > c.1040G > c.1040G c.1121T c.1045- 3C > > c.210G c.1150G > c.1139C c.1061delC 2.5 years na , , 8 na na na na ◦ f f f na na 9, 9, 9 10 10 13 14 1 2 1 8 1 1 al. by Pt 08) , 08) , 08) | Continued alho et al. (2008) Gambineri et al. (2008) , McMurchy et al. (2010) Gambineri et al. (2008) , Passerini et al. (2011a,b) Reported Gambineri et al. (20 Gambineri et al. (20 Gambineri et al. (20 Costa- Carv Redding et (2009) Passerini et al. (2011b) McMurchy et al., (2010) Passerini et al. (2011a,b) (2008) Zhan et al. (2008) Yong et al. Table 1

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 211 | 13 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy (Continued) eeks na na na na na na na na na na na Exitus 7 w na na na na na na na na na na na na na na na na na na na na na na TPN, rapa infections chodystrophy, y Distress, rent infections, recurrent infections, AIN, tubulointerstitial – recurrent infections, sepsis Cheilitis, HA, MGN, recur Cheilitis, ony HA, recurrent infections infections, sepsis HA, recurrent sepsis Hypothyroidism, Respirator sepsis sepsis Seizures, Renal Failure, nephritis Congenital ichthyosis, HA, HA, MGN, recurrent HA, Recurrent infections, tubulointerstitial nephritis na AHA, AIT, MGN AIT Allergic Asthma AHA, AIN AHA, AIT AIT, na na na na na na na na; N na; high na; N na; high na; N na na; N - Psori- Psori- + Erythro- derm + Erythro- derm + + asiform rash + asiform rash − − Exfoliative der matitis Dermatitis − Dermatitis na; highDermatitis − AIT, AIN Dermatitis na; N − Dermatitis na; high AIT, Dermatitis na; N e e e e e e e + − + − + − + + + − − − + + + + + + + + + + + + + + + + + + na na na na na na na < 7 weeks na na na na na months na na na na na birth 6.5 years 4 months 4 months 7 months 6 months na 1 year 1.5months na A A T T 11 > > G na > G 2 months na 0T > G 1 year na 1C > G na 3T > G uncated > Gc.1113T na c.736-1G > c.110 c.560C > c.1121T c.751_753 delGAG c.751_753 delGAG c.1150G truncated protein c.110 p.E251del c.1121T c.111 c.560C > p.E251del p.E251del Protein Tr Truncated na na na na na na na na na na na na na na na na na 1 2 4 8 1 2 4 6 10 12 3 5 9 3 5 9 11 1 (2009) al. (2009) Halabi-Tawil et D’Hennezel et al. Patey-Mariaud de Serre et al. (2009)

www.frontiersin.org July 2012 | Volume 3 | Article 211 |14 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy  ears years years ears Outcome Exitus, Exitus 8 months 5 years 15 12 years 12 13 months Exitus, 5.5 months 22 y 1 years 4 y 9 months 17 years Alive Alive Alive Alive Alive Alive Alive, na CSA, MPD Alive na na na na na PDN Rapa, MTX, PD, HSCT HSCT HSCT Therapy atrophy, aneous -globulinemia, hronic diabetes clinical findings syndrome, infections, , TIA, c complications mucocutaneous candidiasis, infections mucocut candidiasis, infections thrombocytopenia, dysthyroidism, infections pancreatic γ hypo- infections, bronchiectasis, Hemolytic anemia, infections steroid-dependent interstitial disease, membranous nephropathy, hypothyroidism, infections AHA, sepsis Anemia, neutropenia, Autoimmune gastritis, Additional Thyroiditis, Thyroiditis ); 3 > 5000 Sepsis 0–2000; (/mm IgE (IU/mL) na; 1141 Food allergy, nephrotic na, N na; N na; N N; N 100 na; N Thyroiditis, na; 2266 – 132 850; 183 2000; 842 Anemia, − − + − − + + + + + + + + § + − − ; + − + − + − Diarrhea T1DM Eczema− Eos − + Mal digestion + Gastritis + + + + + Vomiting ears diagnosis na na na 19 y 2 months 11years Age at s onset 2 months 5 years 2 day 3.5 months 1 week 8 months 2 months 1 day na A A A 3 weeks na A A 30 days na 7G > C na 2delT + + 5C > G Mutation Age at c.748_750 delAAG > c.1222G > c.1222G c.101 > c.1040G c.***878A > G neonatalc.210 4.5 months > c.1222G c.816 c.227delT f f f ◦ , 4 na f na f na I IIb IV 1 1 1 1 IIa III> c.1010G 2 V by Pt 09) , | Continued (2009) (2010) 09) , Dorsey et al. (2009) Reported Hashimura et al. (20 Rubio-Cabezas et al. Scaillon et al. (20 Burroughs et al. Otsubo et al. (2011) McMurchy et al. (2010) Table 1

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 211 | 15 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy (Continued) years years ears na na na na Exitus na Exitus 8 months Exitus < 5 6/6 3 years Exitus 8 y Exitus 10 Exitus 7 months na na na na na 4.5 months Exitus 1 month 14 months Steroids, TPN Exitus na na na na PN FK506, HSCT FK506, Rapa, HSCT FK506 FK506 HSCT na na na na na Supportive treatment FK506, Rapa Exitus, na nephropathy, infections anemia, w – – – – sepsis Basedo hyperthyroidism, Hp gastritis, allergy Hypothyroidism, interstitial nephritis, hemolytic Hemolytic anemia nephropathy nephropathy recurrent infections Recurrent Proteinuria, Sepsis thrombocytopenia, allergy hepatitis, hypothyroidism, infections Thrombocytopenia, Thyroiditis, Thrombocytopenia, > 4200 Sepsis + na na High; 5500 High; 12,500 na; 2150na; 650 AIH, hemolytic anemia, na; 3700na; 3210 Thyroiditis, hepatitis, na; 1 na; 842 na; 364na; 2444 Thyroiditis na; 2950 na; 657na – 9910; 75 na; High; 8500 Hemolytic anemia, na; na Skin pathol, + Skin pathol, no eczema + + + + + + Skin pathol na − − + − + − + − + − + − − + + + − − − + + + − + + + + 6/6 + + + + + − + + + + + + + + + + + Vomiting – na na na na na na na na na na na na Post mortem mortem na s s na na Birth 6 weeks 4 weeks na 7 day na na na na na 20 day # C A na A G G A na A na A A T T T na > > 4A > 4A > G 2 months Post 7G > 1G > 1G > > + + + + + 5C > G 57 G > > 190G c.816 g.560C > > Gc.1121T Birthc.751_753 delGAG na c.751_753 delGAG c.101 c.210 c.816 c.1150G c.1169G > c.***876A Intron1 2.5 monthsc.1080_1081 2.5 months insA c.210 c.543C > c.817G c.11 3, 4, – II3, – 1 c.816 f f na na na f na na na 5, 6 1 F1 II4, IV4, IV5, 3 5 7 2 4 8 10 12 1 F2 4 6 3 5 9 111 c.1 al. (2011) et al. 10) 10) (2010) Harbuz et al. (20 Moes et al. (20 (2010) An et al. Wang et Tsuda

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 16 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy  ears ears ears Outcome 6 years Exitus 11months 26 years 8 years Exitus 5 months 13 years age na 3 y 7 months 3 y 28 y Alive Alive Alive Alive, Alive Alive Alive A, , Rapa PD, CSA, AZA, HSCT Supportive treatment CSA, HSCT Supportive treatment CSA, CS Rituximab, CS PD, HSCT MPD, FK506, HSCT CSA, MPD TPN, steroids, Therapy sepsis, S. aureus osteomyelitis, clinical findings lymphadenopathy, , pneumonia Pneumonia Nephrotic syndrome Infections, sepsis Hypotonia bronchitis arthritis, AHA, infections AIT, Additional ); –1000 3 + ; 45 AIH (/mm IgE (IU/mL) 22; 681 Nephrotic syndrome, N; na PRCA, MGN, infectionsna; na PD Nephrotic syndrome CSA, steroids Alive 2300; > 2000 3450; 3 na + ; 157 467; 1278 20; 424 − − + Seborrhoeic dermatitis + + + − − − − + + − + − − + + Diarrhea T1DM Eczema+ Eos + + + + + + + + + diagnosis mortem Post mortem na < 7 months < 4 months 27 years Age at s onset 26 day na 1.5months 2 months na Neonatal 5 months A 11months 11years T 6 months na A C A 14 days Post T > 2delT 1G > 1G > + + + 0G > Mutation Age at c.210 c.111 c.970T > C > c.1-23G c.1150G delAAG > c.1037T c.816 c.210 c.748_750 ◦ f f f f 5 1 18> G c.***876A Neonatal na 2 3 1 2 1 17 20 ; na, not available; N, within normal ranges; e, unspecified endocrinopathy; ITP,idiopathic thrombocytopenic purpura; AIT, autoimmune thrombocytopenia; AIN, autoimmune neutropenia; al. al. by Pt al. et al. | Continued ashi ae et al. The age written in the outcome column refers to the age of the patients at the latest follow-up from each publication. Hypo or hyperglycemia. Patient ID refers to the enumeration of patients as reported in the original publications listed in column 1. The mutation has not been studied in this patient but in other relatives with an IPEX phenotype belonging to the same gender. Positive familial history. (2011) B et al. (2011) Otsubo (2011) Lopez et al. (2011) PRCA, pure red cells aplasia; MGN,mune membranous hepatitis;TIA, glomerulonephritis; transient AHA, ischemic autoimmune attack; MSSA, hemolytic Methicillin-sensitive anemia;CSA, Staphylococcus aureus; HA, cyclosporine; PN, hematological FK506, parenteral abnormalities nutrition;TPN, , (cytopenias, total MTX, hepatosplenomegaly,diabetes; parenteral or methotrexate; nutrition; GI, lymphadenopathy); NGT, AZA, nasogastric gastrointestinal. AIH, tube; azathioprine; autoim- PD, ; Cx, PDN,*In cyclophosphamide; ; this VCR, case, vincristine; tubulonephropathy HSCT,◦ could hematopoietic be stem due cells both to transplantation; the FU underlying§ follow-up; disease TNDM, or transient to neonatal tacrolimus. f #  Reported Kobay Kasow et Passerini et (2011b) Passerini et (2011a) Pt, patient; Eos, (2011) Table 1

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later, in two unrelated kindred with IPEX phenotype, Chatila et al. protein. Such mutations lead to an impaired transcriptional regu- (2000) identified mutations in JM2 (later called FOXP3) in the latory activity by altering the binding sites to DNA, the interaction centromeric region of the X chromosome (Xq11.3-q13.3). Shortly with other molecules (e.g.,NFAT,AP1,RORα),or the dimerization after, Bennett et al. (2001b) and Wildin et al. (2001) confirmed of FOXP3 (Figure 1). that IPEX syndrome is the human equivalent of the scurfy mouse, Independently from the type or site of the FOXP3 mutation, the natural mouse model of the disease, and identified mutations all patients described but five (Ferguson et al., 2000; Fuchizawa in the FOXP3 gene in additional IPEX patients. Of note, in the et al., 2007; Rubio-Cabezas et al., 2009; Scaillon et al., 2009; Tsuda first family described in 1982, the disease mapped to the pericen- et al., 2010; Otsubo et al., 2011) developed gastrointestinal symp- tromeric region of the X chromosome (Bennett et al., 2000), but toms (mainly diarrhea).The exact nature of genotype-phenotype no identifiable mutation on FOXP3 was found, so that it was sus- correlation has been difficult to pinpoint, especially considering pected to have a non-coding mutation that affects transcriptional the age at onset and the disease outcome. For example, in 13 regulation or RNA splicing (Bennett et al., 2001b). patients presenting with the same mutation (c.1150G>A), the The highly conserved FOXP3 gene is composed of 12 exons onset ranged from birth to 7 months (Table 1). In addition the encoding a protein of 431 amino acids in . Among outcome was influenced by other factors such as timing of the ther- the 63 mutations reported thus far (Figure 1), the majority of apeutic intervention, concomitant infections, and each individual them (27/63) alter the C-terminal forkhead (FKH) DNA-binding patient’s response to therapy. domain of the protein, while the remaining of the mutations The histopathological lesions also differ among the patients occur outside the FKH domain. The latter include mutations carrying the same mutation, further suggesting that the genotype affecting the N-terminal proline-rich (PRR) domain (14/63), the does not strictly correlate with phenotypical changes of the tar- leucine-zipper (LZ) domain (5/63), the LZ-FKH loop (9/63), the get organs (Patey-Mariaud de Serre et al., 2009). This inconsistent region upstream the initial ATG (3/63), and the C-terminal (3/63; correlation between genotype and phenotype may reflect the com- Figure 1). plex intracellular interactions of FOXP3 (Allan et al., 2005) and Moreover, mutations of the polyadenylation site of the gene also strongly suggests the role of environmental or epigenetic fac- (2/63) have been described, which lead to the expression of an tors that might participate in determining the clinical picture and unstable FOXP3 mRNA and usually result in severe, early onset outcome (Gambineri et al., 2008). disease (Bennett et al., 2001a; Dorsey et al., 2009; Tsuda et al., 2010; Passerini et al., 2011b). Patients with mutations that abro- CLINICAL MANIFESTATIONS gate expression of functional FOXP3 protein (i.e., missense or Most IPEX patients are born at term after an uneventful preg- frameshift mutations or splicing defects resulting in a premature nancy from unrelated parents. A careful family history may reveal stop codon) tend to have severe presentation as well (Gavin et al., the presence of male subjects in the maternal lineage with similar 2006; Gambineri et al., 2008; Burroughs et al., 2010; An et al., clinical phenotype, early death, or multiple spontaneous abor- 2011). Nonetheless, the severity of the disease is not always depen- tions. Notably, these patients may have other affected brothers, dent on the absence of protein expression. The majority of affected but females belonging to the same lineage are usually healthy. individuals have missense mutations (usually point mutations) At birth, they may have a normal weight and length without resulting in a normal or reduced level of expression of mutant pathological findings. The onset of IPEX syndrome usually occurs

FIGURE 1 | Schematic representation of the FOXP3 gene reporting all the CCDS14323.1). *c543C>T is a polymorphism. E, exon; Color code: orange, mutations published so far. Annotations refer to both coding sequence and N-terminal domain; green, zinc finger domain; blue, leucin-zipper domain; red, protein, when applicable (www.ncbi.nlm.nih.gov/CCDS, accession number forkhead domain.

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in males within their first months of life, but in some cases even initially diagnosed and treated as severely allergic individuals after few days or weeks, and can be rapidly fatal if not diagnosed (Torgerson et al., 2007). Given this, severe allergic conditions in and treated. The most severe cases are characterized by the early association with other autoimmune symptoms should raise the onset of a triad of clinical manifestations: intractable diarrhea, suspicion of IPEX syndrome. type-1 diabetes mellitus (T1DM), and eczema. The clinical picture can be complicated by the presence of other Autoimmune enteropathy is a hallmark of IPEX syndrome. autoimmune symptoms (Table 1): thyroiditis (27/136) with either Patients present with neonatal, watery, and sometimes mucoid or hyperthyroidism or, more commonly, hypothyroidism (Kobayashi bloody acute diarrhea. This acute severe enteropathy often begins et al., 2001;Wildin et al., 2001, 2002; Nieves et al., 2004; Myers et al., in the first days of life or during breast-feeding, thus showing to 2006; Moudgil et al., 2007; Costa-Carvalho et al., 2008; Gambineri be independent from cow milk or introduction in the diet. et al., 2008; Halabi-Tawil et al., 2009; Rubio-Cabezas et al., 2009; However, it could be worsen by switching from breast-feeding to Wang et al., 2010; Otsubo et al., 2011) cytopenias (42/136) such regular formula. It typically persists despite dietary exclusions and as hemolytic anemia, thrombocytopenia, and neutropenia, and bowel rest. Since it results in severe malabsorption and significant hepatitis (8/136) that may be autoimmune with positive auto- failure to thrive, parenteral nutrition is often required. In addition (Table 1). Renal disease can be related either to autoim- to diarrhea, other gastrointestinal manifestations can present, such munity or to prolonged administration of nephrotoxic drugs. They as vomiting (Ferguson et al., 2000; Hashimura et al., 2009; Har- are generally described as tubulonephropathy (Kobayashi et al., buz et al., 2010; Otsubo et al., 2011), gastritis (Nieves et al., 2004; 2001; Otsubo et al., 2011) and nephrotic syndrome (Gambineri Gambineri et al., 2008; Scaillon et al., 2009), ileus (Levy-Lahad and et al., 2008; Rubio-Cabezas et al., 2009; An et al., 2011; Otsubo Wildin, 2001), and colitis (Lucas et al., 2007; Otsubo et al., 2011; et al., 2011), although interstitial nephritis (Bindl et al., 2005; Table 1). Patey-Mariaud de Serre et al., 2009; Moes et al., 2010) and mem- Type-1 diabetes mellitus can precede or follow enteritis. T1DM branous glomerulonephritis (Moudgil et al., 2007; Halabi-Tawil is present in the majority of patients including newborns, and et al., 2009; Burroughs et al., 2010; Bae et al., 2011) have also is usually difficult to control (Peake et al., 1996; Baud et al., been found in some patients’ histopathological examinations. A 2001; Gambineri et al., 2008). There have been rare cases (6/136) rare manifestation associated with the milder forms of IPEX with presenting with diabetes mellitus without auto-antibodies (Rubio- delayed diagnosis is arthritis involving one or more joints (Wildin Cabezas et al., 2009; Scaillon et al., 2009). Imaging studies or et al., 2002; De Benedetti et al., 2006). Splenomegaly and lym- autopsy and histological examination often reveal destruction of phadenopathy may progress as a result of an ongoing autoimmune the pancreas and intense lymphocytic infiltrate, suggesting that lymphoproliferation, as evidenced by the extensive lymphocytic an immune mediated damage of this organ may have a role in infiltrates in secondary lymphoid organs found in several patients the pathogenesis (Wildin et al., 2002; Costa-Carvalho et al., 2008; during autopsy (Wildin et al., 2002; Ochs and Torgerson, 2007; Rubio-Cabezas et al., 2009). Costa-Carvalho et al., 2008). Despite multiple and early autoim- Cutaneous manifestations appear in the first months of life. mune manifestations typical of IPEX syndrome, it is important to Similar to diarrhea and diabetes, cutaneous manifestations are underline that their number may increase with age. IPEX patients’ very common (95/136) and can be the first sign of the disease presentation typically begins early with some of these autoimmune (Table 1). symptoms, and progresses with new manifestations over years. Dermatitis can be eczematiform (mainly ) The clinical spectrum can be worsened by infections, although (Wildin et al., 2002; Owen et al., 2003; Ruemmele et al., 2008), they are less frequent than the more prominent signs described ichthyosiform (Baud et al., 2001; Rao et al., 2007), psoriasiform above. The onset of IPEX syndrome is often associated with infec- (Nieves et al., 2004; De Benedetti et al., 2006), or any combina- tions, however a clear causative role of pathogens in the onset of tion of the above (e.g., atopic dermatitis and coexisting autoimmunity has not been demonstrated and infections can often on different areas of the skin) (Halabi-Tawil et al., 2009). Skin be the consequence of multiple immunosuppressive (IS) therapy involvement is severe and diffuse, characterized by erythematous and poor clinical conditions. exudative plaques that could evolve into more lichenfied plaques The most frequent infections are pneumonia, airway infections, (Halabi-Tawil et al., 2009). Pruritus can be a major complain gastrointestinal, and skin super-infections that may lead to life- in these patients since it is intense and difficult to control with threatening sepsis from spp. and Staphylococcus spp. anti- drugs. Cutaneous lesions often show resistance to (Halabi-Tawil et al., 2009). Other common pathogens are Clostrid- classic treatments such as topical steroids or tacrolimus and can ium difficile, albicans, Pneumocystis jiroveci, CMV, and be complicated by bacterial infections (most commonly Staphy- EBV. lococcus aureus and epidermidis) with potential development of sepsis (Halabi-Tawil et al., 2009). Other manifestations affecting LABORATORY FINDINGS the integumentary system include: painful and fissurary cheilitis Laboratory tests can be normal at onset. There are no specific diag- (Halabi-Tawil et al., 2009), onychodystrophy (Halabi-Tawil et al., nostic findings in IPEX syndrome although the laboratory abnor- 2009), and alopecia (Nieves et al., 2004; Moudgil et al., 2007; malities consistent with T1DM and severe enteropathy are com- Gambineri et al., 2008). mon. Moreover, other alterations may suggest ongoing autoim- Two patients presented with severe allergies to food or other mune manifestations in other target organs, such as hypothy- causing asthma, skin rashes, and gastrointestinal symp- roidism, cytopenias, hepatitis, or nephropathy. Markedly elevated toms in the absence of endocrinopathies. These patients were IgE levels and counts are observed in the majority

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of patients as an early hallmark of the disease (Table 1). Serum impairment; Coombs antibodies, anti-platelets antibodies, and IgA, IgG, and IgM levels are generally normal or low due to the anti- antibodies are often present in autoimmune protein-losing enteropathy. cytopenias; anti-smooth muscle (ASMA) and anti-liver-kidney- Patients in the acute phase of the disease, prior to IS therapy, muscle (anti-LKM) antibodies are positive in autoimmune hepati- can have normal or elevated counts. , tis. Recently, Huter et al. (2010) reported that sera from IPEX if present, is due to an increase in lymphocytes but the percent- patients react against keratins, especially keratin 14, suggesting age of the different subpopulations (CD3, CD4, CD8, this molecule as a target for autoreactive lymphocytes in the skin CD16, CD19) remains unchanged despite immune dysregulation. of IPEX patients. The CD4/CD8 ratio is maintained or increased and the T cell Although there is no pathognomonic finding specific to IPEX, repertoire is polyclonal. The percentages of naive and memory T biopsies of the affected organs can help in excluding other etiolo- cells are mostly comparable to their age-matched controls. The gies. Main histological findings in the gastrointestinal tracts are CD4+CD25+FOXP3+ Treg cells are present (Gavin et al., 2006; total or subtotal villous atrophy with mucosal lymphocytic and Gambineri et al., 2008), but FOXP3 expression can be reduced if eosinophil infiltration, but they are not specific for the disease. In FOXP3 mutation prevents the expression of the protein (Bacchetta a recent work,Patey-Mariaud de Serre and colleagues described the et al., 2006) or if the patient is exposed to IS therapy (Gam- intestinal morphological changes of twelve IPEX patients (Patey- bineri et al., 2008). In addition, in vitro proliferative responses Mariaud de Serre et al., 2009). Three different kinds of lesions to mitogens are normal unless the patient is treated with IS drugs were found in the : (1) the graft-versus-host (Bacchetta et al., 2006). The in vitro production shows a disease-like pattern was the most frequent form observed; (2) the decrease in Th1 cytokines and an increase in Th2 (Chatila et al., celiac disease-like pattern, found in two patients; (3) depletion of 2000; Nieves et al., 2004; Bacchetta et al., 2006). The karyotype is the intestinal goblet cells along with the presence of anti-goblet normal. cell auto-antibodies, reported in one child. Hence, one of these A variety of auto-antibodies are detected in most patients and histopathological patterns in the proper clinical context and an their presence usually correlates with signs of in the tar- association with circulating anti-AIE-75 auto-antibodies would get organs, but their production may also be a sign of immune suggest the diagnosis of IPEX syndrome. dysregulation without an obvious pathological linkage (Tsuda In addition, one case reported the autoimmune destruction et al., 2010). of pancreatic exocrine cells contributing to the diarrheal disease There is increasing evidence that anti-enterocyte antibodies (Heltzer et al., 2007). are characteristic of IPEX patients, although not all patients The histopathological changes at the skin biopsies are usually have been tested because the assay is not widely accessible. The non-specific for IPEX syndrome since there is a wide range of autoimmune enteropathy-related 75 kDa (AIE-75), pre- possible dermatological pictures. The clinical and histopathologi- dominantly expressed in brush border of the small intestine and cal features of skin pathology of 10 IPEX patients were described proximal tubules of the kidney, has been identified as a specific tar- by Halabi-Tawil et al. (2009). Either subacute /chronic spongiotic get of the auto-antibodies present in IPEX patients sera (Kobayashi dermatitis or psoriasiform changes, also consistent with a chronic et al., 1998, 1999, 2011; Gambineri et al., 2003; Patey-Mariaud de lichenified eczema, have been shown. One out of the four biopsies Serre et al., 2009; Moes et al., 2010). showed a slight perivascular lymphocytic infiltrate in the upper In addition, a recent study of Kobayashi et al. identified villin, dermis, while the others showed a moderate to intense superfi- a 95-kDa -binding protein, as another brush border anti- cial dermal infiltrate with the simultaneous presence of eosinophil gen aberrantly targeted in IPEX syndrome. Like AIE-75, villin and lymphocyte infiltrates. Although the majority of skin alter- is also expressed both in the microvilli of the small intestine ations were compatible with atopic or psoriasiform dermatitis, and in the proximal renal tubules. In this study, five out of five IPEX patients may present with uncommon allergic (Nieves et al., IPEX patients showed anti-AIE-75 antibodies and four out of five 2004),autoimmune (Ferguson et al.,2000; McGinness et al.,2006), displayed anti-villin antibodies. None of the control sera from or infectious (McLucas et al., 2007) dermatological complications. healthy subjects or patients affected by non-IPEX pathologies (e.g., autoimmune of different origin, enterocolitis, and DIFFERENTIAL DIAGNOSIS colon ) were positive for anti-AIE-75 antibodies and only a A neonate presenting a single severe manifestation of IPEX syn- few were weakly positive for anti-villin antibodies. High levels of drome such as enteropathy, diabetes, or newborn erythroderma anti-villin auto-antibodies have been found only in children with may pose a diagnostic challenge for the . For each of IPEX syndrome (Kobayashi et al., 2011). These findings confirm them, the suspicion of IPEX syndrome should be raised once other the specificity of both anti-AIE-75 and anti-villin antibodies for more common diseases have been excluded. IPEX syndrome. Their link to the tissue damage, the correlation In a neonate presenting with isolated diarrhea, an autoimmune to the progression of the disease, and their predictive value have pathogenesis of the enteropathy is a rare event. Table 2 provides to be clarified. a summary of the possible causes of enteropathy in newborns Early presence of detectable auto-antibodies against , and infants. IPEX enteropathy, like other diarrheal diseases, may pancreatic islet cells, or anti-glutamate decarboxylase correlates have either an aggressive or insidious onset. When the onset of with occurrence of neonatal T1DM. Moreover, anti-thyroglobulin the diarrhea is acute, microbial origins need to be excluded first. and anti-microsome peroxidase antibodies are detected in When the diarrhea persists, a wide range of differential diagnosis autoimmune thyroiditis even in the absence of functional has to be considered (Murch, 2001). The most common cause is

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Table 2 | Differential diagnosis of early onset persistent diarrhea. 2006). In some of these cases,diarrhea would be abrogated by with- drawing oral feeding. Moreover, malabsorption could be in some Infectious and post-enteritis diarrhea cases related to pancreatic disease rather than to an intestinal trans- port or enzymatic alteration. Nevertheless, the intestinal biopsies FOOD-SENSITIVE ENTEROPATHY OR ENTEROCOLITIS in both cases show a normal architecture with intact villous- Cow’s milk sensitive enteropathy (most frequent) crypt axis, unlike in IPEX. On the contrary, primary epithelial Celiac disease enteropathies, such as microvillous inclusion disease and tufting Non-celiac gluten sensitivity enteropathy, are characterized by blunting villi at the intestinal Food protein induced enterocolitis biopsy and usually appear in the first days after birth. They should Eosinophilic gastroenteropathy be excluded if diarrhea is prolonged and continues during total ANATOMICAL DEFECTS AND DYSMOTILITY DISORDERS parenteral nutrition (Sherman et al., 2004). Immunodeficiencies, Hirschsprung disease such as severe combined immunodeficiency (SCID) or interme- Intestinal diate forms of combined immunodeficiency (CID), may present Short bowel syndrome (post ) first with gastrointestinal symptoms, often fatal in early childhood Stagnant loop syndrome (post surgery) if untreated (Geha et al., 2007). In the latter cases, diarrhea may Chronic intestinal pseudo-obstruction be due to a prolonged impairment to clear enteric pathogens or TRANSPORT DEFECTS to a primary concomitant autoimmunity. Even metabolic diseases Chloride-bicarbonate exchanger defect (chloride-losing diarrhea) or endocrinopathies could manifest with chronic diarrhea. Fur- Sodium hydrogen exchanger (congenital sodium diarrhea) ther metabolic and hormonal assessment should be considered Ileal bile acid receptor defect in such cases. Autoimmune enteropathy is usually a diagnosis of Sodium-glucose cotransporter defect (glucose-galactose malabsorption) exclusion. Once the aforementioned diseases have been excluded Abetalipoproteinemia by appropriate clinical or laboratory evaluations, the presence of Hypolipoproteinemia the following clinical and histological findings indicative of the Acrodermatitis enteropathica (zinc deficiency) autoimmune pathogenesis, should be considered: an unrespon- ENZYMATIC DEFECTS siveness to dietary restriction and total parenteral nutrition, an Enterokinase deficiency association with other autoimmune conditions (Unsworth and Disaccharidase congenital defect (lactase, sucrase-isomaltase) Walker-Smith, 1985), small intestinal villous atrophy with hyper- PANCREATIC MALABSORPTION plastic crypt, mononuclear cells infiltrate within the intestinal Cystic fibrosis mucosa (Murch, 1997). Autoimmune enteropathy can be also one Shwachman syndrome of the symptoms of complex forms of immune dysregulation, but PRIMARY EPITHELIAL CAUSES OF INTRACTABLE DIARRHEA other clinical or laboratory features usually help to distinguish Microvillous inclusion disease them from IPEX syndrome (Table 4). Tufting enteropathy The onset of permanent diabetes mellitus in the neonatal age Heparan sulfate deficiency is described as a rare event (Rubio-Cabezas et al., 2010). Although IMMUNODEFICIENCIES (USUALLY UNMASKED BY A ) autoimmune T1DM is diagnosed in over 95% of children present- Severe combined immunodeficiency (SCID) ing with diabetes after 6 months of age (Porter and Barrett, 2004), alternative etiologies should be considered in newborns and young Class II major (MHCII) deficiency infants presenting with diabetes before 6 months of age (Hatter- CD40 ligand deficiency sley et al., 2009). Most of these patients have a monogenic form Neutrophilic specific granule defect of disease, even if the responsible gene remains unknown in up to Acquired immunodeficiency syndrome (AIDS) 40% of patients (Edghill et al., 2008). The main monogenic causes IBD (very rare in infancy, to be considered as a part of a PID) of early onset diabetes are mutations in Kir6.2 gene (the inward METABOLIC DISEASES rectifier subunit of the ATP-sensitive potassium channel of the β Mitochondrial cells), in SUR1 gene (the regulatory subunit of the K channel Wolman disease ATP in pancreatic β cells) and in the preproinsulin gene. Mutations of AUTOIMMUNE ENTEROPATHY chromosome 6q24 and mutations of the insulin gene may also be considered (Valamparampil et al., 2009; Greeley et al., 2010). The presence of auto-antibodies specific for pancreatic antigens before 6 months of age should however pose the question of FOXP3 food-sensitive enteropathy, so appropriate exclusion diets should mutation (Greeley et al., 2010). A recent study reported that 4% be initiated for an adequate period. Anatomical abnormalities of male patients with permanent neonatal diabetes were found to such as malrotation and pseudo-obstruction may cause bacterial have FOXP3 mutations (Rubio-Cabezas et al., 2009). The diag- overgrowth with chronic diarrhea and malabsorption. If chronic nosis of IPEX becomes more obvious when diabetes is preceded diarrhea is associated to protein-losing enteropathy, lymphangec- or followed by other symptoms related to immune dysregulation, tasia should also be considered. Transport or enzyme disorders such as enteropathy and eczema. induce selective malabsorption of glucose-galactose, lipids, fat- Skin pathology is a common finding in infants diagnosed with soluble vitamins, amino acids, electrolytes, and zinc (Murch, 2001, IPEX syndrome. The absence of other clinical signs may delay

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Table 3 | Differential diagnosis of erythroderma presenting in the IPEX syndrome. As recently pointed out by Leclerc-Mercier et al. neonatal period. (2010),early skin biopsy has a central role in excluding the majority of these pathological conditions. INFECTIONS The clinical characteristics that are common in PID with Staphylococcal scalded skin syndrome (SSSS) autoimmunity and unique to IPEX are summarized in Table 4. Congenital cutaneous candidiasis The differential diagnoses with primary immunodeficiencies asso- IMMUNODEFICIENCY ciated with immune dysregulation and subsequent autoimmune Graft-versus-host disease (GvHD) with underlying SCID phenomena,such as CD25 deficiency,STAT5bdeficiency,Omenn’s Omenn’s syndrome syndrome, Wiskott–Aldrich syndrome, Hyper IgE syndrome, ICHTHYOSES autoimmune lymphoproliferative syndrome, autoimmune poly- Non-syndromic ichthyoses (non-bollous ichthyoses, bollous ichthyoses) endocrinopathy candidiasis ectodermal dystrophy, should always Syndromic ichthyoses (Netherton’s syndrome, Conradi–Hünermann be considered. syndrome) METABOLIC DISEASES FOXP3 DYSFUNCTION AND DISEASE PATHOGENESIS Multiple carboxylase deficiencies Forkhead box p3 is a , master regulator for Essential fatty acid deficiency the function of thymic-derived regulatory T (nTreg) cells (Wildin DRUGS et al., 2001; Fontenot et al., 2003; Bacchetta et al., 2007; Gambineri Ceftriaxone et al., 2008). These cells are among the main subsets of CD4+ T Vancomycin cells appointed to maintain peripheral self-tolerance. OTHER SKIN PATHOLOGIES CD4+CD25+FOXP3+ T cells can be present in normal per- Infantile seborrheic dermatitis centage in the peripheral blood of the IPEX patients. This was Atopic dermatitis demonstrated not only by immunophenotype, but also by analy- Psoriasis sis of the Treg-cell-specific-demethylated-region (TSDR; Passerini Cutaneous mastocytosis et al., 2011b; Barzaghi et al., 2012), whose demethylation ensures cell-specific stable expression of FOXP3 (Baron et al., 2007; Wiec- zorek et al., 2009).Therefore, in IPEX patients FOXP3mut Treg the diagnosis, especially in neonates and infants (Nieves et al., cells are physically present but functionally impaired, and this is 2004). The presentation ranges from mild eczema to severe gen- considered the primary direct cause of autoimmunity in IPEX eralized erythroderma or other unusual skin manifestations with (Bacchetta et al., 2006; D’Hennezel et al., 2009; Moes et al., 2010). poor response to steroids (Halabi-Tawil et al., 2009; Redding et al., In this respect, IPEX syndrome is the best example of monogenic 2009). Focusing on the neonates and infants presenting erythro- autoimmune disease due to Treg deficiency. However, autoimmu- derma as single diffuse manifestation of IPEX syndrome at onset, nity in other immunodeficiencies, such as ADA-SCID and WAS, Table 3 summarizes the possible clinical pictures that should be has been recently associated with altered function of Treg cells, considered for differential diagnosis (Hoeger and Harper, 1998; regardless of FOXP3 expression (Marangoni et al., 2007; Sauer Fraitag and Bodemer, 2010). Erythroderma is an inflammatory et al., 2012). skin disorder affecting the majority of the body surface, with sub- Despite the general consensus on the fact that FOXP3 is funda- acute or chronic evolution accompanied by scaling skin. In the mental for acquisition and maintenance of suppressive function neonatal period, it can also be the primary manifestation of several by nTreg cells (Gavin et al., 2007; Wan and Flavell, 2007; Williams conditions. Perinatal or neonatal infections such as Staphylococcal and Rudensky, 2007), it is unclear how the different mutations scalded skin syndrome (SSSS) and congenital cutaneous candidi- affect their function. Functional in vitro studies on Treg cells of asis may result in diffuse skin involvement. Skin swab and/or skin IPEX patients revealed that the degree of functional impairment of biopsy is usually diagnostic. the suppressive activity varies among the patients, with complete Immunodeficiencies may present with extended skin alter- abrogation of suppressive function in patients with null mutations ations as a result of the immune aggression sustained by autoreac- (Bacchetta et al., 2006). Similarly, mutations in the FKH DNA- tive newborn’s lymphocytes (as in Omenn’s syndrome) or mater- binding domain of FOXP3 that caused severe IPEX (p.R347H and nal lymphocytes expanding after birth in the immunodeficient p.F373A) were only partially blocked in their ability to reprogram host (graft-versus-host disease with underlying SCID). Immuno- conventional T cells into Treg cells (McMurchy et al., 2010). It may logical assessment confirms the diagnosis of PID in these cases therefore be hypothesized that some mutated forms of the pro- (Table 4). If ichthyoses is suspected, skin biopsy is diagnostic. tein retain residual protein activities, thus only partially impairing Metabolic disorders can be associated with erythroderma,but usu- FOXP3 functions. The molecular mechanisms of Treg-mediated ally it is not the only complain and other systemic signs can support suppression remain controversial, hence our understanding of the the diagnosis. Ceftriaxone or Vancomycin, if recently adminis- impact of different FOXP3 mutations on Treg cell function is tered, should be stopped immediately to rule out drug-induced incomplete. skin reactions. Other common skin pathology of infancy, e.g., In addition to the well-accepted loss of suppressive function, atopic eczema and psoriasis, may evolve into erythroderma, but we recently described that FOXP3 mutations cause high insta- the early presentation, the persistency of the lesions, and the lim- bility of the Treg cell compartment, with a marked shift to the ited response to topical treatment may increase the suspicion of Th17 cell phenotype of bona fide nTreg cells expressing a mutated

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 22 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy T1DM, insuffi- , young ± arian or testicular Rare Rare thyroiditis Infancy adulthood , adrenal ciency Candidiasis Rare Ov failure, gastritis, hepatitis, keratoconjunctivitis Absent AR AIRE APECED y, 1 year Frequent Urticarial rash Alopecia, Neonatal, Not frequent Frequent Hepatosplenomegaly, lymphadenopath other autoimmune manifestations FAS/FASL/CASP8/ CASP10/unknown Absent AD/unknown ALPS ) rent cathedral year Rare STAT3/TYK2/ eczema Neonatal, 1 Recur pulmonary, cutaneous ( S. aureus Characteristic face, palate, bone fractures unknown / unknown HIES Absent Absent AD/AR/ to ys ear, early Possible Not present Not frequent Not frequent PossibleRare Absent Frequent Eczema Newborn rash, 1 y Frequent Failure thrive, hemorragies, other autoimmune manifestations, tumors X-linked infancy Present Present Absent Absent Alwa WASP WAS 1 year to thrive, c/unknown ere Frequent Rare alopecia Neonatal, Sev Possible Failure hepatosplenomegaly, lymphoadenopathy, inflammatory pneumonitis and enteritis IV/ γ Absent AR/unknown Artemis/IL7RA/, ADA/DNAligase failure, rent ear, eness Eczema Erythroderma, < 1 y GH unrespon- siv Recur Rare Growth STAT5b def OMENN’S infancy (viral), severe varicella chronic lung disease, interstitial pneumonia Present Present Frequent Frequent Rare Rare AR to thrive, def rent/ ear, early infancy yroiditis Eczema, erythroderma < 1 y Recur Possible Failure CD25 persistent (particularly CMV) hepatosplenomegaly, lymphoadenopathy Present Possible Possible Frequent IL-2RA (CD25) STAT5b RAG1/2 (90%), AR Th y to IS 1 year thyroiditis to thrive, ± XP3 ossible erythroderma hepatosplenomegaly, lymphoadenopathy, other autoimmune manifestations IPEX Neonatal, Rare/secondar Possible Failure Present X-linked Possible FO P T1DM y pattern 4 | Differential diagnosis of primary immunodeficiencies (PID) presenting with autoimmunity. S, Wiskott–Aldrich syndrome; HIES, hyper IgE syndrome; ALPS, autoimmune lymphoproliferative syndrome; APECED, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; def, deficiency; IS, Skin lesions Eczema, Enteropathy Always Neutropenia /hyper IgE Gene Onset Endocrinopathy Infections Others Hereditar immunosuppression; T1DM, type-1 diabetes mellitus; AR, autosomal recessive; AD, autosomal dominant. Anemia WA Thrombocytopenia Table

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form of FOXP3 (Passerini et al., 2011b). Indeed, the plasticity proportion of IL-17 producing cells in the patients’ PBMC, which between different CD4+ T cell subsets is a new and dynamic con- could be derived in part from converted Treg, as mentioned above, cept, particularly pronounced between the Th17 and Treg cell or in part from Teff cells. compartments (Lee et al., 2009), although the in vivo relevance Overall,our current view of the pathogenesis of IPEX syndrome of such phenomenon is controversial (Zhou et al., 2009; Rubtsov is that, even if impairment of Treg function is the major step, other et al., 2010). Thus, in addition to the loss of suppressive function, factors such as inflammation and Th17 elevation can cooperate in FOXP3 mutations are associated with inflammation-driven con- maintaining and perpetuating the immune-dysfunction. version from a regulatory to an effector (i.e., IL-17-producing) phenotype of mutated Treg cells, which may directly contribute to THERAPY the autoimmune damage in the target organs. Due to the limited and sporadic number of cases reported in While the necessity of FOXP3 for suppressive function of Treg literature, it has been difficult up to now to compare different cells is undisputed, it is unclear whether functional FOXP3 is therapeutic strategies and relative outcomes. Therefore, the ther- essential for thymic development of Treg cells in humans. Data apeutic approaches for the treatment of IPEX patients are still from murine models of FOXP3 deficiency indicate that FOXP3 based on the experiences in single patients. Moreover, given the is dispensable for thymic development of Treg cells, but rather unclear genotype-phenotype correlation, the clinical course of the essential for their maintenance in the periphery, as demonstrated disease and the response to therapy can be variable and not always in Foxp3gfpko female mice (Gavin et al., 2007) and in FILIG mice, satisfying. Therapy is therefore targeted to the clinical manifesta- which display reduced Foxp3 expression in Treg cells (Wan and tions and severity of the individual patient. The current treatments Flavell, 2007). On the other hand, data from healthy carriers available for IPEX syndrome include replacement and supportive of FOXP3 mutations and transplanted IPEX patients with low therapy, IS therapy, and hematopoietic stem cell transplantation peripheral donor chimerism clearly indicate that only Treg cells (HSCT). Nutritional support and IS therapy should be promptly expressing a wild type FOXP3 survive long term in the periphery, started to counteract the initial acute manifestations. A wasting although leave it unclear whether the selective advantage is already syndrome can acutely affect the outcome of these patients, calling active during thymic differentiation or occurs later on in life (Di for a collaborative multi-disciplinary effort among clinicians from Nunzio et al., 2009; Seidel et al., 2009). Our recent observation that different specialties such as , infectious disease, bona fide Treg cells can be detected by TSDR demethylation analy- and immunohematology. sis in the peripheral blood of IPEX patients both at the onset of the disease and several years after IS treatment, regardless of FOXP3 REPLACEMENT AND SUPPORT THERAPY expression, demonstrates that functional FOXP3 is not necessary At onset, the patient should be hospitalized and receive a broad- for thymic differentiation of Treg cells in humans, as previously spectrum supportive care (fluids, TPN, albumin) with replace- demonstrated for murine Treg cells (Gavin et al., 2007), and that ment therapy for endocrine disorders (e.g., insulin and/or thyroid FOXP3mut Treg cells can survive and be detected long term, in the hormones), autoimmune cytopenias (e.g., hemocomponents), or peripheral blood of patients with IPEX syndrome (Passerini et al., hypogammaglobulinemia (e.g., intravenous immunoglobulins). 2011b; Barzaghi et al., 2012). Prophylactic should be used considering the multi- Evidences from studies on human and murine models show ple potential sources of infection such as skin lesion, damaged that Type-1 regulatory T (Tr1) cells can contribute to suppress- gastrointestinal lining, and central venous catheter. Infectious ing the development of autoimmunity in addition to nTreg cells episodes can drastically exacerbate or complicate the existing (Roncarolo et al., 2006; Sakaguchi, 2006). We recently demon- clinical symptoms, endangering the patient’s life. strated that Tr1 cells can develop in IPEX patients regardless of FOXP3 expression (Passerini et al., 2011a). This observation sug- IMMUNOSUPPRESSIVE THERAPY gests that FOXP3-independent immune regulation can potentially Monotherapy or combination immunosuppression reported so far contribute to controlling the disease, although Tr1 cells alone do has shown to be only partially effective in controlling the autoim- not seem adequate to suppress the initial acute phase of the dis- mune manifestations. Multiple IS therapies are often required to ease. Thus, it is tempting to conclude that FOXP3 is not necessary control symptoms (Gambineri et al., 2008). for function and development of adaptive Treg cells, the IL-10 (prednisone and methylprednisolone) are producing Tr1 cells. used as the first line therapy to limit progression of organ dam- In humans, FOXP3 is also expressed transiently upon activa- age (Gambineri et al., 2008). If the response to prednisone is tion, in conventional Teff cells (Allan et al., 2007; Tran et al., 2007; inadequate, betamethasone (the equivalent oral dose) could have Passerini et al., 2008), in which a still unknown function has been significantly better efficacy (Kobayashi et al., 2001; Taddio et al., postulated (Ziegler, 2006; McMurchy et al., 2010). This implies 2007). Then other IS drugs can be added onto the steroids regimen. that FOXP3 mutations may also impinge on Teff cell function Cyclosporine and/or tacrolimus have been most commonly used and suggests that FOXP3-dependent Teff impaired function may in conjunction with steroids (Baud et al., 2001; Wildin et al., 2002; directly contribute to the pathogenic mechanism underlying the Mazzolari et al., 2005; Taddio et al., 2007; Gambineri et al., 2008). disease. In support of this hypothesis are the data demonstrating Azathioprine also has been used with steroids and/or tacrolimus an impaired Th1 cytokine production from IPEX T cells, with rel- with partial control of the disease (Bindl et al., 2005). The ideal ative increase of Th2 cytokines (Chatila et al., 2000; Nieves et al., dose of medication should be determined to maximize clinical 2004; Bacchetta et al., 2006). In addition, we observed an increased benefit of the individual patient while minimizing .

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Thanks to a better understand the disease pathogenesis, clini- very susceptible to the side effects of because of cians nowadays tend to choose more specific IS drugs, based on their poor clinical conditions. The use of a non-myeloablative the medication’s mechanism of action. Calcineurin inhibitors have conditioning can more easily result in a partial chimerism. partial efficacy with high toxicity and simultaneously suppress Teff Both related and unrelated matched donors were used success- cells,expression of FOXP3,and Treg cell function. On the contrary, fully. Only one patient received HSC from cord blood (Lucas et al., rapamycin selectively target Teff cells and do not interfere with the 2007) and three from mobilized peripheral blood (Zhan et al., function of Treg cells, which are insensitive to mTOR inhibitors 2008; Seidel et al., 2009; Burroughs et al., 2010), otherwise bone (Battaglia et al., 2006; Allan et al., 2008). Even if it is not clear if marrow was used as source of HSC (Baud et al., 2001; Wildin et al., FOXP3mut Treg cells respond to rapamycin in the same way as 2002; Mazzolari et al., 2005; Rao et al., 2007; Dorsey et al., 2009). FOXP3wt, the use of rapamycin (alone or in combination with The longest follow-up reported is approximately 8 years post- azathioprine or steroids) has given promising clinical results in HSCT for three patients, including one patient transplanted at four IPEX cases (Bindl et al., 2005; Gambineri et al., 2008; Yong our Institute (unpublished observations: E. Mazzolari; M. Sei- et al., 2008). In these reports, rapamycin was used not as a first line del; R. Bacchetta). Only one of these patients reached full-donor therapy, but as a second choice when calcineurin inhibitor failed. chimerism, however other cases with favorable outcome despite The dosage used (approximately 0.15 mg/kg/day) was adjusted to partial chimerism have been described. Therefore, complete donor maintain serum levels between 8 and 12 ng/mL. In three patients engraftment in all hematopoietic lineages may not be necessary, with IPEX syndrome, the combination of rapamycin, methotrex- but the preferential engraftment of donor Treg cells does indicate ate, and steroid (in one case) and rapamycin, steroid, and azathio- that at least the replacement of this cell subset is essential to cure prine (in the other two) allowed to obtain clinical remission in all the disease (Seidel et al., 2009). In light of this observation, the cases and maintain it over time (follow-up of 5 years, 6 months, choice of drugs for GvHD prophylaxis should aim for the survival and 1.5 years, respectively; Bindl et al., 2005). The same positive of donor Treg cells. effect was achieved in one patient with rapamycin and steroid, Since wild type Treg cells seem to be sufficient to control the and with rapamycin monotherapy in another. Both showed clin- disease, future cell/gene therapy approaches designed to selectively ical remission with a follow-up of 21 and 15 months, respectively restore the repertoire of Treg cells represent a promising oppor- (Yong et al., 2008). Based on these positive responses to rapamycin, tunity. Constitutive lenti-viral mediated overexpression of FOXP3 its use as the first line IS drug in conjunction with steroid might be into CD4+ T cells can convert Teff into Treg cells both in healthy considered instead of calcineurin inhibitors. Of note, administra- subject (Allan et al., 2008) and in IPEX patients with different tion of rapamycin should be accompanied by frequent monitoring mutations (Passerini, in preparation). When a HLA compatible of serum drug level with appropriate dose adjustment, since the donor is not available, treatment with engineered T cells could enteropathy may affect the drug intestinal absorption. be envisaged. Whether these cells would survive long enough to In IPEX patients who survived the first years of life, immuno- provide a stable life-long immune regulation without generalized suppression may stabilize the existing symptoms, but flares of immunosuppression remains to be clarified. the disease may occur and new symptoms may arise despite the therapy. CONCLUSION Immune dysregulation, polyendocrinopathy, enteropathy, X- HEMATOPOIETIC STEM CELL TRANSPLANTATION linked syndrome can be suspected on the basis of clinical and Currently, the only cure for IPEX syndrome is allogeneic HSCT. A laboratory features, and the timely recognition of the disease summary of the published data regarding HSCT in IPEX patients leads to significant therapeutic benefits. A multicentre collab- is provided in Table 5. Early HSCT leads to the best outcome,as the orative effort is desirable to implement studies in a wider organs are yet to be damaged from autoimmunity and the adverse cohort of patients, in order to achieve a complete knowledge effects of therapy. For this reason it is fundamental to ensure an of the disease, to better understand the factors that influence early diagnosis. Twenty-eight cases reported received HSCT, 6 out the outcome, and to identify new therapeutic targets. Func- of these 28 patients died despite HSCT or during conditioning tional impairment of Treg cells has been recognized as the pri- (Table 1). mary defect at the basis of the immunodeficiency leading to Among the 15 cases of transplanted IPEX patients reported autoimmunity in IPEX syndrome. However, there is evidence in detail (Table 5), half of them (8/15) received the transplant that FOXP3 mutations can contribute to a complex immune- before 1 year of age, one of whom died. Among the other half, dysfunction, also involving Teff cells, and possibly other cell sub- two patients who received the transplant at 9 and 13 years of age sets. Immunological studies on IPEX syndrome have been instru- died of infections shortly after. More recently a 16-year-old patient mental in other PID to identify Treg dysfunctions, independent underwent HSCT and a 1-year follow-up was reported. Despite the from FOXP3 mutations, as cause of autoimmunity and will unfortunate outcome in some patients,the HSCT should be always most likely advance the knowledge and the therapeutic perspec- recommended as the therapy of choice. tives of other diseases with immune dysregulation of different Both myeloablative and non-myeloablative conditioning regi- origin. mens were used in order to limit complications associated with transplantation. The non-myeloablative regimens may enable ACKNOWLEDGMENTS reduction of both the post-transplant infectious complications The authors thank the members of the Italian Study Group of and the toxicity of high dose chemotherapy. IPEX patients are IPEX (www.ipexconsortium.org). Our work is supported by the

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 211 | 25 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy 30 → 50 A months 0 Kasow et al. (2011) 10/10˚ 1.5 c.11 G > BM 10 No Thio, Melph 7 + 0 > C na c.816 G BM 10 Gut 2˚ 2 Flu, TBI ALM, Flu, + Burroughs et al. (2010) 0 MUD MRD MUD***, 2 months c.210 delT mPB 10 Skin 2˚ 0 Dorsey et al. (2009) MUD, 10/10˚ neonatal c.***878 Flu, Melph BM 10 Skin 2˚ A > G ALM, A reg, 10 Seidel et al. (2009) 10/10˚ neonatal c.3G > Melph mPB 90 T Skin 2˚ ALM, Flu, WBC 39 T 0 Zhan et al. (2008) 4 months c.11 mPB MUD10 MUD, Skin-gut, 2–3˚ C > Cx ALM, Flu, > G MUD, 8/8˚ na c.1226 BM na No A A > na 4 years 6 months 5c.1271 monthsG 11months 7 months 9 months 16 years 7 months BM MSD, 8/8˚ na No § ALM, Flu, Melph Rao et al. (2007) 2 months 5 months c.303_304 delTT BM MUD, 7/8˚ na No na Intron 9 BM MUD, 8/8˚ na Gut 2˚ 10 ear Lucas et al. (2007) < 1 y Exon CB 5/6˚ 98 Gut 2˚ Flu, Bu, ATG reg Mazzolari et al. (2005) 4 months Promoter region BM 70 (T No 100) ATG 70 G → 0 2 months c.748_750 delAA BM 10 No A > Moudgil et al. (2007) ; ˚degree of HLA-match; *transient remission; **partial remission; ***T and B depleted. 50 Cx, TBI, ATG Flu, Bu, Cx, → 0 Wildin et al. (2002) 3 months c.1040G BM 10 No MRD MUD MRD MUD, 30 → 13 us Exitus Exitus Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive 01) 0 Baud et al. (20 1 month c.11 BM 10 na Cx T > G available; ATG, anti-thymoglobulin; Bu, busulfan; Cx, cyclophosphamide; TBI, total body irradiation; Flu, fludarabine; ALM, alemtuzumab; Melph, melphalan; Thio, thiotepa; BM, bone marrow; CB, cord blood; 5 | Hematopoietic stem cell transplantation in IPEX patients. + at The same case is reported also in mPB, mobilized peripheral blood; MRD,§ matched related donor; MUD, matched unrelated donor; GvHD, graft-versus-host disease. Donor MRD Remission Yes* YesOutcome Exit Yes** Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes onset Mutation CD34 source Chimerism (%) GvHD na, not Conditioning ATG, Bu, Age Age at BMT 6 months 13 years 9 years 9 months 6 years 7 years 1 years Table

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 26 Barzaghi et al. IPEX: diagnosis, pathogenesis, and therapy

Telethon Foundation (Tele 10A4 to Rosa Bacchetta), the Italian international stay at HSR-TIGET; Massimiliano Cecconi, Human Ministry of Health (Grant RF-2009-1485896 to Rosa Bacchetta), Genetic Laboratory at Galliera Hospital, for helping in revision of and the Seventh Framework project (FP7) of the European Com- the reported mutations, and Dr. Graziano Barera for useful dis- munity (Cell-PID to Rosa Bacchetta).The authors also thank cussion on neonatal enteropathies. Lastly, we are grateful to the Minso Kim, a medical student at New York University, for help patients and their families for their trust and participation in our in discussing and editing the present manuscript during her studies.

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(2009). Quantitative DNA methy- polyendocrinopathy, enteropathy, dysregulation, polyendocrinopathy, Citation: Barzaghi F, Passerini L and lation analysis of FOXP3 as a new X linked (IPEX) syndrome. J. Med. enteropathy, X-linked) syndrome. Bacchetta R (2012) Immune dysreg- method for counting regulatory T Genet. 39, 537–545. 121, e998–e1002. ulation, polyendocrinopathy, enteropa- cells in peripheral blood and solid Williams, L. M., and Rudensky, A. Y. Zhou, L., Chong, M. M., and Littman, thy, X-linked syndrome: a paradigm tissue. Cancer Res. 69, 599–608. (2007). Maintenance of the Foxp3- D. R. (2009). Plasticity of CD4+ T of immunodeficiency with autoim- Wildin, R. S., Ramsdell, F., Peake, J., dependent developmental program cell lineage differentiation. Immu- munity. Front. Immun. 3:211. doi: Faravelli, F., Casanova, J. L., Buist, in mature regulatory T cells requires nity 30, 646–655. 10.3389/fimmu.2012.00211 N., Levy-Lahad, E., Mazzella, M., continued expression of Foxp3. Nat. Ziegler, S. F. (2006). FOXP3: of mice This article was submitted to Frontiers in Goulet, O., Perroni, L., Bricarelli, Immunol. 8, 277–284. and men. Annu. Rev. Immunol. 24, Primary Immunodeficiencies, a specialty F. D., Byrne, G., Mceuen, M., Yong, P. L., Russo, P., and Sullivan, K. E. 209–226. of Frontiers in Immunology. Proll, S., Appleby, M., and Brunkow, (2008). Use of sirolimus in IPEX and Copyright © 2012 Barzaghi, Passerini M. E. (2001). X-linked neonatal IPEX-like children. J. Clin. Immunol. Conflict of Interest Statement: The and Bacchetta. This is an open-access diabetes mellitus, enteropathy and 28, 581–587. authors declare that the research was article distributed under the terms endocrinopathy syndrome is the Zhan, H., Sinclair, J., Adams, S., Cale, conducted in the absence of any com- of the Creative Commons Attribution human equivalent of mouse scurfy. C. M., Murch, S., Perroni, L., mercial or financial relationships that License, which permits use, distribution Nat. Genet. 27, 18–20. Davies, G., Amrolia, P., and Qasim, could be construed as a potential con- and reproduction in other forums, pro- Wildin, R. S., Smyk-Pearson, S., W. (2008). Immune reconstitution flict of interest. vided the original authors and source and Filipovich, A. H. (2002). and recovery of FOXP3 (forkhead are credited and subject to any copy- Clinical and molecular features box P3)-expressing T cells after Received: 03 May 2012; accepted: 01 July right notices concerning any third-party of the immunodysregulation, transplantation for IPEX (immune 2012; published online: 31 July 2012. graphics etc.

www.frontiersin.org July 2012 | Volume 3 | Article 211 | 30 REVIEW ARTICLE published: 18 July 2012 doi: 10.3389/fimmu.2012.00209 Autoimmunity in Wiskott–Aldrich syndrome: an unsolved enigma

Marco Catucci 1, Maria Carmina Castiello1,2, Francesca Pala1, Marita Bosticardo1 and Anna Villa1,3*

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 2 Vita-Salute San Raffaele University, Milan, Italy 3 Milan Unit, Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Milan, Italy

Edited by: Wiskott–Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that Luigi Daniele Notarangelo, Harvard affects 1–10 out of 1 million male individuals. WAS is caused by mutations in the WAS Medical School, USA Protein (WASP) expressing gene that leads to the absent or reduced expression of the Reviewed by: Gerben Bouma, UCL Institute of Child protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in Health, UK hematopoietic cells. WASP deficiency causes many immune cell defects both in humans Mike Recher, University Hospital and in the WAS murine model, the Was−/− mouse. Both cellular and humoral immune Basel, Switzerland defects in WAS patients contribute to the onset of severe clinical manifestations, in par- Stefano Volpi, Children’s Hospital Boston, Harvard Medical School, USA ticular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to *Correspondence: develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% Anna Villa, San Raffaele Telethon of WAS patients and the most common manifestation is autoimmune hemolytic ane- Institute for Gene Therapy mia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA (HSR-TIGET), Via Olgettina 58, 20132, nephropathy. Many groups have widely explored immune cell functionality in WAS partially Milano, Italy. e-mail: [email protected] explaining how cellular defects may lead to pathology. However, the mechanisms under- lying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients.

Keywords:Wiskott–Aldrich syndrome, autoimmunity, primary immunodeficiency,T lymphocytes, B lymphocytes

WISKOTT–ALDRICH SYNDROME: CELLULAR DEFECTS AND of actin filaments at the side of pre-existing filaments,thus creating CLINICAL MANIFESTATIONS a branching network of actin at the plasma membrane (Symons Wiskott–Aldrich Syndrome (WAS) is a rare X-linked Primary et al., 1996; Machesky and Insall, 1998; Miki et al., 1998; Machesky Immunodeficiency (PID) that affects 1–10 out of a million male and Gould,1999; Blanchoin et al.,2000; Pantaloni et al.,2000). The individuals (Ochs and Thrasher, 2006), whose life expectancy is activity of the ARP2/3 complex was shown to contribute to a vari- about 15 years in severe cases (Imai et al., 2004). Affected patients ety of cellular functions, including change of cell shape, motility, demonstrate both cellular and humoral immunodeficiency, high endocytosis, and (Welch and Mullins, 2002). susceptibility to infections, eczema, microthrombocytopenia, and The severity of disease, measured on the basis of the classifica- increased risk of autoimmune disorders and (Bosti- tion proposed by Zhu et al. (1997) and subsequently modified cardo et al., 2009). WAS is caused by defective expression of (Ochs and Thrasher, 2006; Ochs et al., 2009), is schematically WAS Protein (WASP), a key regulator of cytoskeletal organiza- reported in Table 1. tion in hematopoietic cells (Figure 1). The WAS gene is located A score from one to two identifies patients affected from a on the X chromosome and encodes a 502 amino acid protein milder form of the disease, named X-Linked Thrombocytopenia (Derry et al., 1994), which is constitutively expressed in the (XLT;Villa et al.,1995),and characterized by reduced expression of cytoplasm of hematopoietic cells (Kim et al., 2000). WASP is full-length mutated protein and microthrombocytopenia. Local- present in an auto-inhibited conformation and its activation is ized eczema and occasional respiratory infections, in addition to mainly induced by the binding with GTPase Cell division Cycle microthrombocytopenia, identify score 2 of the disease. Patients 42 (CDC42; Abdul-Manan et al., 1999). Other factors, such as the who develop microthrombocytopenia, associated with persistent Non-Catalytic region of tyrosine Kinase adaptor protein (NCK; but therapy-responsive eczema or infections receive a score of 3, Tomasevic et al., 2007), and the phosphorylation of WASP tyro- whereas a score of 4 is given if eczema or infections do not respond sine residue 291 (Y291) can activate WASP independently of to treatments. Finally, score 5 is assigned to patients developing CDC42 (Cory et al., 2002; Badour et al., 2004). The binding of autoimmunity or tumors. Phosphatidylinositol-4,5-bisphosphate (PIP2) is also an impor- Wiskott–Aldrich Syndrome gene mutations are scattered tant regulator of WASP activation by inducing a stable acting form throughout the entire length of the WAS gene, although some (Imai et al., 1999). WASP, in the active form, binds the Actin- hot spots have been identified (Ochs and Thrasher, 2006). Muta- Related Protein (ARP)2/3 complex, which gives rise to nucleation tions that abolish WASP expression are mainly associated with a

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FIGURE 1 | Wiskott–Aldrich syndrome structure and interacting (Kolluri et al., 1996; Symons et al., 1996; Miki et al., 1998). The . TCR, BCR, chemokine receptors, TLRs, integrins, and the Fc proline-rich region (PPP) provides binding sites for the receptor γ-chain can promote the release of GDP from Rho family Vasodilator-Stimulated Phosphoprotein (VASP), and also for SRC family GTPases, allowing GTP to bind. In immune cells, the major Rho GTPase tyrosine kinases and SRC Homology 3 (SH3) domain-containing proteins is the Cell Division Cycle 42 (CDC42). The WASP-Homology 1 (WH1) such as the adaptor proteins GRB2, FYN, PI3K, and NCK. The domain mediates the binding to WASP-Interacting Protein (WIP; Ramesh Verprolin-homology (V) domain binds to actin monomers, and the

et al., 1997). The Phosphatidylinositol-4,5-bisphosphate (PIP2) links to the Cofilin-homology (C) and Acidic (A) domains bind to the Actin-Related Basic (B) domain and stabilizes WASP active form. The binding of the Protein (ARP)2/3 complex. The V/C/A region functions as the platform to GTPase-Binding Domain (GBD) with CDC42 induces WASP activation initiate actin polymerization (Park et al., 2010). severe clinical phenotype (full blown WAS) and a life expectancy are micro-thrombocytopenic, although intermittent X-Linked below 20 years of age (Jin et al., 2004). On the contrary, missense Thrombocytopenia (iXLT) is observed in some patients with sub- mutations, which result in residual expression of a full-length stantial protein expression (Notarangelo et al., 2002). Importantly, point-mutated WASP, are often associated with XLT (Villa et al., up to 11% of patients can present somatic mosaicism due to spon- 1995; Notarangelo et al., 2002; Albert et al., 2010), correspond- taneous in vivo reversion of the original mutation or second-site ing to a disease score of 0.5–2 and a longer life expectancy (Imai compensatory mutations that restore production of the WAS gene et al., 2004). All patients harboring mutations in the WAS gene product (Stewart et al., 2007). The revertant mutation can occur

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Table 1 | WAS scoring system according to Zhu et al. (1997), with subsequent refinements (Ochs andThrasher, 2006; Ochs et al., 2009).

Clinical scores iXLT XLT WAS

<1 1 2 3 4 5

Thrombocytopenia −/+ + + + + + Small platelets + + + + + + Eczema − − (+) + ++ (+)/+/++ Immunodeficiency − −/(+) (+) + + (+)/+ Infections − − (+) + +/++ (+)/+/++ Autoimmunity or malignancy − − − − − +

Scoring system: −, absent; (+), mild; +, present; ++, present and severe. iXLT,intermittent X-linked thrombocytopenia; WAS, Wiskott–Aldrich syndrome; XLT,X-linked thrombocytopenia. at various stages of hematopoietic differentiation thus conferring could be an abnormal platelet clearance induced by an increased high selective advantage to revertant cells over mutated cell popu- exposure of phosphatidylserine on the outer plasma membrane of lations not expressing WASP. Although many reports describe the WASP-deficient platelets (Shcherbina et al., 2009). Another mech- occurrence of this phenomenon, it is still not clear whether the anism of platelet reduction that needs to be investigated more presence of somatic mosaicism might correlate with a better clin- in detail, is the elimination mediated by autoimmune reaction. ical course of the disease (Davis and Candotti, 2009; Trifari et al., In fact, the presence of platelet-associated antibodies in Was−/− 2010). mice (Marathe et al., 2009) and in some patients has been reported Absence or residual WASP expression causes functional defects (Corash et al., 1985; Semple et al., 1997). The second most com- in all immune cells (Figure 2). mon manifestation in WAS patients is the eczema. It is observed in The formation of the Immunological Synapse (IS) in T cells and 80% of patients and its severity inversely correlates with the expres- T Cell Receptor (TCR)-dependent activation (Dupre et al., 2002; sion of WASP. Indeed, it has been shown that WAS patients with Trifari et al., 2006; Nikolov et al., 2010), the cytotoxic activity of residual WASP expression develop moderate or transient form CD8+ T cells and Natural Killer (NK) cells (Orange et al., 2002; of the disease, whereas most of WASP-negative patients develop de Meester et al., 2010) and the suppressor activity of Naturally severe, treatment-resistant eczema (Imai et al., 2004). High IgE occurring Regulatory T (nTreg) cells (Adriani et al., 2007, 2011; levels (more than 1000 IU/mL) were observed in 62% of WASP- Humblet-Baron et al., 2007; Maillard et al., 2007; Marangoni et al., negative patients and in 25% of WASP-positive. Although higher 2007) are all impaired in WASP-deficient cells. Motility, adhesion IgE levels may represent a possible cause of eczema, the correlation and migration of B cells are also defective (Westerberg et al., 2005; between increased IgE levels and eczema has not yet been demon- Meyer-Bahlburg et al.,2008).Additionally,the lack of WASPaffects strated. WAS patients are highly susceptible to infections by bacte- podosome formation (Burns et al., 2001; Calle et al., 2004), motil- ria, , and fungi (Imai et al., 2004). Of note, WASP-negative ity (Binks et al., 1998; de Noronha et al., 2005) and T cell priming patients are more frequently affected by bacterial infections (otitis in Dendritic Cells (DCs; Pulecio et al., 2008; Bouma et al., 2011), as media, skin abscess, pneumonia, enterocolitis, , sepsis, well as podosome and phagocytic cup formation in , and others), viral infections (Herpes sim- (Linder et al., 1999; Tsuboi and Meerloo, 2007). Invariant Natural plex and Cytomegalovirus) and fungal infections (Candida spp., Killer T (iNKT) cell functionality (Astrakhan et al., 2009; Locci Aspergillus spp., and Pneumocystis carinii) as compared to WASP- et al., 2009), adhesion, and migration of neutrophils (Zhang et al., positive patients (Imai et al., 2004). Patients with clinically most 2006) are also altered in the absence of WASP. Moreover, WASP is severe WAS develop malignancies and/or autoimmune manifes- also involved in signal transduction (Figure 3). In particular, TCR- tations. Malignancies can affect adolescent and young adult WAS dependent nuclear recruitment of Nuclear Factor of Activated T patients more than infants (Sullivan et al., 1994; Imai et al., 2004). cells (NFAT)-1 in CD4+ T cells and both NFAT-1 and NFAT-2 Epstein–Barr virus (EBV)-positive B cell lymphoma is most fre- in CD8+ T cells are reduced in WAS patients and correlate with quently reported, but also myelodysplasia can be observed in defective Th1 cytokine production (Cianferoni et al., 2005; Trifari some patients (Imai et al., 2004). Autoimmune complications et al., 2006). Additionally, WASP is involved in B Cell Receptor are frequently observed in WAS, affecting 22–72% of patients (BCR) signaling by binding to the Src homology three domains (Dupuis-Girod et al., 2003; Imai et al., 2004). WAS patients with of several tyrosine kinases, such as the Bruton’s Tyrosine Kinase autoimmune diseases constitute a high-risk group with poor prog- (BTK; Cory et al., 1996; Sharma et al., 2009). nosis. Moreover, autoimmunity is associated with a higher risk of The most common finding in WAS patients is microthrombo- a later development of tumors and an increased risk of mortality cytopenia which causes frequent hemorrhages in more than 80% (Sullivan et al., 1994). A better understanding of the mechanisms of patients (Ochs, 2002; Imai et al., 2004) and severe underlying autoimmunity in WAS would be crucial for the devel- episodes that lead to death in 4–10% of patients (Sullivan et al., opment of more effective therapies for the management of these 1994; Imai et al., 2004). The mechanism underlying thrombocy- manifestations in WAS and could also provide new insights in the topenia is not completely understood. One possible explanation pathogenesis of autoimmunity in PIDs.

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FIGURE 2 | Schematic view of cellular defects described in WASP-deficient cells. MΦ, ; Neut., .

AUTOIMMUNE MANIFESTATIONS IN WAS PATIENTS AND inflammatory bowel disease (9%), and IgA nephropathy (3%). CURRENT TREATMENTS Henoch–Schönlein-like purpura, , recurrent The most common autoimmune manifestation in WAS is , and uveitis have also been reported in some patients hemolytic anemia (36%), followed by vasculitis (including (Dupuis-Girod et al., 2003; Imai et al., 2004). Moreover, multiple cerebral vasculitis; 29%), arthritis (29%), neutropenia (25%), autoimmune manifestations can be observed. In most cases, and

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FIGURE 3 | Role of WASP inTCR and BCR signaling pathways. The these pathways is indicated by stars close to interacting molecules: red main signaling molecules (blue and green ovals) acting downstream of stars show the interactions that are demonstrated, whereas the blue stars TCR (A), BCR, and TLRs (B) are depicted. The red boxes indicate the main with question marks show hypothetical involvement of WASP in TLR pathways induced downstream of these receptors. The role of WASP in signaling pathways. in all cases of hemolytic anemia, the onset of autoimmune compli- 2 (hrIL-2) appeared to be effective in reducing herpes virus cations occurs early in life (0–5 years; Dupuis-Girod et al., 2003). infections and improving dermatitis in a WAS patient (Azuma Interestingly, it has been recently found that Was−/− mice develop et al., 1993). Administration of hrIL-2 ameliorated proliferation proliferative glomerulonephritis with increased IgA in the serum of cultured T cells from one patient (Azuma et al., 2000) and and IgA production by splenic B cells (Nikolov et al.,2010; Shimizu restored and actin accumulation at the IS in NK et al., 2012). Moreover old Was−/− mice showed aberrant glycosy- cells from another treated patient (Orange et al., 2011). Since lation of IgA (Shimizu et al., 2012), feature that has been associated WAS T cells are less efficient in producing IL-2, NK cells do to the development of nephropathy-like glomerular lesions with not receive sufficient IL-2, thus resulting in reduced NK acti- IgA deposition (Nishie et al., 2007). Although these studies have vation and failure to respond effectively to infections. A clinical been performed on WAS mouse model, they clearly suggest a pos- trial with hrIL-2 is currently ongoing in WAS (ClinicalTrials.gov sible mechanism for the pathogenesis of glomerulonephritis in identifier NCT00774358). The treatment of choice for autoim- WAS patients. mune manifestations in WAS patients consists of steroids, alone, Clinical management of WAS patients is a significant chal- or in association with cyclosporine (Dupuis-Girod et al., 2003). lenge since, with the exception of Bone Marrow Transplantation Steroids are the first-line treatment for all patients with hemolytic (BMT), most available therapies are not curative. Intravenous anemia and efficiently induce remission in 10% of cases, are par- immunoglobulins (IVIG) and prophylaxis are often tially effective in 60% of cases, while are ineffective in 30% of used to reduce the risk of infections in WAS patients, but it is cases. Cyclophosphamide and azathioprine are also used in some not clear whether these treatments effectively reduce the incidence cases and are effective in a small percentage of cases. Patients of life-threatening infections (Conley et al., 2003). with severe autoimmune thrombocytopenia after splenectomy are significantly increases and often normalizes the platelet counts usually treated with IVIG, high-dose steroids, azathioprine, and (Corash et al., 1985; Mullen et al., 1993). However, it does not fully cyclophosphamide. Other autoimmune or inflammatory compli- overcome the risk of bleeding and further predisposes to sepsis, cations are generally treated with steroids, in association with obliging the patients to life-long antibiotic prophylaxis (Mullen cyclosporine, and are effective in the majority of skin vasculi- et al., 1993). Relapse of thrombocytopenia has been described tis, arthritis, bowel inflammatory disease and renal disease cases in a fraction of splenectomized WAS patients (Corash et al., (Dupuis-Girod et al., 2003). Anti-CD20 1985; Dupuis-Girod et al., 2003). Of note, in some cases, throm- therapy has been also performed for the treatment of autoim- bocytopenia was found to be autoantibody-mediated and also mune hemolytic anemia in some patients. This treatment results associated with hemolytic anemia or cerebral vasculitis (Dupuis- effective in correcting the anemia,but it may need repeated courses Girod et al., 2003). Therefore, splenectomy is indicated only in due to relapse of the disease (Ship et al., 2002; Kim et al., 2007). severe cases, for which there is no prospect for other curative Currently, the only resolutive therapeutic option for WAS interventions. Treatment with human recombinant Interleukin patients is BMT. When a Related HLA-Identical Donor (RID) is

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available, BMT leads to 73–100% survival (Mullen et al., 1993; (Press Release, Hannover Medical School, http://www.asgct.org/ Ozsahin et al., 1996, 2008; Filipovich et al., 2001; Antoine et al., UserFiles/file/Genetherapy_WAS_final_english.pdf). This adverse 2003; Kobayashi et al., 2006; Pai et al., 2006; Moratto et al., 2011). event gives rise to some concerns on the safety of RV-mediated GT On the other hand, transplantation using the bone marrow of a for WAS. A multicenter clinical trial using a third generation LV Mismatched Related Donor (MMRD) results in a poor survival carryingWAS gene driven by the endogenous promoter is on going ranging from 29 to 52% (Mullen et al., 1993; Filipovich et al., in Milan, Paris, and London. Preclinical data in the murine model 2001; Kobayashi et al., 2006; Ozsahin et al., 2008). In addition, this indicate that the LV-mediated GT approach is effective in restoring type of transplant is associated with an elevated risk of developing immune cell functionality (Blundell et al., 2008; Marangoni et al., life-threatening EBV+ lymphoproliferative syndrome, infections, 2009; Bosticardo et al., 2011; Catucci et al., 2011). GT treated autoimmunity, and graft-versus-host disease (GVHD; Filipovich Was−/− mice did not show any adverse events or tumors even et al., 2001), therefore it is not recommended except in case of in long-term follow up studies (Marangoni et al., 2009). Finally, emergency. When a suitable related donor is missing, transplan- we and others demonstrated the efficacy of LV-mediated GT in tation using the bone marrow or cord blood from a Matched CD34+ cells obtained from WAS patients (Charrier et al., 2007; Unrelated Donor (MUD) is a valid therapeutic option, leading to Scaramuzza et al., 2012). Nevertheless, data from the clinical study 71–81% survival (Filipovich et al., 2001; Kobayashi et al., 2006; Pai are needed to provide definitive evidence of the efficacy and safety et al., 2006). Two recent retrospective studies have analyzed long- of this novel therapeutic approach. term outcome and donor cell engraftment in WAS patients who have been treated by Hematopoietic Stem Cell Transplantation REGULATION OF T CELL TOLERANCE IN WAS (HSCT; Ozsahin et al., 2008; Moratto et al., 2011). They observed T cells are significantly reduced in peripheral blood of WAS that 20% of patients developed autoimmune manifestations after patients and show a defective proliferation in response to TCR HSCT independently of chronic GVHD (Ozsahin et al., 2008) stimulation by CD3-specific antibody, although this defect is and some patients had more than one manifestation. Autoim- present only at low doses of agonistic antibody (Molina et al., mune manifestations appeared at a median of 1.5 years after HSCT 1992, 1993). TCR-dependent activation in WASP-deficient T cells (range: 4 months to 10 years). The median duration of autoimmu- results in a reduced IL-2 production (Molina et al., 1993), that is nity was 4 years (range: 1–20 years). Autoimmune manifestations associated with delayed NFAT-1 nuclear translocation and defec- were more frequent in recipients of MUD (28%) and MMRD tive T-bet induction (Cianferoni et al., 2005; Trifari et al., 2006; (26%) than RID HSCT (11%). Ozsahin and colleagues investi- Taylor et al., 2010). T cell activation is regulated by the formation gated whether patients developing autoimmunity after HSCT had of the IS, a polarized cluster of TCR, costimulatory molecules, sig- autoimmune manifestations also before treatment. Overall, 17 naling molecules, and integrins at the T cell:antigen presenting cell patients had autoimmune manifestations before transplantation (APC) interface. Topromote their lateral movement on the plasma that persisted thereafter in seven of them. Conversely, autoimmu- membrane, the molecules being recruited to the IS are associ- nity occurred de novo in 11–23% of transplanted patients. A very ated with specific cholesterol-enriched membrane microdomains, interesting observation in both retrospective studies was the strong called lipid rafts. In the absence of WASP, IS can be formed only correlation between autoimmunity occurrence and the chimerism after strong TCR stimulation (Cannon and Burkhardt, 2004). In pattern. Overall, incomplete reconstitution of lymphocyte counts particular, WASP-deficient T cells fail to upregulate GM1 on the and incidence of autoimmunity were higher in patients with a cell surface, cluster GM1 in the lipid rafts during IS formation lower degree of chimerism in both lymphoid and myeloid com- (Dupre et al., 2002) and maintain IS stability after migration (Sims partments as compared to patients with full chimerism (Ozsahin et al., 2007). et al., 2008; Moratto et al., 2011). It is commonly assumed that autoimmunity is a consequence of A very promising alternative to HSCT,when a matched donor is the breakdown of central or peripheral tolerance to self-antigens. missing, is the infusion of gene corrected autologous Hematopoi- nTreg cells are fundamental to maintain tolerance to self-antigens etic Stem Cells (HSCs). Two different Gene Therapy (GT) clinical and suppress excessive immune responses. nTreg cell develop- trials have been approved: a Retroviral Vector (RV)-mediated gene ment and function depend on TCR signaling, together with CD28 transfer (Boztug et al.,2010) and a LentiviralVector (LV)-mediated recruitment, FOXP3 expression, and presence of IL-2 (Sakaguchi GT approach, developed by our and other groups (Dupre et al., et al., 2008). Several groups, including ours, have described the 2006; Galy et al., 2008). In the RV-mediated clinical trial, sustained defects of nTreg cells in WAS patients and Was−/− mice in local- expression of WASP in HSCs, lymphoid and myeloid cells, and izing and suppressing T effector cell response (Adriani et al., platelets was shown in two treated patients 3 years after GT (Boztug 2007; Maillard et al., 2007; Marangoni et al., 2007), although their et al., 2010). T and B cells, NK cells, and were also func- number in blood of WAS patients is comparable with healthy tionally corrected resulting in improved clinical conditions. Signs donors (Marangoni et al., 2007). It is not clear whether a defec- and symptoms of autoimmunity disappeared in both patients tive thymic development of Was−/− nTreg cells could account within the first year after GT. In one of the two reported patients, for their impaired in vivo suppressive function, since one group severe autoimmune hemolytic anemia, autoimmune thrombo- has shown reduced nTreg cell percentage in the thymus (Maillard cytopenia, and autoimmune neutropenia disappeared; whereas et al., 2007), while three other groups observed normal frequency severe eczema resolved in the second patient. However, in this while showed a reduced function in vivo (Adriani et al., 2007; trial, occurred in one out of ten GT patients, prob- Humblet-Baron et al.,2007; Marangoni et al.,2007),but all showed ably due to insertional mutagenesis caused by RV integration a reduced in vivo suppression. Was−/− nTreg cell failure to control

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aberrant T cell activation has been also demonstrated in vivo in cytoskeletal functions of B cells, including adhesion, migration, a mouse model of autoimmunity (Humblet-Baron et al., 2007). and homing (Westerberg et al., 2005, 2008; Meyer-Bahlburg et al., Moreover, selective advantage of WASP-expressing nTreg cells was 2008). These defects may compromise the capacity of B cells to be shown in a WAS patient with revertant mutation, demonstrating properly activated and reach the site of infection contributing to that WASP has a role in nTreg cell fitness (Humblet-Baron et al., the inability of the immunodeficient host to completely eradicate 2007). Although the requirement of WASP for nTreg cell func- infectious agents. In this respect, it has been accepted, in particu- tionality has been demonstrated, the role of WASP in these cells lar for PIDs, that chronic due to an incomplete is still unclear. Indeed, differently from effector T cells, WASP is pathogen clearance may favor breakdown of peripheral tolerance. not recruited to the IS (Marangoni et al., 2007), thus suggesting Of note, the complement receptors CD21 (CR2) and CD35 (CR1) a possible role of WASP in TCR signaling of nTreg cells. More- are expressed at lower levels on B cells of patients with WAS (Park over, WASP-deficient nTreg cells are also defective in suppressing et al., 2005) contributing to a suboptimal B cell capacity to capture B cell activation. In fact, it has been shown in in vitro studies that and present opsonized antigens. Additionally, given the critical nTreg cells from Was−/− mice are less efficient in turning off B cell role of CD21 and CD35 in the negative selection of self-reactive proliferation and this defect is associated with a reduced killing B cells (Prodeus et al., 1998), the altered expression or function of B cells and significantly decreased secretion of by of these receptors may affect the maintenance of B cell tolerance nTreg cells (Adriani et al., 2011). Susceptibility of WAS patients to in (IC)-mediated autoimmune diseases such as develop autoimmune diseases can be at least in part explained by SLE and RA (Erdei et al., 2009). nTreg cell dysfunction. The fate of self-reactive B cells within the bone marrow and Recent findings have demonstrated that also T effector cells are peripheral lymphoid compartment is largely determined by the implicated in tolerance breakdown in WAS. Indeed, in response strength of signal mediated by BCR in response to antigen cross- to restimulation through the TCR, activated T cells can undergo linking (Nemazee and Burki, 1989; Erikson et al., 1991; Goodnow, , and this event is called restimulation-induced cell death 1996). To this regard, reports of a defective BCR activation are (RICD; Lenardo, 1991; Siegel et al., 2000). RICD process con- controversial. Activation of WASP-deficient B cells was found to tributes to the maintenance of peripheral by be defective after BCR engagement in terms of calcium mobi- eliminating T cells responding to prolonged presence of antigens, lization in primary B cells isolated from WAS patients and also such as self-antigens and persistent pathogen antigens (Critchfield in WASP-deficient EBV-transformed B cell lines (Simon et al., et al., 1994; Ettinger et al., 1995; Weant et al., 2008). In CD4+ T 1992). However, this defect was not confirmed by Henriquez et al. cells, RICD is induced by the Tumor Necrosis Factor (TNF) family (1994). More recently, studies performed on Was−/− mice showed member Fas ligand (FasL) that is released and binds its recep- a normal proliferative response of B cells after stimulation with tor Fas in an autocrine fashion (Critchfield et al., 1994; Ettinger anti-IgM, LPS, or anti-CD40 (Snapper et al., 1998; Zhang et al., et al., 1995; Siegel et al., 2000; Green et al., 2003; Weant et al., 2008). 1999) and a normal or increased class switch (Westerberg et al., Nikolov and colleagues have shown thatWASPis required for T cell 2005). However, the presence of circulating autoantibodies in apoptosis by RICD. In the absence of WASP,the release of FasL by WAS patients (Dupuis-Girod et al., 2003; Schurman and Candotti, CD4+ T cells is reduced and this is associated to a decreased TCR- 2003) and in Was−/− mice (Humblet-Baron et al., 2007; Nikolov mediated apoptosis (Nikolov et al., 2010). Together with nTreg cell et al., 2010; Becker-Herman et al., 2011; Bosticardo et al., 2011) defects, these recent findings highlight the role played by effector represents the first evidence of a perturbed B cell tolerance. Very T cells in the maintenance of T cell tolerance in WAS. recently, two studies have shown the contribution of B cell intrin- sic defects to the pathogenesis of autoimmunity in two different REGULATION OF B CELL TOLERANCE IN WAS murine models. Indeed, Becker-Herman et al. (2011) observed In the last years, many studies have assessed the role of B cells that female Was+/− mice generate anti-nuclear antibodies at rates in driving autoimmune diseases such as and titers equivalent to Was−/− mice even though heterozygous (RA), (MS), and Systemic Erythematosus have a normal nTreg cell compartment. Based on this evi- (SLE; Townsend et al., 2010). These data revealed the complex role dence, they demonstrated in mixed BM chimeras, in which only B of B cells that work independently or synergistically with other cells lacked WASP expression, that the selective defect in B cells is components of the innate and to drive sufficient for the generation of autoantibodies. Additionally, they autoimmune pathogenesis. suggested that BCR/Toll-Like Receptor (TLR) co-engagement in For many years, the functionality of B cells in WAS patients Was−/− B cells from chimeras could mediate tolerance breakdown, was poorly investigated. The presence of a skewed distribution of since the loss of Myeloid Differentiation primary response gene serum Ig isotypes (reduced IgM, normal IgG, and elevated IgE 88 (MyD88) signaling abolished the production of anti-dsDNA and IgA levels) and a reduced or absent antibody production to antibodies, formation, and development of sys- and other T cell-independent antigens (Golding temic autoimmune disease (Becker-Herman et al., 2011). More et al., 1984; Ochs and Thrasher, 2006) represent the first evi- recently, by conditional WAS gene deletion in B cells (B/WcKO dences of a defective B cell effector function in WAS patients. This mice), Recher et al. (2012) observed that WASP deficiency limited prompted many researchers to investigate more in detail the B to B cells is sufficient to promote autoantibody production and cell compartment in WAS, mainly taking advantage of the murine kidney tissue damage in B/WcKO mice. model of the disease (Was−/− mice). In the last decade, it has Overall, these findings highlight the contribution of B cells to been clearly defined that the lack of WASP causes defects in the the pathogenesis of autoimmunity in WAS and suggest that the B

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cell autonomous defect could represent a sufficient factor to break 2001) and uveitis (Oh et al., 2011). Although the mechanisms tolerance in WAS. However, in addition to the supposed role for have not been fully understood, it has been shown that iNKT TLR signaling in the autoreactivity of Was−/− B cells, other mech- activation reduces autoimmune symptoms by limiting the devel- anisms are potentially involved and need to be further investigated opment of Th17 cells in a cell contact- and cytokine-dependent both in mice and humans. manner (Mars et al., 2009). Moreover, in mice, iNKT cells sup- press anti-DNA antibody production and reduce autoreactive B FUTURE DIRECTIONS cells (Yanget al., 2011), whereas iNKT reduction leads to increased WAS is characterized by a very complex spectrum of cellular autoreactive B cell activation (Wermeling et al., 2010). Since iNKT defects, many of which can predispose patients to the development cells are reduced in WAS patients and functionally defective in of autoimmunity (Figure 4). Was−/− mice (Astrakhan et al., 2009; Locci et al., 2009) it can As described above, defective control of the strength of immune be hypothesized that such impairment may also contribute to response by nTreg cells, the presence of autoantibodies and poten- autoimmunity. tially autoreactive B cells have been demonstrated in WAS (Bosti- A mechanism contributing to the tolerance breakdown in PIDs cardo et al., 2009). However several mechanisms shown to be is related to the inability of innate immune cells, in particular DCs, involved in the pathogenesis of autoimmune diseases still need to properly activate adaptive immune response (Arkwright et al., to be investigated in WAS. iNKT cells have been shown to prevent 2002). Since DCs have a role in the induction of nTreg cells (Mani- autoimmune disease in a mouse model of experimental Autoim- cassamy and Pulendran,2011) and DCs lackingWASPare defective mune Encephalomyelitis (EAE; Miyamoto et al., 2001; Singh et al., in T cell priming (Bouma et al., 2007; Pulecio et al., 2008), it is

FIGURE 4 | Schematic view of immunodeficiency and between immunodeficiency and autoimmunity. Defective suppression autoimmunity in WAS. The impairment of both innate and adaptive of WASP-deficient nTreg cells toward both T and B cells contributes to immune systems is responsible of immunodeficiency (blue box) and the tolerance breakdown in WAS. Defect in RICD process, resulting in autoimmunity (red box) in WAS. Immune cell defects described in defective effectorT cell apoptotic death afterTCR restimulation, concurs WASP-deficient cells are shown in red. The incomplete pathogen in the persistence of T cell response to pathogens or self-antigens. clearance is sustained by cytoskeleton and functional defects of Additionally, intrinsic B cell defects contribute to autoimmunity in WAS, macrophages, DCs, T cells, B cells, and their defective interactions. The probably via a TLR-mediated mechanism. Dashed lines represent reduced expression of CD21 and CD35, two complement receptors hypothetical mechanisms involved in WAS-related autoimmunity. MHC, involved in antigen uptake and presentation and also in negative Major Histocompatibility Complex; RICD, Restimulation-Induced Cell selection of self-reactive B cells, places B cells at the interface Death; autoAbs, autoantibodies; Ag, Antigen.

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possible to hypothesize that a defect in nTreg cell induction by DCs peripheral checkpoints during B cell maturation which require might occur in the absence of WASP.Immunodysregulation can be proper BCR and TLR signaling together with extrinsic factors also sustained by overload of pathogen antigens or apoptotic mate- (Meffre, 2011). The cytoskeleton controls the distribution of the rial due to defective clearance by innate immune cells. Antigen BCR and shapes its signaling (Batista et al., 2010). In particular, the overload in fact results in a prolonged immune response, which density of actin network inversely correlates with the rate of BCR promotes expansion of Th17 cell subset, playing a central role in diffusion and the restriction of BCR diffusion limits signaling. many autoimmune diseases, such as MS, RA, and Crohn’s disease Since WASP is required for actin polymerization and cytoskeletal (Langrish et al., 2005; Fouser et al., 2008; Isaksson et al., 2009; organization in B cells (Facchetti et al., 1998; Westerberg et al., Sharma et al., 2009). Furthermore, reduced clearance of apoptotic 2005), it is reasonable to speculate that the threshold of activation material has been associated to the accumulation of autoantibod- might be altered in WASP-deficient B cells. In the bone marrow, ies in SLE (Gaipl et al., 2005; Fransen et al., 2012). WASP-deficient receptor editing is the major mechanism aimed at eliminating self- DCs are impaired in antigen uptake and migration to secondary reactive B cells during differentiation (Monroe and Dorshkind, lymphoid tissues (Westerberg et al., 2003; de Noronha et al., 2005) 2007; von Boehmer and Melchers, 2010) by editing autoreactive suggesting an inefficient pathogen clearance, process that needs to receptors through secondary rearrangements in light chain loci be investigated in in vivo models of infection. Moreover, WASP- (Halverson et al., 2004). Abnormal receptor editing is involved in deficient macrophages are less efficient in uptaking apoptotic cells the loss of central B cell tolerance (Ng et al., 2004). Interestingly, both in vitro and in vivo (Leverrier et al., 2001). All together, these alterations in the regulation of secondary recombination events findings suggest that dysregulation of Th17 cell activation might have been reported in BTK-, Interleukin-1 Receptor-associated contribute to autoimmunity induction in WAS patients, although Kinase 4 (IRAK4)-, and MyD88-deficient patients and in a group no evidence has been provided so far to sustain such hypothesis. of Common Variable Immunodeficiency (CVID) patients with Recent studies have demonstrated the key role played by a spe- expanded autoreactive CD21−/low B cells (Ng et al., 2004; Isnardi cific subset of DCs, namely plasmacytoid DCs (pDCs), in the et al.,2008; Meffre,2011). Given the interaction of WASPwith BTK pathogenesis of systemic autoimmune diseases. In particular, IFN- (Cory et al., 1996; Sharma et al., 2009), the involvement of MyD88 α produced by pDCs upon recognition of foreign nucleic acids via signaling in B cell tolerance (Becker-Herman et al., 2011) and the TLR7 and TLR9 contributes to tolerance breakdown in several increased frequency of CD21− B cells in WAS patients (Park et al., autoimmune diseases, such as SLE, SS, and psoriasis (Ronnblom, 2005), it would be worth to investigate whether receptor editing is 2011). In these clinical settings, self-nucleic acid-containing ICs defective also in the absence of WASP.Furthermore, in the periph- trigger TLR7 or TLR9 leading to an uncontrolled pDCs activation. ery, survival of autoreactive B cells is supported by high levels of In PIDs, an increased susceptibility to viral infection, in combina- BAFF and APRIL, members of the TNF superfamily, found to be tion with a defective clearance of pathogens,could be the triggering increased in several autoimmune diseases (Townsend et al., 2010) factor of the over-activation of type I IFN pathway. Moreover, cell and lymphopenic conditions (Cassani et al., 2010). This represents death induced by viral infection leads to the release and accumula- an important mechanism involved in the regulation of peripheral tion of self-antigens in the extracellular matrix. Since PID patients, human B cell tolerance that would be interesting to investigate in including WAS patients, are highly susceptible to infections and WAS. Finally, a new function as regulator of immune response has fail to completely eradicate the pathogens, high levels of ICs and been described for B cells and is mainly mediated by the secretion activation of the type I IFN system can be expected. Furthermore, of IL-10 (Matsushita et al., 2008; Yanaba et al., 2009; Watanabe increasing evidences highlight the role played by neutrophils in et al., 2010). Although the origin of regulatory B cells is unclear, SLE in the induction of type I IFN production. Mature neutrophils MZ B cells (Lenert et al., 2005; Evans et al., 2007) seem to have reg- are primed in vivo by type I IFN and die upon exposure to anti- ulatory functions. Thus, considering the reduction of MZ B cells ribonucleoprotein antibodies, releasing neutrophil extracellular in Was−/− mice (Westerberg et al., 2008; Bosticardo et al., 2011), it traps (NETs) which in turn activate pDCs to produce high lev- would be interesting to investigate whether a defect in regulatory els of type I IFN (Garcia-Romo et al., 2011). Overall, these studies B cell function is a factor contributing to autoimmunity. have demonstrated an important role of neutrophils and pDCs in In conclusion, together with the defects already described in the promoting autoimmune diseases and it can be envisaged that these literature, these future lines of enquiry underline the greater than mechanisms may act in the complexity of WAS autoimmunity. expected extent to which the WASP deficiency affects the immune Triggering of autoreactive B cells by self-nucleic acid- system. Further research is necessary to define the underlying mol- containing ICs can be another possible mechanism underlying ecular and cellular mechanisms leading to autoimmunity, which the production of autoantibodies in WAS. In fact, it is known represents the main collateral damage caused by WASP deficiency. that self-nucleic acid-containing ICs can activate B cells through synergistic engagement of BCR and TLR7 or TLR9 (Leadbetter ACKNOWLEDGMENTS et al., 2002; Lau et al., 2005; Chaturvedi et al., 2008), and the loss This work was supported by grants from Italian Telethon Founda- of MyD88 signaling in Was−/− mice abolish the production of tion (toAnnaVilla),and Ministero della Salute RF 2007-2008-2009 anti-dsDNA antibodies (Becker-Herman et al., 2011). The recent Giovani Ricercatori Grant (to Marita Bosticardo). Maria Carmina findings of B cell intrinsic defect (Becker-Herman et al., 2011; Castiello conducted this study as partial fulfillment of her PhD. in Recher et al., 2012) open a new scenario in tolerance breakdown , Program in Basic and Applied Immunology, in WAS although the underlying mechanisms are still unclear. It San Raffaele University, Milan, Italy. Figures were produced using is known that B cell tolerance is established through central and Servier Medical Art (www.servier.com).

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CD1d-positive B cells. J. Exp. Med. in marginal zone B-cell home- Zhang, J., Shehabeldin, A., da Cruz, could be construed as a potential con- 207, 943–952. ostasis and function. Blood 112, L. A., Butler, J., Somani, A. K., flict of interest. Westerberg, L., Larsson, M., Hardy, S. 4139–4147. Mcgavin, M., Kozieradzki, I., Dos J., Fernandez, C., Thrasher, A. J., Yanaba, K., Bouaziz, J. D., Mat- Santos, A. O., Nagy, A., Grinstein, S., Received: 23 April 2012; paper pending and Severinson, E. (2005). Wiskott- sushita, T., Tsubata, T., and Ted- Penninger, J. M., and Siminovitch, published: 06 May 2012; accepted: 01 July Aldrich syndrome protein deficiency der, T. F. (2009). The develop- K. A. (1999). Antigen receptor- 2012; published online: 18 July 2012. leads to reduced B-cell adhesion, ment and function of regulatory B induced activation and cytoskele- Citation: Catucci M, Castiello migration, and homing, and a cells expressing IL-10 (B10 cells) tal rearrangement are impaired in MC, Pala F, Bosticardo M and delayed humoral immune response. requires antigen receptor diversity Wiskott-Aldrich syndrome protein- Villa A (2012) Autoimmunity Blood 105, 1144–1152. and TLR signals. J. Immunol. 182, deficient lymphocytes. J. Exp. Med. in Wiskott–Aldrich syndrome: an Westerberg, L., Wallin, R. P., Greicius, 7459–7472. 190, 1329–1342. unsolved enigma. Front. Immun. 3:209. G., Ljunggren, H. G., and Sev- Yang, J. Q., Wen, X., Kim, P. J., Zhu, Q., Watanabe, C., Liu, T., doi: 10.3389/fimmu.2012.00209 erinson, E. (2003). Efficient anti- and Singh, R. R. (2011). Invari- Hollenbaugh, D., Blaese, R. M., This article was submitted to Frontiers in gen presentation of soluble, but ant NKT cells inhibit autoreactive Kanner, S. B., Aruffo, A., and Primary Immunodeficiencies, a specialty not particulate, antigen in the B cells in a contact- and CD1d- Ochs, H. D. (1997). Wiskott- of Frontiers in Immunology. absence of Wiskott-Aldrich syn- dependent manner. J. Immunol. 186, Aldrich syndrome/X-linked Copyright © 2012 Catucci, Castiello, drome protein. Immunology 109, 1512–1520. thrombocytopenia: WASP gene Pala, Bosticardo and Villa. This is an 384–391. Zhang, H., Schaff, U. Y., Green, C. E., mutations, protein expres- open-access article distributed under the Westerberg, L. S., de la Fuente, M. Chen, H., Sarantos, M. R., Hu, Y., sion, and phenotype. Blood 90, terms of the Creative Commons Attribu- A., Wermeling, F., Ochs, H. D., Wara, D., Simon, S. I., and Low- 2680–2689. tion License, which permits use, distrib- Karlsson, M. C., Snapper, S. B., ell, C. A. (2006). Impaired integrin- ution and reproduction in other forums, and Notarangelo, L. D. (2008). dependent function in Wiskott- Conflict of Interest Statement: The provided the original authors and source WASP confers selective advantage Aldrich syndrome protein-deficient authors declare that the research was are credited and subject to any copy- for specific hematopoietic cell pop- murine and human neutrophils. conducted in the absence of any com- right notices concerning any third-party ulations and serves a unique role Immunity 25, 285–295. mercial or financial relationships that graphics etc.

Frontiers in Immunology | Primary Immunodeficiencies July 2012 | Volume 3 | Article 209 | 44 REVIEW ARTICLE published: 27 August 2012 doi: 10.3389/fimmu.2012.00265 Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency

Aisha Vanessa Sauer 1, Immacolata Brigida1, Nicola Carriglio1,2 and Alessandro Aiuti 1,2*

1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy 2 Università degli Studi di Roma Tor Vergata, Rome, Italy

Edited by: Genetic defects in the adenosine deaminase (ADA) gene are among the most common Rosa Bacchetta, San Raffaele causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from Scientific Institute, Italy lymphopenia, severely impaired cellular and , failure to thrive, and recur- Reviewed by: Rosa Bacchetta, San Raffaele rent infections. Currently available therapeutic options for this otherwise fatal disorder Scientific Institute, Italy include bone marrow transplantation (BMT), enzyme replacement therapy with bovine Mirjam van Der Burg, Erasmus MC, ADA (PEG-ADA), or hematopoietic stem cell gene therapy (HSC-GT). Although varying Netherlands degrees of immune reconstitution can be achieved by these treatments, breakdown of *Correspondence: tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune Alessandro Aiuti, San Raffaele Telethon Institute for Gene Therapy, hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are Via Olgettina 58, Dibit 2A2, Milan frequently observed in milder forms of the disease. However, several reports document 20132, Italy. similar complications also in patients on long-term PEG-ADA and after BMT or GT treat- e-mail: [email protected] ment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic dis- ease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregu- lation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.

Keywords: adenosine deaminase, severe combined immunodeficiency, ADA-SCID, autoimmunity, gene therapy

THE ADA METABOLISM the brain, and gastrointestinal tract. The ADA enzyme is ubiqui- THE ADA ENZYME tously expressed both intracellularly and on the cell surface where As an enzyme of the purine salvage pathway, adenosine deam- it complexes with two molecules of CD26 as a combined protein inase (ADA) catalyzes the deamination of adenosine and 20- (Kameoka et al., 1993). deoxyadenosine, as well as several naturally occurring methylated 0 adenosine compounds (Hirschhorn and Ratech, 1980; Ratech THE ADA SUBSTRATES ADENOSINE AND 2 -DEOXYADENOSINE 0 20-Deoxyadenosine is a component of DNA and primarily derives et al.,1989). The deamination of adenosine and 2 -deoxyadenosine 0 gives rise to and deoxyinosine, respectively (Hirschhorn from its breakdown. Therefore, 2 -deoxyadenosine concentration and Candotti, 2006). Further conversion of these deaminated is expected to be highest at sites of cell death, such as the bone marrow and thymus, where lymphocytes undergo apoptotic death nucleosides leads to hypoxanthine, which can be either trans- 0 formed irreversibly into uric acid or salvaged into mononucle- during differentiation and selection. 2 -Deoxyadenosine behaves osides (Figure 1). as a cytotoxic metabolite and is generally considered the pri- Although ADA is present in all cell types, its enzyme activ- mary cause of lymphotoxicity in ADA-severe combined immun- ity differs considerably among tissues. The highest amounts in odeficiency (SCID; Hirschhorn and Candotti, 2006). The most humans are found in lymphoid tissues, particularly the thymus, striking metabolic alteration in ADA deficiency is the accumu- lation of massive amounts of dATP in erythrocytes and lym- phocytes (Hirschhorn et al., 1992). This results from uptake Abbreviations: ADA, adenosine deaminase; Adora, adenosine receptor(s); ANA, of increasing 20-deoxyadenosine present in surrounding body anti-nuclear antibody; BCR, B cell receptor; BMT, bone marrow transplanta- fluids with subsequent intracellular phosphorylation and trap- tion; HLA, ; HSC, hematopoietic stem cell; HSC-GT, hematopoietic stem cell gene therapy; PEG-ADA, pegylated bovine ADA; SCID, ping. severe combined immunodeficiency; TCR, T-cell receptor; TLR, Toll-like receptor; Adenosine on the other hand is a component of adenine Tregs, naturally occurring regulatory T cells. nucleotides including ATP and RNA (Hirschhorn and Candotti,

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FIGURE 1 |The adenosine deaminase (ADA) metabolism. ADA is non-reversible pathway to uric acid or salvaged back into other an enzyme of the purine salvage pathway, which catalyzes the mononucleosides. In the absence of ADA, the presence of these irreversible deamination of adenosine and 20-deoxyadenosine into alternative “bypass” pathways results in normal concentrations of the inosine and 20-deoxyinosine, respectively. Most adenosine derives catabolic products of the enzyme reaction in patients with ADA-SCID. from endogenous breakdown of ATP and degradation of RNA, or is Conversely, the levels of ADA substrates, adenosine and taken up exogenously by ubiquitously expressed nucleoside 20-deoxyadenosine, are not only found in increased amounts in transporters. Unlike adenosine, 20-deoxyadenosine is formed by DNA extracellular body fluids, but they also “spill over” into additional degradation is predominantly catabolized by ADA. Further conversion pathways normally only minimally utilized, thus contributing to the of inosine nucleoside leads to hypoxanthine, which can either enter a pathogenic mechanisms of the disease.

2006). Elevated adenosine levels, as occurring in ADA deficiency IMMUNE DEFECTS contribute to apoptosis and block in the differentiation of thy- Lymphopenia and attrition of immune function over time are the mocytes, causing severe T lymphopenia in mice and humans two findings common to all presentations of ADA deficiency. It (Apasov et al., 2001; Gaspar et al., 2009; Poliani et al., 2009). More- is associated with thymic hypoplasia and a severe depletion of over adenosine, acting through cell surface G protein-coupled all three major categories of lymphocytes, T-, B-, and NK-cells receptors, functions as an extracellular signal transducer in a (Buckley et al., 1997). Absence of cellular and humoral immu- variety of physiological processes (Olah and Stiles, 1995). Apart nity and a rapidly fatal course due to infections with fungal, viral, from T-cell receptor signaling (Huang et al., 1997), adenosine and opportunistic agents are characteristic of early onset forms is involved in the control of heart rate and blood pressure of ADA deficiency (Giblett et al., 1972; Buckley et al., 1997). (Fukunaga et al., 1982; Belardinelli et al., 1989), renal function Total immunoglobulin levels may be only slightly depressed at (Churchill and Bidani, 1982), inflammatory responses (Black- birth due to the maternal contribution of IgG, whereas both burn,2003),and in neurotransmission (Fredholm and Dunwiddie, IgM and IgA, which ordinarily do not cross the placental bar- 1988). rier, are often absent. However, once IgG levels decline as maternal antibodies are cleared, a pronounced hypogammaglobulinemia ADA-SCID signals the absence of humoral immunity (Morgan et al., 1987; Adenosine deaminase deficiency is the second-most prevalent Hirschhorn and Candotti, 2006). About 20% of ADA-SCID cases form (approximately 20%) of SCID. The overall incidence in occur later in childhood (delayed) or beyond (late/adult onset). is estimated to range between 1:375,000 and 1:660,000 live Delayed or late-onset patients have significant immunodeficiency, births. ADA-deficient patients suffer from lymphopenia, severely but variable clinical manifestations (Ozsahin et al., 1997). These impaired cellular and humoral immune function, failure to thrive, forms show progressive immunological and clinical deteriora- and a rapidly fatal course due to infection (Hirschhorn and tion, often associated with autoimmune manifestations, includ- Candotti, 2006). Moreover, autoimmune manifestations are com- ing hemolytic anemia, and immune thrombocytopenia (Parkman monly observed in milder forms of the disease. Currently available et al., 1975; Aiuti et al., 2003). Serum immunoglobulin levels therapeutic options include bone marrow transplantation (BMT), are altered in late-onset patients, with IgG2 levels being highly enzyme replacement therapy with bovine ADA (PEG-ADA), or reduced or absent. IgE levels are elevated and often associated to hematopoietic stem cell gene therapy (HSC-GT). eczema and asthma. An inability to produce antibodies against

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and pneumococcal antigens was frequently found donors (43%). Indicating that despite recent progress in trans- in ADA-SCID patients with milder forms of the disease (Levy et al., plantation, the use of alternative donors is still associated with 1988). a reduced overall survival (Gaspar et al., 2009). This is further complicated by the fact that ADA-SCID patients are more difficult NON-IMMUNE DEFECTS to transplant especially from unrelated and haploidentical donors The initial and most devastating presentation of ADA-SCID is possibly due to their need for conditioning and the underlying due to the immune defects (Gaspar et al., 2009). Nonetheless, metabolic nature of the disease (Gaspar et al., 2009; Sauer et al., several non-immune abnormalities have been described in ADA 2009). While superior survival was seen in patients who received deficiency, indicating that this disease should be considered a unconditioned transplants in comparison to myeloablative proce- systemic (Aiuti et al., 2003; Hirschhorn and dures (81 and 54%), non-engraftment was a major problem after Candotti, 2006). ADA is ubiquitously expressed in all cell types; unconditioned haploidentical transplants (Hassan et al., 2012). when absent, the systemic metabolic toxicity is frequently asso- Long-term immune recovery showed that regardless of trans- ciated with organ damage (Sauer and Aiuti, 2009). These include plant type, overall T-cell numbers were similar although a faster hepatic and renal disease (Bollinger et al.,1996),skeletal alterations rate of T-cell recovery was observed following matched sibling (Sauer et al., 2009), neurological abnormalities (Honig et al., 2007; or matched unrelated BMT. Humoral immunity and donor B Titman et al., 2008), and behavioral impairments (Rogers et al., cell engraftment was achieved in nearly all evaluable surviving 2001). Because complications from infections usually predomi- patients and most patients were able to discontinue immunoglob- nate in the clinical presentation of infants with ADA deficiency, ulin replacement, suggesting that immune recovery is relatively the full spectrum of non-immunologic manifestations and their complete (Hassan et al., 2012). According to the available data, natural course may be obscured (Honig et al., 2007). It is impor- the immunological and metabolic recovery after transplant is well tant to note, that several abnormalities have been described in few maintained even after 10 years or longer in some patients (Gaspar patients only, and might reflect effects of infectious agents rather et al., 2009). than primary defects due to ADA deficiency: i.e., renal and adrenal Nevertheless delayed or suboptimal immune reconstitution as abnormalities, phyloric stenosis, and hepatic disease (Hirschhorn a result of poor early engraftment or gradual decline in immune and Candotti, 2006). functions is observed in a significant fraction of surviving patients (Gaspar et al., 2009). Complications such as graft-versus-host dis- THERAPIES FOR ADA DEFICIENCY ease, autoimmune and inflammatory manifestations, persistent Bone marrow transplantation with allogeneic HSC has long been infections, and disease-related issues have been described (Honig considered the mainstay of ADA-SCID treatment. However, unlike et al., 2007; Titman et al., 2008; Mazzolari et al., 2009). other SCID forms, two other treatment options are available for In summary, the results obtained with transplantation from ADA-SCID: enzyme replacement therapy with pegylated bovine HLA-identical siblings or family donors indicate superior ADA (PEG-ADA) and autologous HSC-GT (Hershfield et al.,1987; donor/host compatibility and outcome both in terms of survival Aiuti et al., 2009). The availability of different treatment modal- and sustained immune recovery. Whereas the current evidence ities presents an opportunity for improved patient care but also suggests that haploidentical donor transplants (performed with or difficulties in deciding upon the specific choice of treatment for without conditioning) have a poor chance of success and are there- individual patients (Figure 2). Making the correct choice is fur- fore only undertaken if no other treatment options are available ther complicated by the fact that ADA deficiency is not purely an (Gaspar et al., 2009). immune defect, and that the systemic manifestations, which can be of major clinical consequence, must also be managed (Gaspar ENZYME REPLACEMENT THERAPY WITH PEG-ADA et al., 2009). Enzyme replacement therapy with PEG-ADA was developed as lifesaving, not curative treatment for patients lacking an HLA- HEMATOPOIETIC STEM CELL TRANSPLANTATION compatible donor. Attachment of PEG through lysine residues Hematopoietic stem cell-transplantation (BMT) from allogeneic confers several therapeutically beneficial properties to ADA (Abu- human leukocyte antigen (HLA)-compatible sibling donors chowski et al., 1977; Davis et al., 1981). This chemical modification resulting in long-term survival and effective immune reconsti- of the bovine enzyme reduces its immunogenicity and prevents its tution is the treatment of choice for patients with ADA-SCID and degradation by plasmatic proteases as well as the binding of neu- other severe variants of primary immunodeficiencies. Since less tralizing antibodies (Abuchowski et al., 1977; Davis et al., 1981). than 20% of ADA-SCID patients have access to HLA-matched Thereby the circulating life of the compound is prolonged from family donors, transplants are often performed from mismatched minutes to days as clearance from the circulation is inhibited family or matched unrelated donors (Antoine et al., 2003; Gaspar (Booth and Gaspar,2009). Cellular uptake of PEG-ADA is insignif- et al., 2009; Ferrua et al., 2010). A recent retrospective analysis icant and its distribution is limited to the plasma. Enzymatically on the specific outcome of transplants for ADA-SCID collected active ADA continuously circulates and eliminates accumulating data from several multicenter studies and analyzed the survival adenosine and 20-deoxyadenosine metabolites (Chan et al., 2005). of 106 patients who received a total of 119 transplants (Hassan The principle of exogenous PEG-ADA administration is based et al., 2012). BMT from matched sibling and family donors had on the direct conversion of accumulating ADA substrates in the a significantly better overall survival (86 and 81%) in compari- plasma and the indirect reduction of intracellular toxic metabolites son to BMT from matched unrelated (66%) and haploidentical by diffusion.

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FIGURE 2 | Current therapeutic options in ADA-SCID and reported of human gene therapy. It is based on the reinfusion of autologous HSC autoimmune manifestations after treatment. Immune reconstitution transduced with a retroviral vector containing the ADA cDNA. Variable in ADA deficiency can be achieved by bone marrow transplantation, degrees of immune reconstitution can be achieved by these treatments, enzyme replacement, or gene therapy, nonetheless recovery of immune but onset of autoimmunity is of concern in post-treatment ADA-SCID functions may vary depending on the applied treatment and patient’s patients. Reported autoimmune manifestations include: autoimmune characteristics. Treatment of choice remains bone marrow hypothyroidism, diabetes mellitus, thrombocytopenia, hemolytic anemia, transplantation from an HLA-identical sibling donor, while transplants and development of anti-ADA antibodies. HLA, human leukocyte antigen; from alternative donors are associated with high morbidity and mortality. BMT, bone marrow transplantation; MUD, matched unrelated donor; Enzyme replacement therapy using pegylated bovine ADA is a PEG-ADA, pegylated bovine ADA; HSC, hematopoietic stem cell; PSC, non-curative treatment requiring weekly intramuscular injections with pluripotent stem cell; CLP,committed lymphocyte precursor; NK, natural PEG-ADA. ADA-SCID has been the pioneer disease for the development killer cell.

To date more than 150 patients worldwide have received this lymphocyte counts of all PEG-ADA treated patients were below the treatment (Booth and Gaspar, 2009; Gaspar et al., 2009). PEG- normal range at all times. A gradual decline of mitogenic prolifer- ADA is usually administered weekly or bi-weekly by intramuscular ative responses occurred after a few years of treatment and normal injections throughout life. In general, PEG-ADA treatment seems antigenic responses occurred less than expected (Kohn, 2008; to be well tolerated, with clinical benefits appreciable after the first Serana et al., 2010). No toxic or reactions have month of therapy (Figure 3). Studies have shown that upon the been reported with PEG-ADA administration. However, several initiation of PEG-ADA therapy, the absolute numbers of circulat- other side effects have been reported including manifestations of ing T- and B-lymphocytes and NK-cells increase and protective immune dysregulations including autoimmunity (type I diabetes, immune function develops (Weinberg et al., 1993). Although only hypothyroidism, immune thrombocytopenia, hemolytic anemia) limited information is available,some analysis indicated that about and allergic manifestations (Notarangelo et al., 1992; Ozsahin half of PEG-ADA treated patients discontinued IVIg (Gaspar et al., 1997). An additional concern with PEG-ADA beyond about et al., 2009), whereas long-term follow-up suggests that immune 8–10 years is the emergence of serious complications, including recovery is often incomplete (Booth and Gaspar, 2009). Two lymphoid and hepatic malignancies, and progression of chronic retrospective studies showed that despite initial improvements, the pulmonary insufficiency (Gaspar et al., 2009).

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FIGURE 3 | Immune reconstitution and development of followed by a gradual increase in T-cell numbers and reconstitution of autoimmunity after PEG-ADA treatment. Enzyme replacement therapy immune cell functions. However, the long-term consequences of with pegylated bovine ADA is a lifesaving but non-curative treatment for PEG-ADA treatment are unknown. Immune recovery in B and T- cells is ADA-SCID patients. It provides metabolic detoxification and protective below normal levels. Major concerns are the susceptibility to immune function with patients remaining clinically well, but immune opportunistic infections and the development of autoimmunity due to reconstitution is often suboptimal and may not be long-lived. Shortly after lymphopenia with gradual decline of immune functions and perturbation initiation of PEG-ADA treatment, patients show recovery of B-cell counts, of T- and B-cell tolerance.

The main side effect associated with the use of PEG-ADA Since 2000, 37 patients have been treated in Italy, UK, and USA, is the development of anti-ADA antibody. The development of achieving substantial clinical benefit in the majority of them. All specific IgG antibody to bovine peptide of PEG-ADA patients received reduced intensity conditioning and PEG-ADA has been reported by several groups and often coincides with an was discontinued to exploit the selective growth advantage for improvement in humoral immunity (Chaffee et al., 1992; Lainka gene corrected over defective cells. At present, all patients are et al., 2005; Booth et al., 2007). In about 10% of treated patients, alive and in 26 patients PEG-ADA is no longer required (Aiuti inhibitory antibodies lead to the enhanced clearance of PEG-ADA et al., 2009; Gaspar et al., 2011; Montiel-Equihua et al., 2012). with subsequent decline in metabolic parameters and immune Gene therapy resulted in sustained engraftment of transduced function (Chaffee et al.,1992; Hershfield,1995; Lainka et al.,2005). cells, increased lymphocyte counts, improvement of cellular and humoral responses, and effective metabolic detoxification (Aiuti GENE THERAPY et al., 2009; Gaspar et al., 2011). Gene corrected cells were detected Hematopoietic stem cell gene therapy is a promising therapeutic in myeloid and lymphoid subsets, the latter being more repre- option for genetic disorders of the immune system (Bordignon sented due to their survival advantage (Aiuti et al., 2009; Gaspar and Roncarolo, 2002; Fischer et al., 2005). ADA-deficient SCID et al., 2011). In the HSR-TIGET study, all children maintained has been under intensive preclinical and clinical investigation and stable engraftment of vector ADA-transduced CD34+ cells with nowadays represents a paradigmatic model of gene therapy for sustained systemic detoxification (Aiuti et al., 2009). At present, 15 inherited disorders (Aiuti et al., 2003, 2007). The strong rationale of the 18 treated children do not require enzyme replacement ther- for somatic gene therapy and the need for alternative treatments apy, with the longest follow-up at 11 years after treatment (Aiuti led to the design of clinical trials based on retroviral-mediated gene et al., 2009; Ferrua et al., 2010). These findings demonstrated the transfer of the normal ADA gene into autologous HSCs (Aiuti, clinical efficacy of ADA gene transfer in restoring normal immune 2004). Replication-deficient, recombinant retroviruses derived function and metabolic functions of ADA-SCID patients. from the backbone of Moloney murine leukemia virus (MLV) Unlike trials with gammaretroviral vectors in other diseases like were selected for these trials because of the available long-term X-linked SCID (Hacein-Bey-Abina et al., 2008; Howe et al., 2008), experience and their ability to efficiently insert the therapeutic chronic granulomatous disease (Ott et al., 2006) and Wiskott– gene into the genome of dividing hematopoietic cells. Aldrich Syndrome (Trobridge, 2011), the cumulative experience

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of these studies for ADA-SCID (Aiuti et al., 2009; Ferrua et al., showed immune dysregulation while on PEG-ADA, developed 2010; Montiel-Equihua et al., 2012) did not reveal leukemic or signs of autoimmunity, such as hemolytic anemia, thrombo- oncogenic events, indicating that ADA-SCID gene therapy has a cytopenia, autoimmune hepatitis, and autoimmune thyroiditis favorable risk/benefit profile. Unique risk factors may have con- (Aiuti et al., 2009 and unpublished observation). tributed to the differential outcome of the other trials, such as vector constructs or promoters, inappropriate expression of trans- ADA-DEFICIENT MOUSE MODEL genes involved in cell signaling (Kohn, 2008), cooperation between The availability of a genetic model for ADA deficiency transgene and cellular oncogenes (Dave et al., 2009), or the disease allowed a wide range of biochemical and immunological exper- background itself (Shou et al., 2006). iments that are not feasible in humans. The first attempts to generate ADA-deficient mice lead to their perinatal death due to AUTOIMMUNITY IN ADA-SCID severe liver damage (Blackburn et al., 1998). Subsequent studies Immunodeficiency and autoimmune phenomena may occur con- suggested that ADA expression in trophoblast cells of the comitantly in the same individual (Etzioni,2003). Immune dysreg- is critical for fetal development in the mouse. Thus, ADA-deficient ulation, which often manifests as multiple forms of autoimmunity, mice were successfully generated by specifically targeting expres- can affect both the adaptive and , indicating sion of an ADA minigene to the trophoblast lineage of ADA+/− that all these immune components are required for the appropri- mice and by inter-crossing these mice. This gave rise to litters that ate development of tolerance in humans (Cunningham-Rundles, contained mice expressing the ADA minigene in their placenta 2011). Since varying degrees of immune reconstitution can be that were also homozygous for the ADA null allele (ADA−/−; achieved by the available treatment options for ADA-SCID, break- Blackburn et al., 1998). down of tolerance and development of autoimmunity can repre- sent a major concern. Autoimmune dysregulation are frequently UNTREATED ADA−/− MICE observed in patients with milder forms of the disease or late-onset The ADA−/− mouse reproduces not only the biochemical but patients. They may manifest as autoimmune hypothyroidism, dia- also the immunological abnormalities of the human disease phe- betes mellitus, hemolytic anemia, and immune thrombocytopenia notype. They manifest both combined immunodeficiency as well (Notarangelo et al., 1992; Ozsahin et al., 1997; Figure 2). as metabolic abnormalities and are therefore commonly used to Similar complications, such as autoimmune hemolytic anemia assess the effect of ADA deficiency not only on the lymphoid and autoimmune thyroiditis, have also been reported in at least organs and peripheral blood, but also its systemic organ toxic- nine patients after long-term PEG-ADA treatment (Ratech et al., ity. ADA−/− deficient mice die at approximately 3 weeks of age 1989; Notarangelo et al., 1992; Ozsahin et al., 1997; Gaspar et al., from severe respiratory distress (Blackburn et al., 1998). 2009; Serana et al., 2010). Refractory hemolytic anemia was fatal in Initial examinations of the thymus and revealed a sub- three patients (Gaspar et al., 2009). Two additional studies assessed stantial decrease in organ size. The cellular proportion from the defects in the lymphoid compartments of ADA-SCID patients fol- thymus of ADA−/− mice showed a significant increase in the per- lowing PEG-ADA. Different degrees of abnormalities in the B-cell centage double-negative immature , accompanied by compartment and inability to respond to , despite the a decrease in the percentage of CD4+ or CD8+ single-positive presence of normal serum-Ig or hypogammaglobulinemia were thymocytes. T-cell apoptosis was abundant in the ADA-deficient reported (Malacarne et al., 2005). Moreover, a retrospective lon- thymi (Blackburn et al., 1998). ADA−/− splenic B lymphocytes gitudinal analysis in ADA-SCID patients treated with PEG-ADA showed defects in proliferation and activation with high propen- showed that decreased levels of newly produced B cells underlie sity to undergo B cell receptor-mediated apoptosis. As a result, the progressive and significant decrease in circulating B cells in profound loss of germinal center architecture was noted, which these patients (Serana et al., 2010). Since long-term PEG-ADA may be responsible for impaired B cell development (Aldrich et al., treatment is associated with abnormalities in B cell subsets, but 2003). Lymphopenia was also seen in the peripheral circulation, often also with a decrease in T-cell functions (Malacarne et al., confirming that this model of ADA deficiency exhibits a SCID 2005), a limited B or T-cell repertoire combined with alterations phenotype. in peripheral tolerance could further favor breakdown of tolerance At death, the severe immune deficiency and organ alterations (Figure 3). are the most prominent features, whereas no apparent autoim- No specific reports on immune dysregulation or autoimmu- mune manifestations can be observed. The almost complete nity in BMT-treated ADA-SCID patients are available in liter- absence of effector T- and B-cell populations in these mice and ature (Serana et al., 2010). Nevertheless, autoimmune manifes- the high levels of anti-inflammatory adenosine might prevent their tations have been reported in larger single-center studies on development in the first 3 weeks of life. Reconstitution of effector BMT-treated patients with various kinds of immunodeficiencies, T- and B-cells as well as metabolic detoxification after treatment including ADA deficiency (Mazzolari et al., 2009; Neven et al., might therefore be requirements for the onset of autoimmunity 2009). The major immune dysregulations observed in both studies (Sauer et al., 2012a). included thyroid autoimmunity, autoimmune hemolytic anemia, and glomerulonephritis (Mazzolari et al., 2009; Neven et al., 2009). MODEL FOR AUTOIMMUNITY IN ADA-DEFICIENT MICE Most recently autoimmune manifestations have also been Similarly to ADA-SCID patients, ADA−/− mice can be treated described in patients treated with HSC-GT (Aiuti et al., 2009). with PEG-ADA, HSC-GT with transduced BM ADA−/− cells, or Four ADA-SCID patients, including one patient that already BMT with wild type donor cells (Mortellaro et al., 2006; Sauer

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et al., 2009). A dose of 1000 U/kg/week of PEG-ADA starting from and rheumatoid arthritis (Sebastiani et al., 2006; Saccucci et al., postnatal day 10 provides rescue and metabolic detoxification in 2009). ADA−/− mice (Blackburn et al., 2000). HSC-GT is performed using a SIN-lentiviral vector driving ADA expression from the ROLE OF ADA METABOLITES IN IMMUNE CELL phosphoglycerate kinase (PGK) promoter (Mortellaro et al.,2006), DEVELOPMENT AND FUNCTION instead of the gammaretroviral vector used in the clinical trial. A Although autoimmunity is frequently observed in certain immun- long-term comparative approach between these three treatment odeficiencies, there is accumulating evidence that ADA deficiency options revealed important new information on their efficacy and predisposes to this phenomenon not only through general mech- established a model for autoimmunity in the context of long-term anisms of immune dysregulation but also through specific alter- PEG-ADA treatment (Sauer et al., 2012a). ations caused by the accumulating ADA metabolites. Main fea- The long-term survival of PEG-ADA, HSC-GT, and BMT- ture of ADA deficiency is the gradual accumulation of adenosine treated mice was comparable between the three groups (60–70% and 20-deoxyadenosine nucleosides. In the absence of ADA, these with respect to wildtype). This outcome was the result of an early nucleosides are metabolized differently into AXP or dAXP,respec- mortality in the BMT and HSC-GT treated groups, while PEG- tively, and exert distinct biochemical action (AXP: AMP, ADP, ADA treated mice had a less stable long-term survival. As expected or ATP; dAXP: dAMP, dADP, or dATP). Several pathophysio- from the fact that PEG-ADA remains in circulation without enter- logical mechanisms have been proposed to describe the role of ing in cells, ADA activity in PEG-ADA treated mice was exclusively ADA substrates in cytotoxicity as well as their immunomodu- detectable in the plasma. Reconstitution of enzymatic activity in latory properties in patients and in the ADA-deficient mouse RBC, BM, spleen, and thymus from BMT-treated mice was com- model (Hirschhorn and Candotti, 2006). The major effects of parable to wildtype, while only slightly lower in HSC-GT treated adenosine, 20-deoxyadenosine and their nucleotide byproducts are mice (Sauer et al., 2012a). summarized in Table 1. Strikingly, ADA−/− mice treated with PEG-ADA developed multiple autoantibodies and hypothyroidism in contrast to mice CYTOTOXICITY OF 20-DEOXYADENOSINE AND dATP treated with HSC-GT or BMT.Proliferation of various lymphocyte Based upon in vivo and in vitro findings, several mechanisms are subpopulations, including B cells and highly abnormal antibody believed to account for the block of lymphocyte development in production affecting all types of antibody subclasses was observed ADA-SCID (Hirschhorn and Candotti, 2006). The biochemical in PEG-ADA treated mice. Moreover, autoantibodies that reacted hallmarks of ADA deficiency consist of the general belief, that to ADA, platelets, the thyroid, and the gastrointestinal tract were 20-deoxyadenosine is the primary cause of lymphotoxicity in ADA- detected in the sera from PEG-ADA treated mice. Focal atresia with SCID,which exerts its effects at the nucleoside level or after conver- non-secreting follicles, an increase in apoptotic cells in affected sion to dATP. Although 20-deoxyadenosine is a weak substrate for tissue areas and significantly elevated levels of thyroid-stimulating adenosine kinase and deoxycytidine kinase, in the absence of ADA hormone (TSH) represent signs of autoimmune hypothyroidism. these enzymes can phosphorylate 20-deoxyadenosine. In turn, the The role of autoantibodies against the stomach and intestine devel- resulting dATP pool expansion may interfere with a number of oping in PEG-ADA treated mice, without causing gross patholog- critical metabolic pathways. ical alterations, remained unclear. However, it was hypothesized These ADA substrate accumulations inhibit methyl-transfer that the occurrence of antibody responses to GI tissues not only reactions by suicide inactivation of S-adenosylhomocysteine interferes with nutrient uptake, but also reflect alterations in (SAH) hydrolase (Hershfield et al., 1979). dATP is known to be gastrointestinal immunity (Sauer et al., 2012a). The established a feedback inhibitor of ribonucleotide reductase. Its inhibition mouse model for autoimmunity after PEG-ADA treatment repre- causes an imbalance of deoxynucleotides (dNTP), leading to an sents a valuable model for future studies on the in vivo effects of impairment of DNA synthesis, which is critical for the expan- PEG-ADA on immune cell function and inflammatory responses. sion of lymphocytes in response to antigenic challenge (Benveniste Interestingly, PEG-ADA treated mice produced antibodies to et al., 1995). ADA, platelets, the thyroid, and gastrointestinal tract, but not other organs such as the pancreas or endocrine glands. The strong ROLE OF ADENOSINE AS ANTI-INFLAMMATORY MEDIATOR overlap of autoimmune manifestations observed in this model By binding to G-coupled adenosine receptors present on the of autoimmunity in ADA−/− mice with those reported in ADA- surface of target cells, adenosine acts as an extracellular signal deficient patients suggests that a component of autoimmune sus- transducer to exert suppressive functions (Sitkovsky et al., 2004). ceptibility may map to the target tissue. In both humans and in Physiologically, adenosine-mediated triggering of these receptors mouse models, single genetic loci have been linked with suscep- can promote a fine-tuning of the inflammatory responses. In the tibility to multiple autoimmune diseases. The genes underlying context of defective ADA metabolizing enzyme, where the extra- such loci, including AIRE, FoxP3, CTLA-4, and PTPN22, are likely cellular levels of adenosine are increased, this regulation may be to confer a general predisposition to the failure of immune tol- exaggerated and cause immune dysfunction. This process is mostly erance and development of an auto-aggressive immune response regulated by the aberrant engagement of adenosine 2a receptor (Hill et al., 2007). However, other loci are clearly disease specific, (Adora2a)-mediated signaling. and presumably modify a generalized predisposition to confer Functional studies on T cells from ADA-deficient mice and organ/disease specificity. Interestingly, recent studies have impli- patients showed an increased susceptibility to apoptosis as well as cated ADA polymorphisms in the development of type1 diabetes altered intra- and extracellular signaling leading to impaired T-cell

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Table 1 | Effects of ADA metabolites on lymphocyte development and function.

Mouse/ ADA metabolite Cell type Mode of action Reference human

20-Deoxyadenosine Lymphocytes Human Inhibition of SAHH activity results in accumulation Hershfield et al. (1979) and Benveniste et al. of S-adenosylhomocysteine and inhibition of (1995) transmethylation reactions Fibroblasts Mouse SAHH acts as a physiological modulator of Ratter et al. (1996) Fas-mediated cell death T cells Human Inhibition of T-cell activation by aberrant Adora2a Cassani et al. (2008) signaling and PKA hyperactivation

Lympocytes dATP Human Intracellular ATP depletion Siaw et al. (1980), Simmonds et al. (1982), Koller and RBC et al. (1984), and Simmonds et al. (1984) T cells Human Inhibition of ribonucleotide reductase causes an Waddell and Ullman (1983), Benveniste et al. imbalance of dNTPs and an impairment of DNA (1995) synthesis T cells Human Accumulation of DNA single strand breaks Cohen and Thompson (1986) and Gangi-Peterson et al. (1999) T cells Mouse Inhibition of development past the Van de Wiele et al. (2002) and Van de Wiele CD4−/CD8− double-negative stage et al. (2006) B cells Human Nucleotide pool imbalance affects TdT activity Gangi-Peterson et al. (1999) during V(D)J recombination in the bone marrow

Adenosine T cells Human Compromised TCR/CD28-driven proliferation and Cassani et al. (2008) cytokine production, defective activation of NF-κB transcriptional events Resting T cells Human Upregulation of CD152, CTLA-4, normally involved Vendetti et al. (2002) in the termination of immune responses T cells Mouse Decreased TCR-triggered activation and Apasov and Sitkovsky (1999), Apasov et al. upregulation of activation markers (2001) Activated T cells Mouse Inhibition of IL-2,TNFα, and INFγ secretion Erdmann et al. (2005) and Lappas et al. (2005) B cells Human Adora2a signaling interferes with BCR- and Sauer et al. (2012b) TLR-function, inhibition of B-cell activation after stimulation B cells Mouse Profound loss of GC, susceptibility to apoptosis, Aldrich et al. (2003) defects in B-cell proliferation and activation, block in Ag-dependent B-cell maturation B cells Mouse Increase of intracellular cAMP suppresses the Minguet et al. (2005) activation of NF-κB after BCR and TLR-stimulation Mouse/ Tregs Adora signaling causes alterations in the Sauer et al. (2012a) human CD39/CD73 adenosinergic machinery, upregulation of FoxP3 Adaptive Tregs Mouse Adora2a signaling inhibits the generation of Zarek et al. (2008) adaptive effector T cells and promotes the induction of adaptive Tregs, upregulation of FoxP3

ATP T cells Human Purinergic stimulation through P2X receptors Yip et al. (2009) prolongs TCR-initiated activation and IL-2 secretion T cells Mouse Antagonism of P2X blunts TCR-mediated activation Schenk et al. (2008) and results in unresponsiveness to subsequent stimulation Tregs Mouse Activation of P2X7 inhibits the suppressive Schenk et al. (2011) potential and stability of Tregs

Adora2a, adenosine 2a receptor; ATP, adenosine-50-triphosphate; BCR, B cell receptor; cAMP, 30,50-cyclic adenosine monophosphate; dATP, 20-deoxyadenosine 50-triphosphate; dNTPs, deoxynucleotides; GC, germinal center; INFγ, interferon gamma; NF-κB, nuclear factor kappa B; P2X/P2X7,purinergic receptors; PKA, cAMP- dependent protein kinase A; RBC, red blood cells; SAHH, S-adenosylhomocysteine hydrolase; TCR, T-cell receptor; TdT, terminal deoxynucleotidyl transferase; TNFα, tumor necrosis factor alpha; Tregs, naturally occurring regulatory T cells.

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FIGURE 4 | Immunosuppressive action of adenosine in lymphoid cells. that the inhibitory properties of PKA are mediated via activation of Csk or Upon TCR-antigen-binding and T-cell activation, the translocation of activated inhibition of Raf1. Consistently it has been shown that the severely transcription factors and the expression of anti-apoptotic factors supports compromised effector functions in ADA-deficient T cells associates with an their survival, proliferation, and differentiation. The adenosine 2a receptor intrinsically reduced ERK1/2 signaling, a decreased phosphorylation of IκB (Adora2a) present on T-lymphocytes interacts with coupled G-proteins to and an altered nuclear recruitment of NF-κB. This suggests that the stimulate cAMP formation. The effects of cAMP in T cells are almost entirely accumulating adenosine levels in an ADA-deficient environment can directly mediated by the cAMP-dependent protein kinase A (PKA). It has been shown antagonize biochemical events arising from TCR engagement. function (Apasov and Sitkovsky, 1999; Apasov et al., 2001; Cas- Less information is available about the effects exerted by adeno- sani et al., 2008). As summarized in Figure 4, the TCR-dependent sine on B-cell function. Similarly to the alterations in T cells activation defect in ADA deficiency is augmented by the immuno- described above, adenosine receptor ligation in B cells inhibits suppression through extracellular adenosine receptor triggering. downstream responses to antigen receptor engagement like BCR- Extracellular adenosine induces increased levels of cAMP in T- induced IκB phosphorylation and the NF-κB pathway after BCR or lymphocytes, which inhibits both proximal signaling events after TLR4 stimulation (Minguet et al., 2005). Adenosine may thereby TCR triggering as well as other downstream effector functions drive BCR-stimulated B cells toward an anergic rather than an (Huang et al., 1997; Lappas et al., 2005; Ohta et al., 2009). In accor- immunogenic response. Recent findings showing defects in BCR dance with previous data obtained in thymocytes from ADA−/− and TLR signaling as well as in tolerance checkpoint control in mice (Apasov et al., 2001), IκBα phosphorylation after TCR trig- human B cells are discussed and illustrated separately in section gering was low or undetectable in ADA-deficient cells (Cassani “Defects in B-cell tolerance in ADA-SCID.” et al., 2008). Reduced levels of IκBα phosphorylation and degrada- Overall, these evidences strongly suggest that rather than tion leads to low levels of NF-κB translocation and transcription of controlling a single pathway downstream of the TCR or BCR, target genes in the nucleus, thereby contributing to the functional the immune defects in ADA-deficient lymphocytes may involve impairments of ADA-deficient T cells. multiple pathways converging toward the defective induction

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of lymphocyte activation. They also illustrate how extracellu- which encounter antigen in the absence of the co-stimulatory lar adenosine levels can interfere with the downstream signal- signals that accompany inflammation (Meffre and Wardemann, ing transduction upon activation, thereby exerting its immuno- 2008). suppressive activity on the transcriptional machinery. Because Numerous mechanisms have been proposed to explain the of cell-specific expression and regulation, aberrant adenosine break of tolerance and development of autoimmune manifes- receptor-mediated signaling might also contribute to the occur- tations, such as defective negative selection of autoreactive T- rence of autoimmune manifestations observed in someADA-SCID lymphocytes in the thymus, alterations in the number and/or patients (Kohn, 1996; Ozsahin et al., 1997). function of regulatory T cells, defects of the central and peripheral B-cell tolerance checkpoints, impaired apoptosis of autoreactive ROLE OF ATP AND OTHER PURINERGIC RECEPTORS lymphocytes, break of tolerance due to increased or decreased Stimulation of almost all mammalian cell types leads to the release clearance of apoptotic cells and pathogens,or increased homeosta- of cellular ATP and autocrine feedback through a diverse array of tic lymphoid proliferation and cytokine secretion associated with purinergic receptors (Junger, 2011). ATP binds to two classes of lymphopenia (Carneiro-Sampaio and Coutinho,2007;Westerberg purinergic P2 receptors in the plasma membrane of eukaryotic et al., 2008; Notarangelo, 2009; Meffre, 2012). cells: P2X receptors, which are ligand-gated ion channels, and het- erotrimeric G protein-coupled P2Y receptors (Schenk et al., 2011). T-CELL TOLERANCE Depending on the types of purinergic receptors that are involved, Central T-cell tolerance mechanisms are based on the elimination autocrine signaling can promote or inhibit immune cell activation or negative selection of the majority of T cells recognizing self and fine-tune adaptive immune responses (Junger, 2011; Schenk with high affinity for negative selection in the thymus. Nonethe- et al., 2011). less thymic selection is not a tight process and T cells expressing In addition to the autocrine feedback mechanisms that reg- low-avidity TCR on their cell surface are frequently released in the ulate the function of healthy immune cells, purinergic receptors periphery, where they are potentially dangerous to the host as they allow immune cells to recognize ATP that is released from dam- can be effectively recruited into an autoimmune response (Parish aged or stressed host cells. Thus, the purinergic signaling systems and Heath, 2008). of immune cells serve an important function in the recognition of A major cause of tolerance breakdown is associated with lym- danger signals. ATP that is released by stressed cells guides phago- phopenia (Daikeler and Tyndall, 2007). This typical state of pri- cytes to inflammatory sites and promotes clearance of damaged mary immunodeficiencies may contribute to the induction of and apoptotic cells (Elliott et al., 2009; Junger, 2011). spontaneous homeostatic proliferation of residual T cells allowing To date, little information is available on alterations in the peripheral expansion of autoreactive cells with a skewed reper- ATP-induced regulation of immune cells in ADA deficiency. It toire. Particularly, after conditioning or transplantation these cells is reported that dATP accumulation in the absence of ADA leads may persist, since insufficient thymic reconstitution may affect the to a cellular depletion of ATP (Siaw et al., 1980; Simmonds et al., control of self-reactivity due to defective negative selection in the 1982, 1984; Koller et al., 1984). The pool of extracellular ATP on thymus and/or reduced regulatory T-cell development and func- the other hand might well be augmented in ADA-deficient lym- tion (Hauri-Hohl et al., 2007). In the case of ADA deficiency, it has phoid organs, due to the increased percentage of cells undergoing been hypothesized that the structure and functions of the thymic apoptosis. It can therefore be hypothesized that alterations in ATP microenvironment might be altered, either directly, by toxicity concentrations in ADA deficiency also influence T-cell responses of purine metabolites, or indirectly, by failure of T cells arrested on the level of TCR induced activation and in response to stimuli in their development to deliver supportive signals to the thymic from an inflammatory microenvironment. stroma (Apasov et al., 2001). Peripheral tolerance depends on the balance between immune BREAK OF TOLERANCE AND CONTRIBUTION OF responses to invading pathogens and immune tolerance to self- LYMPHOCYTES TO AUTOIMMUNITY IN ADA DEFICIENCY antigens. In the context of tissue damage and frequently occurring Adaptive immunity requires sophisticated regulatory mechanisms infections in primary immunodeficient patients, apoptotic cells to ensure protection to a variety of pathogenic microbes while represent a major source of autoantigen. Since apoptosis plays a maintaining immune self-tolerance and preventing autoimmunity major role in the deletion of autoreactive lymphocytes and the (Sakaguchi et al., 2008). The main mechanisms for the induction removal of virus-infected cells, defects in cell death have been and maintenance of a self-tolerant repertoire, which is diverse in implicated in the development of autoimmune diseases and per- antigen recognition, are central and peripheral tolerance. Central sistent viral infection (Utz et al., 2000). The release of self-antigen tolerance is the mechanism able to eliminate newly developing into the intracellular space and their presentation mediated by T cells and B cells that have high affinity to self (Mathis and dendritic cells or other antigen-presenting cells may prime naive Benoist, 2004). is distinct from peripheral tol- autoreactive T cells, which were not eliminated by depletion or erance in that it occurs while cells are still present in the primary anergy (Waldner et al., 2004). Several mechanisms exist, includ- lymphoid organs, whereas emigrant cells are controlled through ing a spectrum of CD4+ regulatory T cells (Tregs), to suppress peripheral tolerance mechanisms, after they reach the periphery self-reactive T cells that escape thymic clonal deletion and atten- (Wardemann and Nussenzweig, 2007; Klein et al., 2009). These uate anti-pathogen effector mechanisms from inducing immune include suppression of autoreactive cells by regulatory T cells and pathology (Piccirillo and Thornton, 2004). There is ample evi- the generation of hyporesponsiveness (anergy) in lymphocytes, dence that Tregs actively mediate suppression to control immune

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responses to self- and non-self-antigens and the onset of autoim- might stimulate autoreactive B cells that are normally quiescent, munity (Bach,2003; Sakaguchi,2005). Lessons from other primary through inherent adjuvant activity and trigger the development of immunodeficiencies have provided unequivocal evidence for the systemic autoimmune disease (Marshak-Rothstein, 2006). In ADA essential role of Tregs in suppressing autoreactive T cells in the deficiency, the metabolic basis underlying immune cell deficiency periphery (Westerberg et al., 2008). Rising of autoimmunity may is the cytotoxic effect impact of the ADA substrates deoxyadeno- not only be linked to a reduction in Treg numbers but also to atten- sine and dATP,leading to apoptosis of lymphocytes. It is therefore uation of their suppressive activity (Sakaguchi et al., 2008). While conceivable that developing B lymphocytes in affected lymphoid this is principally mediated by cell–cell contact, recent findings organs encounter massive amounts of nucleic acid. Nucleic acid- revealed additional mechanisms of Treg-mediated suppression, sensing TLRs might therefore represent Achilles’heel in susceptible including secretion of immunosuppressive cytokines, functional ADA-deficient patients by which relative tolerance for nucleic acid- modification or killing of APC, and metabolic disruption (Vig- containing antigens is breached and autoimmunity occurs (Kono nali et al., 2008). Moreover, extracellular adenosine produced by et al., 2009). Tregs, has been identified as one of the mechanisms mediating their suppressive activity (Sitkovsky et al., 2008; Mandapathil et al., NEW INSIGHTS INTO IMMUNE CELL DYSFUNCTION AND 2010). Treg cells possess a unique biochemical signature amongst ONSET OF AUTOIMMUNITY IN ADA DEFICIENCY T cells in that they generate and sustain high adenosine con- Recent in-depth studies have revealed specific defects in ADA centrations. Since Tregs primarily mediate peripheral control of deficiency that may contribute to the onset of autoimmunity in autoreactive T cells, it is conceivable that this compartment might these patients. Herein we discuss alterations in the adenosiner- be specifically affected inADA-SCID (see also Defective Regulatory gic machinery of ADA-deficient regulatory T cells and in B-cell T Cell Function in ADA Deficiency). Consequently the autoim- tolerance in the absence of functional ADA. mune manifestations associated with ADA deficiency might be the result of an altered purine metabolism interfering with normal regulatory T-cell function (Sauer et al., 2012a). DEFECTIVE REGULATORY T-CELL FUNCTION IN ADA DEFICIENCY Although autoimmune manifestations are frequent findings B-CELL TOLERANCE in ADA-deficient patients with milder forms or in patients A variety of mechanisms ranging from clonal deletion to func- under PEG-ADA, mechanisms causing the loss of peripheral tional inactivation by anergy of autoreactive B cells serve to shape tolerance and onset of autoimmunity have remained elusive. the peripheral B-cell repertoire. Nevertheless, dysregulation of B CD4+CD25+FoxP3+ Tregs actively suppress pathological and cell development and autoantibody production is a characteris- physiological immune responses in order to maintain periph- tic of most autoimmune diseases including rheumatoid arthri- eral immune self-tolerance and prevent autoimmunity (Sakaguchi tis, systemic , and type 1 diabetes, but also et al., 2008; Sitkovsky et al., 2008). Extracellular adenosine pro- immunodeficiencies such as CVID, Wiskott–Aldrich Syndrome, duced by Tregs has been described as one of the mechanisms and X-linked agammaglobulinemia (Park et al., 2005; Cuss et al., mediating their suppressive activity (Figure 5A). Concordant 2006; Westerberg et al., 2008). In ADA deficiency, some of the expression of the ectoenzymes CD39 and CD73 has been reported observed immune dysregulation were hypothesized to be associ- both for murine and human Tregs (Borsellino et al., 2007; Deaglio ated with a more restricted B-cell repertoire due to abnormalities et al., 2007; Mandapathil et al., 2010). The CD39 ectoenzyme pro- in central B-cell generation or to a dysregulated expansion of these duces AMP from ATP or ADP, which is subsequently converted cells in the periphery. into extracellular adenosine by the CD73 ectoenzyme (Hasko et al., Autoantibodies appear in the serum many years before the 2008). Treg function requires the coordinated expression of the onset of clinical disease suggesting an early break in B-cell Adora2a on activated T effector cells to enable adenosine-mediated tolerance (Wardemann et al., 2003). Some of B-cell mediated immunosuppression (Sitkovsky et al., 2008). Moreover, Tregs have autoimmune diseases, such as (MG), idiopathic been shown to express low levels of ADA, whereas T effector cells autoimmune thrombocytopenic purpura (AITP), and Graves’ dis- are enriched in ADA but express low levels of CD39 and CD73 ease are characterized by auto-Abs production that destroy target (Mandapathil et al., 2010; Sauer et al., 2012a). This molecular pro- tissues (Barsalou et al., 2011; Cunningham-Rundles, 2011). A file of Tregs (CD39+CD73+ADAlow) has functional importance, remarkably high proportion of autoantibodies associated with sys- as it not only confers Tregs the capability to produce extracellular temic autoimmune diseases binds DNA, RNA, or macromolecular adenosine but also to sustain relatively high concentrations due to complexes that contain DNA or RNA. It has been hypothesized, low ADA expression (Mandapathil et al., 2010). that under certain circumstances these intracellular autoantigens Figure 5B summarizes recently described defects and func- become visible to the immune system when they accumulate dur- tional alterations of the adenosinergic pathway in Tregs from ADA- ing apoptosis. In fact the impaired clearance of apoptotic cell deficient mice and patients (Sauer et al., 2012a). ADA−/− Tregs debris and dsDNA by macrophages might induce TLR signal- showed significantly higher expression of CD39, while expressing ing and differentiation of autoreactive B cells (Gaipl et al., 2006). significantly less CD73. ADA−/− Tregs are sensitive to extra- Response to nucleic acid-containing immunecomplexes relies on cellular adenosine concentrations and the expression of CD73 the coengagement of endosomal members of the TLR family,TLR9 is regulated by this metabolite. With adenosine accumulating in and TLR7 (Marshak-Rothstein, 2006). Therefore, self-antigens ADA−/− mice, possibly to avoid a further increase of extracellu- that can effectively engage both the BCR and either TLR7 or TLR9 lar concentrations, CD73 is reduced and ADA−/− Tregs display a

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FIGURE 5 | Loss of regulatoryT-cell function in ADA-SCID. (A) By levels, possibly to avoid a further increase of extracellular concentrations, concomitant expression of CD39 and CD73, Tregs have the enzymatic CD73 is reduced and ADA−/− Tregs display a decreased suppressive machinery to generate and maintain high levels of extracellular adenosine. activity toward T effector cells. Nevertheless no apparent autoimmune Contrarily to T effector cells, Tregs express low levels of ADA and CD26. manifestations can be observed at onset, likely due to the severely reduced Extracellular ATP or ADP is converted by the ectonucleotidase CD39 into effector T- and B-cell populations. Moreover, the accumulating extracellular AMP,which is further converted into adenosine by the CD73 ectoenzyme. adenosine is likely to maintain an anti-inflammatory environment in these The produced adenosine binds to the Adora2A receptor expressed on mice. (C) After PEG-ADA treatment, the adenosinergic machinery of CD39 activated effector T cells, which are enriched in ADA and the surface-bound and CD73 are upregulated, indicating an increased requirement for ATP glycoprotein CD26. The coordinated expression of CD39 and CD73 on Tregs hydrolysis and enhanced adenosine production. Despite the initial rescue of and Adora2a on T effector cells enables adenosine-mediated suppressive activity by upregulation of CD73 for elevated adenosine immunosuppression. (B) In the absence of ADA, deficient Tregs express production, long-term PEG-ADA treatment interferes with Treg function by high levels of CD39, increasing their capacity for ATP hydrolysis, but augmenting adenosine turnover. PEG-ADA present in the extracellular space reduced levels of CD73 on their surface. Although both CD39 and CD73 are eliminates adenosine produced by the ectoenzymatic chain and hinders rate limiting for extracellular adenosine generation, CD73 is the last adenosine-mediated suppression by interfering between adenosine and component of the ectoenzymatic chain. With accumulating adenosine Adora2a expressed on T effector cells.

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decreased suppressive activity toward T effector cells. The under- plays a crucial role in controlling B-cell survival based on exces- lying mechanism accounting for increased CD39 expression in sively strong or weak BCR signals that identify autoreactive or ADA−/− Tregs remains to be elucidated. However, intracellular functionally unfit B cells (Goodnow, 1996; Nemazee et al., 2000; cAMP levels, which are elevated in the absence of ADA, have been Cancro, 2009; Figure 6). Alterations of BCR signaling thresh- reported to increase CD39 expression (Liao et al., 2010). olds result in a defective central B-cell tolerance checkpoint and In order to dissect the cellular mechanisms leading to loss interfere with the removal of developing autoreactive B cells in of peripheral tolerance, ADA−/− mice were studied after treat- humans (Ng et al., 2004; Menard et al., 2011). In addition to their ment with PEG-ADA, HSC-GT, and BMT. Although short-term BCRs, B cells also express TLRs that were originally described to PEG-ADA treatment initially rescued Treg-mediated suppression bind microbial components but that are also able to recognize in comparison to untreated ADA−/− mice, their functional- self-antigens (Marshak-Rothstein, 2006) and are involved in the ity became exhausted by long-term PEG-ADA treatment. Tregs removal of developing anti-nuclear antibody (ANA)-expressing from PEG-ADA treated animals maintained increased expres- B cells (Isnardi et al., 2008). Both BCR- and TLR-mediated B-cell sion of CD39 and upregulated CD73 expression in comparison responses have been reported to be modulated by adenosine recep- to age-matched wildtype controls. Consistently, CD39 activity tor signaling and intracellular cyclic adenosine monophospate measured by ATP consumption and AMP formation, as well (cAMP), which are increased in ADA deficiency (Apasov et al., as adenosine production by CD73 were significantly increased 2001; Hershfield, 2005; Minguet et al., 2005; Power Coombs et al., in comparison with wildtype Tregs. These results were con- 2011). firmed in a cohort of patients including 7 PEG-ADA treated B-cell tolerance checkpoints in ADA-SCID patients were and 11 retroviral HSC-GT treated patients. The percentage assessed by cloning antibodies expressed by single B cells before of CD4+CD25+FOXP3+CD127−/low Tregs was significantly and after successful HSC-GT (Sauer et al., 2012b). New emi- reduced in PEG-ADA treated patients and their expression of grant/transitional and mature naïve B cells from ADA-deficient CD39 and CD73 ectonucleotidase were significantly increased. patients before HSC-GT contained high frequencies of autore- Unlike Tregs from HSC-GT treated patients and HD, Tregs iso- active and ANA-expressing clones compared to healthy donors, lated from PEG-ADA treated patients were unable to suppress the revealing defective central and peripheral B-cell tolerance check- proliferation of effector cells (Sauer et al., 2012a). points in the absence of functional ADA. The obtained results revealed an elevated adenosine catab- The receptor candidates for the removal of ANA-expressing olism in the presence of PEG-ADA, characterized by alter- clones are nucleic acid-sensing endosomal members of the TLR ations in the adenosinergic machinery producing high levels of family, TLR7 and TLR9 (Marshak-Rothstein, 2006), thereby sug- adenosine and a significantly increased turnover by the enzy- gesting that ADA impinges not only on BCR but more importantly matic activity of PEG-ADA. Upregulation of CD73 in treated TLR signaling. A similar mechanism has also been hypothesized ADA−/− mice and patients can therefore be interpreted as a to contribute to B-cell dysfunctions, defective B-cell proliferation, compensatory mechanism representing a higher requirement for and activation observed in ADA-deficient mice (Aldrich et al., ATP/ADP to adenosine conversion in the presence of extracel- 2003; Hershfield, 2005). The accumulating adenosine blocks NF- lular PEG-ADA. Despite the initial rescue of suppressive activ- κB activation in murine B cells stimulated through BCRs or TLR4 ity by upregulation of CD73 for elevated adenosine production, by LPS (Minguet et al., 2005; Power Coombs et al., 2011). In line long-term PEG-ADA treatment interfered with Treg function by with this hypothesis, we found that stimulation through TLR7 augmenting adenosine turnover. These findings fit the hypoth- and TLR9 were significantly dependent on proper ADA enzymatic esis that PEG-ADA present in the extracellular space eliminates activity and adenosine receptor engagement, further underlin- adenosine produced by this ectoenzymatic chain and hinders ing the inability of these receptors to function in the absence of adenosine-mediated suppression by interfering between adeno- functional ADA (Figure 6). sine and Adora2a expressed on T effector cells (Sauer et al., 2012a; Strikingly, ADA-deficient patients treated with HSC-GT dis- Figure 5C). played quasi-normal early B-cell tolerance checkpoints as evi- denced by restored efficient removal of developing autoreactive DEFECTS IN B-CELL TOLERANCE IN ADA-SCID and anti-nuclear B cells. Hence, ADA plays an essential role in the Although PEG-ADA induces metabolic detoxification, BMT and establishment of early B-cell tolerance and the removal of devel- HSC-GT provide superior restoration of purine metabolism oping autoreactive B cells in humans (Luning Prak, 2012; Sauer and immune functions. However, it had remained unclear how et al., 2012b). patient’s B cells contribute to autoimmune complications and if B-cell tolerance is established properly in ADA-deficient patients CONCLUDING REMARKS ON THE OCCURRENCE OF before and after treatment. AUTOIMMUNITY AFTER ADA-SCID TREATMENT RandomV(D)J recombination produces large numbers of anti- In summary, the available literature provides supporting evidence bodies displaying self-reactive specificities and during normal for a predisposition to autoimmunity in ADA deficiency. Alter- B-cell development the majority of these antibodies are removed ations in both central and peripheral tolerance in T- and B-cells at two distinct checkpoints in the bone marrow and periphery have been described to contribute to the pathogenesis of autoim- (Wardemann et al., 2003). Large numbers of self-reactive antibod- munity. Moreover, it is becoming increasingly clear that tolerance ies are removed from the B cell repertoire during the immature mechanisms and immune responses are specifically altered by the B cell stage in the bone marrow, where BCR-mediated selection lack of ADA and the accumulation of its substrates.

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FIGURE 6 | Development of ANA-expressing B-cell clones in ADA cell development and function. During the bone marrow differentiation of B deficiency. (A) During B-cell development in the bone marrow, BCR signals cells, a major source of autoantigen is developing lymphocytes, which provide a cell-intrinsic measure for negative or positive selection. By undergo apoptosis. Autoantigens are released in the extracellular space excessively weak or strong BCR signals, two thresholds identify and form immune complexes. Binding of nucleic acid-containing immune functionally unfit or autoreactive B-cell clones. B cells that fail to rearrange complexes to an autoreactive BCR produces a strong activation signal and signal through their BCR die of neglect. Negative selection against through simultaneous activation of TLRs and leads to the depletion or autoreactive BCR specificities occurs following high avidity BCR receptor editing of the B cell. It can be hypothesized that in ADA deficiency interactions with antigen. While positive selection of B-cells requires this negative selection is dampened by adenosine present in the persistent intermediate BCR signaling in both developing and mature extracellular place and engagement of the Adora2a. Activation of Adora2a B-cells. (B) Upon antigen-binding the BCR is internalized and transported to elevates intracellular cAMP through activation of adenylyl cyclase. In turn, the cytoplasmic compartments containing Toll-like receptor 9 (TLR9) or cAMP activates PKA that blocks the BCR-induced phosphorylation of IκB to TLR7. Physiological engagement of immunoreceptors leads to the inhibit immunoreceptor-mediated NF-κB activation in the cytoplasm. The phosphorylation and proteasomal degradation of IκB, thereby releasing strong depletion signal coming from BCR and TLR coengagement is NF-κB into the cytoplasm. Subsequently, NF-κB translocates to the nucleus thereby lowered and ADA-deficient B cells carrying an autoreactive BCR and initiates the transcription of NF-κB target genes required for immune egress into the periphery.

Particularly, the impact of accumulating adenosine as anti- metabolism to exert their suppressive activity, since excessively inflammatory mediator has to be underlined in ADA deficiency. high adenosine concentrations, or excessive conversion of extra- The ligation of Adora2a receptors leads to an increase in cAMP lev- cellularly produced adenosine by PEG-ADA interferes with their els, which in cooperation with PKA induces immunosuppression, suppressive function (Luning Prak, 2012; Sauer et al., 2012a). attenuation of proximal signaling events after TCR and BCR trig- The precise role of PEG-ADA alongside other treatment gering, and inhibition of downstream effector functions (Skalhegg options is still undetermined, but it certainly allows rapid detox- et al., 1992; Huang et al., 1997; Lappas et al., 2005; Cassani et al., ification and stabilization of patients awaiting more definitive 2008; Sauer et al., 2012b). It can further be hypothesized that treatment (Booth and Gaspar, 2009). With a progressive loss of dampening of TCR- and BCR-downstream signaling interferes lymphocyte functions, the occurrence of neutralizing anti-ADA with the depletion signals during negative selection in central tol- antibodies and autoimmune manifestations, long-term immuno- erance, thereby allowing the survival of autoreactive cells in ADA logical reconstitution in PEG-ADA patients is often incomplete. It deficiency. In addition to its effects on T- and B-cells, adenosine has been hypothesized that partial ADA correction resulting in low is an important regulator, physiologically involved in inhibiting a enzymatic activity may mimic late-onset patients, which typically variety of activated immune cells and in protecting tissues from display a higher prevalence of autoimmune manifestations (Ochs acute inflammatory damage (Panther et al., 2003; Sitkovsky et al., et al., 1992; Ozsahin et al., 1997; Luning Prak, 2012). Indeed, recent 2004). Indeed we showed that Tregs require a balanced adenosine data underlined the importance of intracellular ADA expression

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and superior efficacy of gene therapy over PEG-ADA treatment for cell functions. However, as suggested by the cloning of single the development of functional T- and B-cell tolerance, including B-cell receptors, non-gene corrected cells may carry autoreactive Tregs (Sauer et al., 2012a,b). specificities (Sauer et al., 2012b). It can be hypothesized that the Both BMT and HSC-GT provide efficient reconstitution of co-existence of non-corrected autoreactive T- or B-cells and the the immune system through endogenous enzymatic ADA activ- restored functional T cell help could allow the development of ity. BMT from an HLA-identical sibling donor remains the autoimmune manifestations in ADA-SCID patients after HSC- treatment of choice, while transplants from alternative donors GT (Aiuti et al., 2009). Modification of preparatory regimen or are associated with high morbidity and mortality. The occur- increased gene transfer efficiency by more robust approaches such rence of mixed chimerism in transplanted patients with other as lentiviral vectors may further improve HSC-GT outcome for primary immunodeficiencies is associated with a higher inci- ADA deficiency (Mortellaro et al., 2006). dence of autoimmune manifestations (Moratto et al., 2011) and Adenosine deaminase-SCID remains a challenging condition might well play a role also in ADA deficiency (Cancrini et al., to treat (Gaspar et al., 2009). With large-scale outcome studies still 2010). Moreover, transplantation-induced lymphopenia is a pos- lacking, the choice between lifelong PEG-ADA, unrelated BMT sible cause for the homeostatic expansion of autoreactive T- and HSC-GT is currently based on the risk/benefit ratio, the avail- and/or B-cells with subsequent loss of self-tolerance (Daikeler ability, and costs of the three different treatment options (Gaspar and Tyndall, 2007). Phenomena of immune dysregulation as et al., 2009). Due to the rarity of the disease and the small cohort occurring in the context of pre-transplant conditioning and numbers, accurate survey and long-term follow-up will be essen- BMT might further trigger the onset of autoimmunity (Etzioni, tial to determine the outcome following different treatments and 2003). their efficacy in restoring immune tolerance. In accordance with the current guidelines of the European group for Blood and Marrow Transplantation (EBMT) and given ACKNOWLEDGMENTS the long-term experience in gene therapy (Aiuti et al., 2009), this Supported by grants from the Italian Telethon Foundation: treatment can now be considered for all ADA-SCID patients lack- TIGET, core grant A1; the European Commission: Advanced Cell- ing an HLA-identical sibling donor (Gaspar et al., 2009). After based Therapies for the treatment of Primary ImmunoDeficiency HSC-GT, high levels (50–90% on average) of gene correction were (Cell-PID), HEALTH F5-2010-261387; and the Ministero della detected in T- and B- and NK-cells (Aiuti et al., 2009), leading Salute: Ricerca Finalizzata, 005/RF-2009-1485896. Figures 2–6 to an efficient systemic detoxification and recovery of immune were produced using Servier Medical Art.

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www.frontiersin.org August 2012 | Volume 3 | Article 265 | 63 REVIEW ARTICLE published: 14 August 2012 doi: 10.3389/fimmu.2012.00234 The STAT5b pathway defect and autoimmunity

Takahiro Kanai †, Jennifer Jenks† and Kari Christine Nadeau*

Division of Immunology and Allergy, Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA, USA

Edited by: The signal transducer and activator of transcription (STAT) 5b is a universal transcription fac- Rosa Bacchetta, Fondazione Centro tor that plays key biological roles in allergic diseases, immunodeficiencies, autoimmunities, San Raffaele Del Monte Tabor, Italy , hematological diseases, growth disorders, and lung diseases. The identification Reviewed by: Michael Jordan, Cincinnati Children’s of distinct pathological manifestations of STAT5b deficiency in humans has highlighted the Hospital/University of Cincinnati, USA critical role of the STAT5b pathway. Proper gene transcription at IL-2Rα, FOXP3, Bcl-2, and Rupali Das, Children’s Hospital of growth hormone (GH) associated loci are thought to be associated with normal STAT5b tran- Philadelphia, USA scriptional activity.These genes are thought to play important roles in allergy/autoimmunity, *Correspondence: immunodeficiency, cancer/anemia, and growth, respectively. The STAT5A and STAT5B Kari Christine Nadeau, Division of Immunology and Allergy, Department genes are collocated on 17q11. Although these two monomeric proteins exhibit peptide of Pediatrics, School of Medicine, sequence similarities of >90%, it is known through observations of STAT5b deficient sub- Stanford University, Stanford, CA jects that STAT5a and STAT5b are not fully redundant in humans. Patients with STAT5b 94305, USA. deficiency have decreased numbers of regulatory CD4+CD25high T cell (Treg) despite their e-mail: [email protected] STAT5a levels being normal. Prior studies on STAT5b deficient subjects have revealed †Takahiro Kanai and Jennifer Jenks contributed equally to this review. immunological aberrations associated with the following disease phenotype: modest lym- phopenia and decreased populations of Treg, γ-δ T cells, and natural killer (NK) cells. Most subjects with STAT5b deficiency show severe eczema, and autoimmune disease (juve- nile idiopathic arthritis, autoimmune thyroiditis, idiopathic thrombocytic purpura) which are thought to be associated withTreg dysfunction. We will review the likely pathophysiological mechanisms associated with STAT5b deficiency.

Keywords: allergy, autoimmunity, IL-2, immunodeficiency, STAT5b, CD25, Foxp3, Bcl-2

INTRODUCTION Although STAT5a and STAT5b show peptide sequence similar- The signal transducer and activator of transcription (STAT) 5b ities of >90%, they differ by six amino acid in the DNA binding is a universal transcription factor that plays key biological roles domain and 20 amino acids in their carboxy termini (Boucheron in allergic disease, immunodeficiencies, autoimmunities, cancers, et al., 1998; Grimley et al., 1999; Soldaini et al., 2000; Wei et al., hematological disease,growth disorders,and lung disease (Buggins 2008). Additional reports of a common disease phenotype specif- and Pepper, 2010; Nadeau et al., 2011). ically associated with STAT5b deficiency in humans (but no such There are several differences between human and mouse in phenotype associated with STAT5a deficiency) indicates that, at the roles of STAT5b (Nadeau et al., 2011). The identification of least in humans, the roles of STAT5a and STAT5b are not fully STAT5b deficiency in humans, and the distinct and destructive redundant (Nadeau et al., 2011). pathology associated with this deficiency has highlighted the criti- Structural dissimilarities between the STAT5a and the STAT5b cal role the STAT5b pathway. Research on the immunologic func- on transactivation domains or subtle differences in the DNA bind- tion of STAT5b has demonstrated its importance for the in vivo ing affinities of STAT5 dimer pairs could influence gene regulation, accumulation of regulatory CD4+CD25high T cells (Treg) with but cell-dependent asymmetries in the availability of phosphory- immunoregulatory function (Cohen et al., 2006; Nadeau et al., lated STAT5a or STAT5b could also another factor. Signal atten- 2011). The specific role that STAT5b plays in the pathogenesis of uation by phosphatase action or classic feedback inhibition, or the aforementioned diseases has led to suggestions that the tran- truncated forms of STAT5b lacking in transactivation capacity, scription factor might have potential as a novel diagnostic and/or may compete upstream for activation and diminish access of full therapeutic target in some disease settings. length molecules to DNA binding sites (Grimley et al.,1999). Thus, In this review, we summarize recent advances in our under- both STAT5 proteins could bind to the same targets, and any dif- standing of the STAT5b pathway in human mainly as well as the ferences between STAT5a and STAT5b may arise from differential autoimmune manifestations induced by the defects within it. expression or difference in kinetics of DNA binding (Grimley et al., 1999). THE STAT5b PATHWAY STAT5b GENE AND PROTEIN, AND NON-REDUNDANCY BETWEEN UPSTREAM OF STAT5b: CYTOKINES AND THEIR RECEPTORS STAT5a AND STAT5b Signal transducer and activator of transcription 5b is a common The STAT5B gene is collocated on 17q11.2 approximately 12 kb downstream effector of the IL-2, -4, -7, -9, -13, -15, -21, growth apart from STAT5A (Figure 1). Both genes are regulated by a Sp-1 hormone (GH; Liu et al., 1997), erythropoietin, thrombopoietin, cis-element (Crispi et al., 2004). and colony-stimulating factor signaling molecules

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As a result of this docking,JAK and STAT molecules are brought into close enough proximity to allow for JAK phosphorylation, and therefore activation, of STAT molecules. In the case of STAT5, phosphorylated STAT5a and/or STAT5b then homo- or hetero- dimerize (sometime tetramaerization; John et al., 1999; Soldaini et al., 2000; Mandal et al., 2011) by each SH2 domain, leave the receptor, and translocate to the nucleus where they act as a transcriptional activator for each target gene (Levy and Darnell, 2002).

DOWNSTREAM OF THE STAT5b PATHWAY Signal transducer and activator of transcription 5b dimers translo- FIGURE 1 |The STAT5B gene is collocated on 17q11.2 approximately cate into the nucleus and bind to specific regions thought to 12 kb apart from the STAT5A gene. The STAT5B gene is on the negative be associated with transcription of FOXP3, CD25, Bcl-2, IGF-1 strand, and the STAT5a gene is on the positive strand. The genomic size of (Nadeau et al., 2011). Reports indicate that STAT5b may preferen- STAT5b is 58,700–77,229.The genomic size of STAT5a is approximately 24,000. tially interact with different DNA binding sites depending on the cell type considered.

(Nadeau et al., 2011). Each cytokine has associated receptors, and Fork-head box P3 (FOXP3): a key transcription factor essential for each receptor has associated Janus kinases (JAK). For example, the Treg cell development and function IL-2 receptor is composed of an α chain (CD25), β chain (CD122), The transcription factor FOXP3 is critical for the thymic devel- and γ chain (CD132; Lin and Leonard, 2000). The β chain is asso- opment of Tregs (Sakaguchi et al., 2008). In mice, CD4+CD25+ ciated with JAK1 and JAK3 (Zhu et al., 1998) and the γ chain is peripheral T cells and CD4+CD25+CD8− thymocytes express associated with JAK3 (Figure 2; Russell et al., 1995). The growth Foxp3 and are considered to be immunoregulatory, whereas other hormone receptor (GHR) is associated with JAK2 (Hwa et al., thymocytes/T cells, either in a resting or activated state, do not 2011). (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003; The CD25 plays an important role as an integral component of Sakaguchi et al., 2008). the high affinity IL-2 receptor. Its ligand, IL-2, is a cytokine known Studies investigating the effects of FOXP3 suppression report for the role it plays in lymphocytic function, especially with rela- complications associated with Treg dysfunction to be a main tion to T cell . There are two functional receptors for IL-2: pathological consequence. Mutations of the FOXP3 gene were one is a heterodimeric complex formed by the β and γ chains,while found to be the cause of an IPEX (immune dysregulation, polyen- the other is a trimeric membrane-spanning complex composed of docrinopathy, enteropathy, X-linked syndrome), which is charac- the α, β, and γ subunits. The latter receptor has a higher affinity for terized by autoimmune disease in multiple endocrine organs (as in IL-2 than the former (Lin and Leonard,2000). Additionally,defects type I diabetes and thyroiditis), inflammatory bowel disease, and in STAT5b expression and function have been shown to result in severe allergy (Chatila et al., 2000; Bennett et al., 2001; Wildin et al., reduced expression of IL-2Rα, thereby potentially limiting cellular 2001). Deletion or dysfunction of FOXP3 causes impaired func- response to IL-2 signaling (Cohen et al., 2006). tion and/or homeostasis of Tregs, and has been implicated in the The engagement between cytokines and their cell surface recep- development of several common autoimmune and inflammatory tors results in subsequent activation of receptor-associated JAK diseases (Campbell and Koch, 2011). tyrosine kinase activity. Activated JAKs phosphorylate specific tyrosine resides in the cytoplasmic domain of their associated The essential role of CD25 in Treg development and function receptor,and these newly phosphorylated residues serve as docking High expression of CD25 is considered to be a marker of Tregs sites for STAT proteins (Figure 2; Grimley et al., 1999). (Sakaguchi et al., 1995) and studies have elaborated on this con- cept, demonstrating that the IL-2Rα serves not only as a marker PHOSPHORYLATION OF STAT5b BY JAKs (MAINLY JAK1 AND 3) for natural Treg, but also, as a protein essential for its development Intracellular signal transduction pathways are essential for trans- and function (Sakaguchi et al., 2008). The importance of CD25 forming extracellular cytokine signaling into appropriate cellular in the development of a normal immune response is emphasized responses. The phosphorylation of STAT molecules is a key com- by the finding that a truncation mutant of CD25 results in an ponent in the JAK/STAT signal transduction pathway (Xu and Qu, immunodeficiency in humans characterized by an increased sus- 2008). ceptibility to viral, bacterial, and fungal infection (Sharfe et al., Cytokine engagement of membrane-associated receptors 1997). In addition, gene targeting analysis also reveals that CD25 brings receptor subunits into proximal relationships necessary deficient mice exhibit autoimmunity (Willerford et al., 1995). for JAK autophosphorylation (Figure 2). Cytoplasmic STAT While CD25 contributes to IL-2 binding affinity and not to the monomers are subsequently able to bind the phosphotyrosine recruitment of signaling molecules (Lin and Leonard,2000) its role residues on engaged cytokine receptors through the highly con- as a component of the high affinity IL-2 receptor makes it indis- served SH2 domain located on all proteins of the STAT family pensable for the activation of cell signaling pathways associated (Figure 3). with IL-2 signal transduction (Sakaguchi et al., 2008).

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FIGURE 2 |This shows the schema of STAT5a and/or STAT5b activation. receptor which in turn serves as the docking sites for STAT5a and/or STAT5b. The engagement between a cytokine and its cell surface receptor results in STAT5a and/or STAT5b are recruited to the phosphorylated receptor and subsequent activation of receptor-associated JAK. Activated JAK subsequently phosphorylated by JAKs. The phosphorylated STAT5a and/or phosphorylates specific tyrosine resides in the cytoplasmic domain of the STAT5b dimerize, leave the receptor, and translocate to the nucleus.

Bcl-2 is an apoptotic inhibitor protein regulation of Bcl-2 has been reported to cause or correlate with Bcl-2 is an apoptosis inhibitor protein. Most cell death in verte- autoimmunity or cancer, particularly leukemia (Buggins and Pep- brates occurs via the mitochondrial pathway of apoptosis,in which per, 2010; Tischner et al., 2010). Deletion of self-reactive immune Bcl-2 and other anti-apoptotic proteins (Bcl-xL, Bcl-w, Mcl-1, and cells occurs through this apoptotic pathway and is necessary for Bfl-1/A1) are key effectors (Llambi and Green, 2011). Aberrant the maintenance of immune tolerance (Tischner et al., 2010).

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FIGURE 3 | Schematic structures of STAT5a and STAT5b. STAT5a and STAT5b differ in the C terminal domain. The dimerization occurs through the interaction between the SH2 domains.

Overexpression of Bcl-2 has been noted in patients with systemic and/or lymphocytic interstitial pneumonitis (Kofoed et al., 2003; lupus erythematosus (Tischner et al., 2010). In malignant dis- Bernasconi et al., 2006; Chia et al., 2006). Immunophenotyping of eases, decreased rate of apoptotic cell death is also found to be these patients have revealed modest lymphopenia and decreased responsible in the proliferative process (Ulukaya et al., 2011). populations of Treg, γ-δ T cells, and natural killer (NK) cells (Bernasconi et al., 2006; Cohen et al., 2006). There are currently Insulin-like growth factor-I and Insulin-like growth factor binding 10 published cases of STAT5b deficiency (Table 1; Nadeau et al., protein-3 play an important role in fat metabolism and skeletal 2011). Ongoing research efforts aim to identify the molecular development mechanisms of STAT5b in postnatal growth and immunity. Insulin-like growth factor-I promotes skeletal development and fat metabolism, and insulin-like growth factor binding protein-3 (IGFBP-3) acts as a negative regulator for IGF-I signaling (Kawai Previous cases and Rosen, 2010). The first case of a STAT5b mutation was reported in 2003, in The activation of IGF-I is initiated by the interaction of circulat- a 16-year-old female with severe growth retardation (−7.5 SD) ing GH with the GHR. The cytoplasmic domain of GHR associates and pulmonary complications (Kofoed et al., 2003). The reported preferentially with JAK2. Activation of JAK2 by GHR engagement missense mutation (p.A630P) disrupted the core of anti-parallel leads to the activation of STAT5b (Hwa et al., 2011). β-sheets that enable phosphate-binding, causing aberrant folding In humans, serum IGF-I concentrations have a positive corre- (Chen et al., 1998) aggregation of mutant STAT5b protein, and lation with skeletal mass (Langlois et al., 1998). A report on the loss of thermodynamic stability (Chia et al., 2006; Fang et al., disease characteristics of STAT5b deficiency in humans highlights 2006). The patient presented with early onset lymphocytic inter- low serum IGF-1 as one defining clinical feature of the disease stitial pneumonitis, chronic lung disease, hemorrhagic varicella, (Hwa et al., 2004; Nadeau et al., 2011). STAT5b deficient patients , and autoimmune disease (Kofoed et al., 2003). At age 7, she also exhibit stunted growth and poor response to GH therapy developed lymphocytic interstitial pneumonia and after receiv- (Nadeau et al., 2011). ing potent immunosuppressive therapy, had two major infectious IGF-I was also reported as a critical factor for adipogenesis complications – severe varicella-zoster virus infection and Pneu- (Kawai and Rosen, 2010). The lack of this factor results in a defect mocystis jiroveci pneumonia. Another biopsy at age 10 also indi- in adipose tissue formation by mitogen-activated protein kinase cated lymphoid interstitial pneumonia, and P. carinii was isolated deactivation in conjunction with GH (Boney et al., 2000; Hwa from the tissue. Later studies revealed decreased numbers of Treg et al., 2011). and reduced Treg suppressive function (Cohen et al., 2006). IGFBP-3 suppresses adipogenesis independent of IGF-I bind- In 2005, a second case of a STAT5b deficiency was identified ing (Chan et al., 2009) and reduces bone density (Kawai in a 16-year-old Turkish female with severe growth failure, GHI, and Rosen, 2010). atopic dermatitis, pruritic skin lesions, primary idiopathic pul- monary fibrosis with diffuse lung involvement, and autoimmune HUMAN STAT5b PATHWAY DEFECT AND AUTOIMMUNITY disease, as well as bleeding diathesis caused by defective thrombo- Human STAT5b deficiency is a recently identified, rare autosomal cyte aggregation, preventing a potential lung biopsy (Hwa et al., recessive disease that involves both severe GH-resistant growth 2005). Sequencing of the STAT5b gene revealed a novel homozy- failure and severe primary immunodeficiency. It was first dis- gous frameshift mutation (c.1191insG) that led to protein termi- covered in patients with dwarfism associated with normal levels nation (p.N398EfsX16) and consequent lack of immunodetectable of serum GH, but very low levels of IGF-I (Kofoed et al., 2003; STAT5b protein (Hwa et al., 2005). Bernasconi et al., 2006; Chia et al., 2006). Affected individu- Another case was identified in 2006 in a 16-year-old female als also exhibited recurrent infections, chronic diarrhea, eczema, with severe postnatal growth failure, GHI, and immunodeficiency

Frontiers in Immunology | Primary Immunodeficiencies August 2012 | Volume 3 | Article 234 | 67 Kanai et al. The STAT5b pathway defects and autoimmunity values 119–483 > 6.0 No #10 Reference NA 18 F 1.7 27.1 dermatitis Scaglia et al. (2012) -5.90 AIT Adopted NA 2 No ND M 1 Brazil Argentina 14 750 840 210-740 750 NA − 1.6− 1.3 NA NA − 3 − / − / Pugliese- Pires et al. (2010) < 25 16 #9 Yes 6 M − 1.6 − 1.3 − 5.6 Pugliese- Pires et al. (2010) − 1.28 − 0.6 − 5.6 #7 #8 Hwa et al. (2007) < 5 34 4 F 6 years 6 years Yes #6 − 1.28 − 0.6 2 SJIA No No F − 5.8 − / − / − / 17.7 5.7 1.7 Hwa et al. (2007) < 5 NA NA 20.6 700 800 520 0.4 0.8 520 Yes Yes ND Abs + #5 14 − 0.8 − 2.8 Delayed NA NA NA 31 Ichytosis to platelets M − 5.9 − / 0.13 et al. (2006) 14.2 180 0.7 Vidarsdottir Abs to + #4 with thymoglobulin − 5.3 Bernasconi et al. (2006) F NA Adopted NA F NA 12.5 NA 500 NA 0.7 − 2.2− 3.3 NA NA No AIT − 9.9 Bernasconi et al. (2006) #3 < 10 0.8 − / − / Abs to + − 0.9 − 0.6 #2 platelets − 7.8 − / Hwa et al. (2005) 16.4 15.3 12 F 14.2 6.6 1.8 NA 543 1.2 7.0 Yes Kofoed Abs to BEC ITP − 0.3 − 1.2 7 years 7 years+ 1 year 6 months No − 7.5 − / #1 (2006) , et al. (2003) Delayed Delayed Delayed NA Eczema Eczema Eczema Eczema Ichytosis No No Atopic Atopic Seborrheic 16.5 F 9.4 Cohen et al. 874 A630P 1191insG R152X R152X 1103insC 1680delG 1680delG 424_427del 424_427del F646S Argentina Turkey Argentina Argentina Caribbean Kuwait Kuwait Brazil Y 1 | Demographics of published cases with STAT5b deficiency. , idiopathic thrombocytopenic purpura; AIT, autoimmune thyroiditis; Abs, antibodies; BEC, bronchial epithelial cells; SJIA, systemic juvenile idiopathic arthritis; ND, not diagnosed; NA, not currently available; PC, AT5b Mutation BIOCHEMISTR Parental consanguinityPaternal height, SDS Mother height, SDS Birth weight/length Yes (cm)Puberty 1400/ND 2350/49 2500/ND 1650/ND 3270/50 2400/ND 3600/ND 1650/39 2400/49 2250/44 Onset of chronic pulmonary disease Skin pathology Place of origin Sex Height, SDS GH stimulated (ng/mL) 53.8 ST GH basal (ng/mL) ITP personal communication. The data of #6 andHeterozygote STAT5b-deficient #7 subjects in who adjacent are columns relatives represent ofof homozygote data their STAT5b-deficient from subjects clinical siblings. currently demographics have and not laboratory been identified studies to has have not any been abnormal done clinical yet. phenotypes, although a specific detailed review References for published case IGF-I stimulated (ng/mL) 38 IGFBP-3 (ng/mL) Age Autoimmune manifestations ALS (acid labile subunit; mg/L) 2.9 Table

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(Bernasconi et al.,2006). Pulmonary-function tests showed mixed, dysfunction. Congenital ichthyosis was diagnosed at birth, and restrictive, and obstructive moderate ventilative insufficiency, but the patient had hemorrhagic varicella at 16 years of age but had no no lung biopsy was performed. Notably, this case was the first history of pulmonary of immunological problems (Vidarsdottir to identify a role for STAT5b not only in the human GH signal- et al., 2006). ing cascade, but also in the cytokine-mediated immune response. Previous immunological studies have established the impor- The STAT5b deficient patient had moderate T cell lymphopenia, tance of STAT5b proteins in the development, homeostasis, and normal CD4/CD8 ratios, and very low numbers of NK cells and proliferation of different lymphocyte populations. Immune reper- γ-δ T cells, and the T cells presented a chronically hyperactivated toires of STAT5b deficient patients have shown moderate lym- phenotype (Bernasconi et al., 2006). phopenia, with very low numbers of NK and T cells, as well as Treg Since 2012, five other mutations have been published on a dysfunction. Furthermore, B cell populations and immunoglobu- total of seven additional subjects (Table 1). Lung pathology has lin G levels in at least patient are normal to elevated, as consistent been common among these patients (8 of 10), but of these with autoimmune disease symptoms (Cohen et al., 2006). remaining seven subjects, only a few have received lung biop- sies. A STAT5b deficient male with the mutation 424_427del Disease management received a biopsy at 6 years of age that indicated severe lympho- In order to improve clinical outcomes for patients with STAT5b cytic interstitial pneumonitis. Considered together, these studies deficiency, optimizing early diagnosis in these patients is critical. have firmly established a correlation between STAT5b deficiency To date, overall management of STAT5b deficiency is still unclear. and immune dysfunction, in addition to GHI and severe growth GH therapy is ineffective due to the patients’ GHI. It is presumed problems. that IGF-I therapy may be an effective treatment, unless the pres- ence of chronic infection limits the growth response. However, to date, no clinical trials of IGF-I therapy have been performed in Clinical manifestations and diagnosis these patients. Signal transducer and activator of transcription 5b deficiency Patients should be closely monitored for should be considered in the differential diagnosis of a patient of immunodeficiency. Infections such as severe varicella or recur- who has normal gestational growth and birth size but acquires rent pneumonias should be aggressively treated with appropriate significantly short stature and recurrent infections. This pattern antimicrobial therapies. Patients with autoimmune conditions, of growth is typical of patients with GHI. Height may range from atopic diseases, or pulmonary fibrosis may also require antipro- − − 3.0 to 9.9 SD in girls and boys, respectively (Table 1). liferatives or immunosuppressants, such as steroids, to address Regarding hormone evaluations, all described patients have overactive effector T cell responses. Because severe chronic lung had normal levels of GH at baseline, but after stimulation, GH disease in this patient population often leads to high morbid- concentrations were often elevated (Table 1). In contrast, serum ity and mortality, patients should be carefully monitored with IGF-I, IGFBP-3, and acid labile subunit concentrations were low, pulmonary-function tests and physical examinations, which may and even upon administration of GH, remained low. Elevated improve treatment options to decrease the lung disease severity. prolactin levels were also observed in patients with recorded Although current management of STAT5b deficiency is pri- concentrations. marily dictated by specific end-organ pathology, current research Most patients have displayed evidence of immune dysfunc- is addressing the possibility of enhancing STAT5b and/or STAT5a tion,including atopic disease,chronic lung disease,viral infections, pathways (Zeiser et al., 2008; Strauss et al., 2009). Future therapy and/or autoimmune diatheses. Often present in childhood, severe may be expected to prevent and reflect rationally based drug design pulmonary disease is of particular concern, as it has affected 8 of to enhance certain drug targets in the STAT5b and/or STAT5a the 10 known STAT5bdeficient patients and two patients have died pathways. of respiratory failure. For all cases of lung pathology except for that of Patient #5, an axial chest CT scan has shown increased inter- CONCLUSION stitial patterns and ground-glass appearance. These pulmonary In this review, we focused on the STAT5b pathway and the mech- lesions are T cell predominant, despite peripheral lymphopenia. anisms by which defects in protein structure and or expression In most cases, severe eczema, thrombocytopenic purpura, and/or might result in autoimmunity. A better understanding of STAT5b autoimmune disease, such as juvenile idiopathic arthritis, were and its distinct biological functions is necessary for the develop- present in addition to severe lung disease. However, it should be ment of new diagnostic and therapeutic approaches for treating noted that 1 of the 10 subjects to date has less severe immune patients suffering from its deficiency.

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of mouse scurfy. Nat. Genet. 27, N., Beilhack, A., Loh, J., Hou1, Il-2Rbeta. J. Biol. Chem. 273, and autoimmunity. Front. Immun. 18–20. J.-Z., and Negrin, R. S. (2008). 10719–10725. 3:234. doi: 10.3389/fimmu.2012.00234 Willerford, D. M., Chen, J., Ferry, J. Differential impact of mammalian This article was submitted to Frontiers in A., Davidson, L., Ma, A., and Alt, target of rapamycin inhibition on Conflict of Interest Statement: The Primary Immunodeficiencies, a specialty F. W. (1995). Interleukin-2 recep- CD4+ CD25+ Foxp3+ regulatory authors declare that the research was of Frontiers in Immunology. tor alpha chain regulates the size T cells compared with conven- conducted in the absence of any com- Copyright © 2012 Kanai, Jenks and and content of the peripheral lym- tional CD4+ T cells. Blood 111, mercial or financial relationships that Nadeau. This is an open-access arti- phoid compartment. Immunity 3, 453–462. could be construed as a potential con- cle distributed under the terms of the 521–530. Zhu, M. H., Berry, J. A., Rus- flict of interest. Creative Commons Attribution License, Xu, D., and Qu, C. K. (2008). Pro- sell, S. M., and Leonard, W. J. which permits use, distribution and tein tyrosine phosphatases in the (1998). Delineation of the regions Received: 04 May 2012; paper pending reproduction in other forums, provided JAK/STAT pathway. Front. Biosci. of interleukin-2 (IL-2) receptor beta published: 27 May 2012; accepted: 15 July the original authors and source are 13:4925–4932. chain important for association of 2012; published online: 14 August 2012. credited and subject to any copyright Zeiser, R., Leveson-Gower, D. B., Jak1 and Jak3. Jak1-independent Citation: Kanai T, Jenks J and Nadeau notices concerning any third-party graph- Zambricki, E. A., Kambham, functional recruitment of Jak3 to KC (2012) The STAT5b pathway defect ics etc.

Frontiers in Immunology | Primary Immunodeficiencies August 2012 | Volume 3 | Article 234 | 71 REVIEW ARTICLE published: 02 August 2012 doi: 10.3389/fimmu.2012.00232 APECED: is this a model for failure of T cell and B cell tolerance?

Nicolas Kluger1, Annamari Ranki1 and Kai Krohn2*

1 Department of , Allergology and Venereology, Institute of Clinical Medicine, Skin and Allergy Hospital, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland 2 Clinical Research Institute HUCH Ltd, Helsinki, Finland

Edited by: In APECED, the key abnormality is in the T cell defect that may lead to tissue destruc- Rosa Bacchetta, Fondazione Centro tion chiefly in endocrine organs. Besides, APECED is characterized by high-titer antibodies San Raffaele Del Monte Tabor, Italy against a wide variety of cytokines that could partly be responsible for the clinical symp- Reviewed by: toms during APECED, mainly chronic mucocutaneous candidiasis, and linked to antibodies Rosa Bacchetta, Fondazione Centro San Raffaele Del Monte Tabor, Italy against Th17 cells effector molecules, IL-17 and IL-22. On the other hand, the same anti- Mario Abinun, Newcastle upon Tyne bodies, together with antibodies against type I interferons may prevent the patients from NHS Foundation Trust, UK other immunological diseases, such as psoriasis and systemic lupus erythematous. The *Correspondence: same effector Th17 cells, present in the lymphocytic infiltrate of target organs of APECED, Kai Krohn, Clinical Research Institute could be responsible for the tissue destruction. Here again, the antibodies against the cor- HUCH Ltd, Salmentaantie 751, 36450 Salmentaka, Finland. responding effector molecules, anti-IL-17 and anti-IL-22could be protective.The occurrence + + e-mail: kai.krohn@nic.fi of several effector mechanisms (CD4 Th17 cell and CD8 CTL and the effector cytokines IL-17 and IL-22),and simultaneous existence of regulatory mechanisms (CD4+ Treg and anti- bodies neutralizing the effect of the effector cytokines) may explain the polymorphism of APECED. Almost all the patients develop the characteristic manifestations of the complex, but temporal course and severity of the symptoms vary considerably, even among siblings. The autoantibody profile does not correlate with the clinical picture. One could speculate that a secondary homeostatic balance between the harmful effector mechanisms, and the favorable regulatory mechanisms, finally define both the extent and severity of the clini- cal condition in the AIRE defective individuals. The proposed hypothesis that in APECED, in addition to strong tissue destructive mechanisms, a controlling regulatory mechanism does exist, allow us to conclude that APECED could be treated, and even cured, with immunological manipulation.

Keywords: AIRE, APECED, endocrine disorders, interleukin 17, interleukin 22, IPEX,T regulatory cells

INTRODUCTION Ahonen et al., 1990) and Sardinians 1 (1:14,500; Rosatelli et al., Autoimmune polyendocrinopathy syndrome type 1 (APS-1) or 1998; Meloni et al., 2012). It is characterized by the variable asso- autoimmune polyendocrinopathy–candidiasis–ectodermal dys- ciation of autoimmune endocrine [hypoparathyroidism (HP), trophy syndrome (APECED; OMIM 240300) is a rare recessively Addison’s disease (AD), hypothyroidism, gonadal insufficiency, inherited disorder (Perheentupa, 2002; Betterle and Zanchetta, insulin-dependent diabetes mellitus, , and Bier- 2003; Perheentupa, 2006; Husebye et al., 2009). It is caused by mer’s disease] and non-endocrine disorders (keratitis, malabsorp- mutations in the autoimmune regulator (AIRE) gene located tion, vitiligo, and alopecia areata) and a specific predisposition to on locus 21q22.3 (Bjorses et al., 1996; Nagamine et al., 1997; chronic mucocutaneous candidiasis (CMC). A definite diagnosis The Finnish–German APECED Consortium, 1997). APECED of APECED is made upon one of the following criteria: (i) the displays a worldwide distribution, but specific clusters of high presence of at least two of three major clinical features: CMC, HP, prevalence of the disease are observed among Finns (1:25,000; and AD, or (ii) one disease component if a sibling has already a definite diagnosis, or (iii) disease-causing mutations in both alle-

Abbreviations: AADC, aromatic L-amino acid decarboxylase; AD, Addison’s disease; les of the AIRE gene. However, APECED being highly variable AE, autoimmune enteropathy; APS-1, autoimmune polyendocrinopathy syn- in its presentation, the classical triad may be complete only after drome type 1; APECED, autoimmune polyendocrinopathy–candidiasis–ectodermal years of evolution and diagnose may be therefore missed. Besides, dystrophy; CMC, chronic mucocutaneous candidiasis; EECs, enteroendocrine cells; GI, gastro-intestinal; HP, hypoparathyroidism; IPEX, immune dysregula- APECED may appear during adolescence or in the young adult tion, polyendocrinopathy, enteropathy and X-linked; IF, intrinsic factor; IL-1, (Husebye et al., 2009). Therefore, criteria for a probable APECED interleukin-1; IL-17, interleukin-17; IL-22, interleukin-22; mTECs, thymic have been defined as follows: (i) presence of one of CMC, HP, medullary epithelial cells; NALP-5, NACHT leucine-rich-repeat protein 5; PE, AD (before 30 years of age) and at least one of the minor compo- promiscuous expression; PTH, parathormone; SLE, systemic lupus erythemato- sus; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; TRIMs, tripartite nents chronic diarrhea, keratitis, periodic rash with , severe motif-containing proteins; TSA, tissue-specific antigens. constipation, autoimmune hepatitis, vitiligo, alopecia, enamel

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hypoplasia, (ii) any component and anti-interferon antibodies, or linked to a recessively inherited gene defect (Ahonen et al., 1990; (iii) any component and antibodies against NACHT leucine-rich Perheentupa, 2002, 2006). The autoimmune endocrinopathies repeat protein 5 (NALP5), AADC, tryptophan hydroxylase (TPH), could thus be grouped on the basis of their genetic background or TH (Husebye et al., 2009). in two distinct categories: those linked to HLA variation and the one, APS-1 caused by a single mutated gene (Table 1). At that FROM CIRCULATING AUTOIMMUNE ANTIBODIES stage, however, the responsible gene, the APECED gene, was not TO AIRE, FOXP3, APECED, AND IPEX yet identified. Once identified, the APECED gene was renamed Our knowledge of the nature of the condition now called APS-1 as AIRE in 1997 (Nagamine et al., 1997; The Finnish–German or APECED has increased simultaneously with the general devel- APECED Consortium, 1997). opment of immunology and autoimmunity. Since the condition Another immunopathy, termed originally as autoimmune was clearly defined in the end of 1950s and early 1960s, the char- enteropathy (AIE) and later identified as immune dysregula- acteristic clinical picture, the immunological abnormalities and tion, polyendocrinopathy, enteropathy and X-linked (IPEX), was the relationship to other autoimmune endocrine diseases were described in the 1980s and 1990s (Powell et al., 1982). This dis- defined in late 1960s and early 1970s. Furthermore, the genetics order was later shown to be caused by a defect in a single gene, of APECED, and the fact that the syndrome was caused by a reces- FOXP3 (Bennett et al.,2000). IPEX and APECED are two examples sive gene defect – as opposed to the HLA-linked genetics seen in of immune deficiency diseases disclosing both disturbed tolerance the other solitarily occurring endocrine diseases – were character- and autoimmune phenomena (Moraes-Vasconcelos et al., 2008). ized in the 1980s and the target antigens in the organs affected by Traditionally, reviews tend to associate both IPEX and APECED APECED were molecularly defined in 1990s. A landmark stage in because of common features. However, both clinical manifesta- the study of APECED was reached in 1997, when the long sought tions and predisposition to infections are rather different when APECED gene was cloned by two independent groups (Nagamine comparing both diseases (Moraes-Vasconcelos et al., 2008). et al., 1997; The Finnish–German APECED Consortium, 1997). Finally, a new phase in APECED research occurred during the AIRE GENE, MUTATIONS, AND MECHANISM OF ACTION first decennium of 2000, when the autoantibodies toward soluble AIRE is expressed in thymus, lymph nodes, and fetal liver, and mediators if immune response were characterized (Meager et al., encodes a protein with two putative zinc fingers and other motifs 2006; Kisand et al., 2011). suggestive of a transcriptional regulator (Nagamine et al., 1997; The notion that several diseases affecting endocrine organs and The Finnish–German APECED Consortium, 1997). The AIRE earlier defined as idiopathic, were in fact caused by an autoim- gene, approximately 13 kb in length, contains 14 exons that mune response toward self antigens, became apparent when novel encode a polypeptide of 545 amino acids. The AIRE protein func- immunological methods became available in 1950s and early 1960s tions as a transcription factor (Fierabracci, 2011; Gardner et al., (Blizzard et al., 1963). The association of the three conditions, 2009). AIRE is expressed in the thymic medullary epithelial cells candidiasis, HP, and AD that were later judged to be the hall- (mTECs, Figure 1) and in cells of the /dendritic cell marks of APECED was clearly stated by the groups of Blizzard and lineage (Kogawa et al., 2002). mTECs through the expression of Maclaren (Blizzard et al., 1963; Brun, 1978; Neufeld et al., 1981). MHC class II express a wide array of tissue-restricted antigens These groups also defined two clearly distinct syndromes with (TRAs) derived from different organs in the body. TRAs include several associated autoimmune diseases: autoimmune polyglan- self-proteins with patterns of expression restricted to a single or dular syndrome type 1 (PGS-1) and polyglandular syndrome type small handful of organs. Thymic expression of TRA serves as an 2 (PGS-2). The nomenclature was later changed to APS-1 and important source of self-antigens to allow the negative selection APS-2, and the former further to APECED (Ahonen et al., 1990; of autoreactive T cells. Collectively, mTEC and thymic mono- Perheentupa, 2002, 2006; Betterle and Zanchetta, 2003). cyte/dendritic cells play a crucial role in establishing self-tolerance Pioneering studies in this field were made especially with the by eliminating autoreactive T cells (negative selection) and/or use of immunohistochemistry, demonstrating antibodies react- by producing immunoregulatory FOXP3+ T cells, which prevent ing with gastric parietal cells in chronic gastritis (Walder et al., CD4+ T cell-mediated organ-specific autoimmune diseases. Col- 1963; Irvine et al., 1965) and intrinsic factor (IF) in pernicious lectively, several studies in mouse and man have shown that AIRE anemia (Schwartz, 1961; Jeffries et al., 1962), with thyroid epithe- regulates thymic expression of several genes of ectopic peripheral lial cells in various forms of thyroid diseases (Witebsky et al., 1957; proteins including many TRAs. Thus, AIRE dysfunction leads to Irvine et al., 1962; Doniach and Roitt, 1964), with the beta cells of a decrease in the expression of TRAs in the thymus, and con- Langerhans islands in diabetes mellitus (Kaldany, 1979; Bottazzo sequently, autoreactive T cell clones escape into the periphery et al., 1980), and adrenal cortical cells in AD (Blizzard et al., 1962). (Derbinski et al., 2005; Moraes-Vasconcelos et al., 2008; Gardner Blizzard’s group noticed that the two polyglandular syndromes, et al., 2009; Fierabracci, 2011) APS-1 and APS-2, differed in their HLA haplotypes (Neufeld et al., The most common AIRE mutation, the “Finnish mutation,” 1981). Further studies on the HLA haplotypes revealed that the R257X, affects 82% of Finnish APECED alleles (Nagamine et al., genetic basis of APS-2, but also of the other isolated forms of 1997; The Finnish–German APECED Consortium, 1997). Inter- endocrine autoimmune diseases found in APS-1, were in the HLA estingly, this mutation occurs also in 70% of the Russian APECED haplotype of the patients. In contrast, APS-1 was shown not to be patients studied (Orlova et al., 2010). The same mutation, R257X linked to HLA, and studies with large patient material, collected was also detected in Swiss patients on a different haplotype with by Perheentupa’s group in Finland, clearly stated that APS-1 was closely linked polymorphic markers (Nagamine et al., 1997) and in

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“fimmu-03-00232” — 2012/7/31 — 17:51 — page2—#2 Kluger et al. APECED syndrome production. , andα TNF- Increased production of IL-17 tory T cells,IFN- γ defective IL-2, Cellular immune response CTL against affected organs? Failure in Treg population gene gene Impaired function of regula- AIRE FOXP3 No single gene defect CTL against affected organs? No single gene defect CTL against affected organs? Gene defect described in the No association Defective Risk haplotypes HLA DR3: DRB1*0301, DQA1*0501, DQB1*0201 DR4 DR1, DR7, DR13, and DR14: protective ( Betterle and Zanchetta, 2003 ) HLA-DRB1-DQA1-DQB1 HLA-DR3 HLA enteropathy-related 75-kDa antigen) pancreatic-islet cells, insulin, and glutamic acid decarboxilase (GAD), and thyroid (antithyroid microsomal antibodies) beta cells, thyroid Against P450c21, P450scc Against enterocytes (autoimmune Against adrenal cortex, pancreatic Against all affected organs No association (?) Close to 100 mutations Autoantibodies High ACTH, low cortisol, high renin, low aldosterone, subnormal cortisol response to ACTH test: hyponatremia, hyperkalemia (mainly eczema), failure to thrive, thyroiditis, recurrent infections type I diabetes or thyroid diseases endocrine organs and non-endocrine autoimmunity Key laboratory findings in the different autoimmune endocrine diseases. | IPEX Enteropathy, diabetes skin disease Diagnosis Clinical findings Addison’s disease Low levels of gluco- and mineralocorticoid APS-2 Addison’s disease with insulin-dependant APECED (APS-1) Candidiasis and multiple failure of most Table 1

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numerous various causes that may be associated or follow each other during the life of the patients.

CANDIDIASIS Chronic mucocutaneous candidiasis infection by is one of the major characteristic of APECED, usually one of first symptoms and most likely the most disabling features of APECED. CMC is naturally not specific for APECED but any child with CMC should be suspected of APECED. According to the Finnish experi- ence, almost all adults with APECED display symptoms of CMC, up to 70% of the patients at the age of 10, up to 94% at age of 20, and 97% at the age of 30 (Perheentupa, 2002, 2006; Betterle and Zanchetta, 2003; Husebye et al., 2009). However, the course and severity vary widely. affects the tongue, the buccal mucosa, the gingival, and the pharynx. It ranges in severity from mild form with redness, soreness, , pseudomem- branous lesions, erosions, ulceration and pain to severe chronic FIGURE 1 | Medullary epithelia cells in thymus, expressing the AIRE inflammation with dysphagia, and development of hyperkera- proteins (reddish brown), in close vicinity of the Hassall’s corpuscles totic plaques. In the absence of active antimycotic treatment and (HC) where auto-reactiveT cells are thought to be destroyed. Note cell debris in HC. Magnification 1:40. AIRE was demonstrated with specific careful follow-up, chronic oral candidiasis may lead to the devel- monoclonal antibody at 1:2,000 dilution. opment of squamous cell carcinoma with potential lethality by metastatic dissemination. Candida has been reported to affect 15–22% of the patients (Perheentupa, 2006; Kisand et al., northern Italian APECED patients. Nonsense mutation R139X was 2011) with pain while swallowing, retrosternal pain, and dysphagia found as the predominant haplotype among Sardinian patients (Ahonen et al., 1990; Husebye et al., 2009). Chronic esophagi- (18/20 independent alleles; Rosatelli et al., 1998). Other hotspots tis can lead to local stricture and exceptional esophageal cancer have been identified such as the Y85C missense mutation in (Rautemaa et al., 2007). an isolated Iranian Jewish community (Zlotogora and Shapiro, Intestinal candidiasis may cause chronic diarrhea. It should 1992; Björses et al., 2000). A 13-bp deletion in exon 8 [1085– be stressed that esophageal and intestinal candidiasis may occur 1097(del)] is ubiquitous and can be found in Norwegians, but without any active oral candidiasis. Genital candidiasis affects also Anglo–Saxons descendant (Zlotogora and Shapiro, 1992) and mainly women with pruritus and vaginal whitish discharge south Americans (Moraes-Vasconcelos et al., 2008). Today, over 60 while genital candidiasis seems less frequent in males, pos- different mutations have been described throughout the coding sibly underreported due to discrete signs of balanitis. Lastly, region of AIRE (Akirav et al., 2011). Candida may affect the nails with chronic paronychia and ony- chomycosis (Collins et al., 2006). Fingernails are more commonly CLINICAL PICTURE OF APECED affected than toenails and the thumbs are the commonest digit The clinical picture of APECED is characterized by sequentially affected. This can be explained as infection occurs during the occurring diseases, with great variation among the patients as “thumbsucking” period. Management of candidiasis in APECED to the severity and time course of the various conditions. In patients implies an excellent oral hygiene with a careful and most cases, the affected individual starts suffering from CMC regular dental follow-up. Candidiasis should be treated aggres- in early infancy or childhood. In most cases, the next organs to sively with antimicrobial therapy and regular prophylaxis should be affected are the parathyroid glands, followed by AD and at be given. puberty, hypogonadism mainly in female teens or young adults. Any clinically suspicious, chronic thickening or erosion of the Additional clinical features are less common, and include dia- mucosa that does not heal should be biopsied to rule out a potential betes type I, hypothyroidism, atrophic gastritis with or without underlying lesion of squamous cell carcinoma. Any difficulties pernicious anemia (Biermer’s disease), cutaneous manifestations in swallowing or eating or retrosternal pain should prompt to (alopecia areata, vitiligo, transient skin rash during fever episodes, perform esophagoscopy (Rautemaa et al., 2007). non-infectious nail dysplasia), ocular symptoms (keratoconjunc- tivitis, dry eye, iridocyclitis, cataract, retinal detachment, and HYPOPARATHYOIDISM optic atrophy; Merenmies and Tarkkanen,2000), enamel dysplasia, Hypoparathyroidism is one of the first endocrine features of hyposplenism/ (implying against Streptococ- APECED. Symptoms are related to hypocalcemia, muscle cramps, cus pneumonia, Haemophilus influenzae, and as well as , clumsiness, seizures, and diarrhea. The diagnosis is antibiotic prophylaxis), autoimmune hepatitis, tubulo-interstitial simply based on blood calcium, phosphorus, and parathormone nephritis, or organized pneumonitis. Involvement of the gastro- (PTH) levels: hypocalcemia, hyperphosphatemia, inadapted nor- intestinal (GI) tract may be responsible for chronic diarrhea, mal/low PTH without any kidney failure. It is considered that constipation, and malabsorption leading sometimes to malnu- APECED should be systematically considered in cases of primary trition. GI involvement is difficult to assess as it can be due to HP (Husebye et al., 2009). Antibodies against NALP5 as well as

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against the calcium-sensing receptor of parathyroid epithelial cell retrospect, the cases described by Thomas Addison were most have been identified in APECED patients (Gavalas et al., 2007; likely caused by . Kemp et al., 2009, 2010). Patients who are free of HP need an The clinical signs and symptoms of AD are mostly similar in annual monitoring of blood calcium and phosphorus levels. Man- APECED and in solitary AD as well as in APS-2 complex. These agement of HP relies on daily oral supplementation of vitamin D include decreased levels of gluco- and mineralocorticoids and ele- derivatives and calcium. vated ACTH concentrations. The most severe consequence of AD is the life-threatening Addisonian crisis, characterized by general GASTRITIS AND PERNICIOUS ANEMIA , dizziness, diarrhea, and death, if the patient is not quickly Chronic gastritis, with or without concomitant pernicious anemia substituted with , mainly hydrocortisol. belongs to the APECED complex but is found only in a fraction of Autoantibodies to adrenal cortex are the characteristic cases. In non-APECED population, two types of chronic gastritis immunological feature in AD, be it part of APECED or APS-2 occur, divided by Strickland into type A and B gastritis. Type A or the solitary form. These antibodies can be easily demonstrated gastritis was known to be caused by autoimmunity while the B by immunofluorescence. However, in APECED, but not in the gastritis was suspected to be the results of environmental factors. other forms of AD, the autoantibodies are precipitating, and this In early 1980s, it was shown by Warren and Marshall (1984) that phenomenon can be demonstrated by Ouchterlony’s immunod- the major was in fact a chronic infection with iffusion (Andrada et al., 1968; Krohn et al., 1974; Heinonen et al., Helicobacterium pylori. 1976). In immunodiffusion with APECED serum against adrenal The type A chronic gastritis, with and without pernicious ane- homogenate three precipitating lines were observed, and one of mia that occur in non-APECED individuals, is clearly linked these were shown to represent a mitochondrial antigen while the to certain HLA risk haplotypes, in analogy to isolated AD. In two others were microsomal. APECED patients, the chronic gastritis differs from the above The nature of the adrenal cortical autoantigens were revealed also in time of occurrence and the speed of the progression. In in early 1990s and shown to be the three main steroidogenic non-APECED patients, the time needed for progression from the enzymes, P450c17, P450c21, and P450scc (Krohn et al., 1974; early stage of gastritis, the superficial form to diffuse gastritis, to Winqvist et al., 1993; Uibo et al., 1994a,b). These three enzymes atrophic gastric and to full gastric atrophy is a slow process, taking were also shown to be the ones that could be precipitated up several decennia. Also, the process usually starts in the adult by immunodiffusion. The autoantibodies against these three life. In contrast, an APECED-associated gastritic process is much steroidogenic enzymes clearly distinguish the three clinical con- faster and can start in the first decennium of life. Thus, one of ditions with adrenal failure: antibodies to all three can be found the authors of this review was able to follow such a gastric process only in APECED, while in solitary AD and in APS-1, only anti- in two 8-year-old girls with sequential gastric biopsies and could bodies to P450c21 are seen. Furthermore, in non-autoimmune see how, within the time period of only 2 months, the superficial AD, caused by tumors or chronic infections, such antibodies process lead to complete gastric atrophy of the fundus and corpus do not occur. (K. Krohn, personal experience). The target molecule for the parietal cell antibodies were shown GONADAL FUNCTIONS to be the sodium-potassium channel molecule of the parietal cells Autoimmune oophoritis is responsible for an ovarian insufficiency on corpus and fundal part of the stomach (Karlsson et al., 1988). that may be dramatic for female patients as insufficiency starting In antral gastritis, the antigen are the gastrin-producing cells in teenagers and young adults. Patients may have either a pri- (Uibo and Krohn, 1984). mary with no or arrested puberty. Other patients Pernicious anemia is the end stage of the gastric immunological develop premature menopause. The diagnosis is confirmed by sex- destruction, caused partly by the lack of IF, that in addition to the ual hormones status; elevated plasma levels of follicle stimulating hydrochloric acid is the main product of parietal cells, but also by hormone (FSH) and luteinizing hormone (LH) and low estrogen the autoantibodies recognizing this -binding protein. levels. Autoantibodies against side-chain cleavage enzyme have There are two types of antibodies to IF: one blocking vitamin been related to ovarian insufficiency (Soderbergh et al., 2004) and B12 binding to IF and another type, binding to the IF molecule also steroidogenic enzymes antibodies against cytochrome p450 without interfering with vitamin B12 binding (Toh et al., 1997). 21-hydroxylase (CYP21A2), cytochrome p450 17α-hydroxylase Both antibody types prevent the binding of IF to its receptor on (CYP17), and cytochrome p450 side-chain cleavage enzyme the ileal mucosa and subsequent translocation of the vitamin B12 (CYP11A1). from ileum to circulation. In female patients, hormonal substitution by estrogen needs to be initiated during puberty. It is strongly advised not to delay ADDISON’S DISEASE . In case of hypogonadism, embryo donation has been Adrenocortical failure or AD, described by Thomas Addison in tried with success. the ninetieth century, is considered one of the three hallmarks In males, testicular failure is less common and occurs later. of APECED, but it occurs also as a solitary disease, or as part of The prevalence of hypogonadism in males is three times lower the APS-2 complex. Today, in western word, most cases of AD (8–28%) than in females (35–70%; Perheentupa, 2006). It leads to are caused by autoimmunity, but adrenal cortical destruction and clinical hypogonadism or isolated azoospermia (Husebye et al., subsequent cortical failure can be caused by several other factors, 2009). It has been hypothesized that the blood–testis barrier notably by secondary tuberculosis or other chronic infections. In protects the Leydig cells from an autoimmune attack. However,

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the physiopathogenic link between circulating autoantibodies and splenic atrophy could thus be disturbance of differentiation, due hypogonadism is far from being clear. The two steroidogenic to lack of AIRE expression. enzymes, p450scc and p450c17, are the main antigens in gonadal Various types of GI manifestations are common in APECED failure linked to APECED, but other potential antigenic targets patients. These include chronic diarrhea that can be related to HP, have been identified such as testis-expressed protein TSGA10 severe constipation. Intestinal infection by candida and giardia (Reimand et al., 2008). However, despite autoantibodies directed especially, pancreatic insufficiency and autoimmune enteropa- against TSGA10 in 7% of the APECED patients, no correlation thy. Several autoreactive circulating antibodies directed toward could be found with gonadal failure (Reimand et al., 2008). One intestinal components have been described. Ekwall et al. (1998) should not forget that the origin of gonadal dysfunction may identified TPH as an intestinal autoantigen in APECED patients. be related to an authentic-specific autoimmune attack but also TPH is expressed in serotonin-producing cells in the central ner- be related to other hormonal dysfunction such as AD, pituitary vous system and in the intestine. In their series of 80 patients, they insufficiency, dysthyroidism, or diabetes for instance. Besides, were able to relate “GI symptoms” to the presence of circulating Schaller et al. (2008) suggested that lack of AIRE might affect fer- TPH antibodies and also to the total absence of enterochromaf- tility by disrupting scheduled apoptosis of testicular germ cells. fin cells in the mucosa of small bowel. These enteroendocrine In this respect, the recent hypothesis presented by Matsumoto cells (EECs) are scattered through the intestinal mucosa, from the (2011) that the function of AIRE in thymus would not be in gastric body and antrum to the rectum. They play a key role in the regulation of transcription but rather in the development growth of the gut, blood flow, motility, secretion of pancreatic and differentiation of the medullary epithelial cells is of primary enzymes, bile, and bicarbonate-rich fluid (Posovszky et al., 2012). interest TPH antibodies were found in 89% of the APECED patients with GI symptoms and in 34% of those without (Ekwall et al., 1998). OTHER ENDOCRINE DISORDERS Antibodies can precede clinical symptoms (Ekwall et al., 1998). Various other endocrine disorders have been described such dia- Conversely, TPH autoantibodies are absent in other inflamma- betes type I mellitus, hypothyroidism, and pituitary failure, the tory or autoimmune intestinal diseases. Additionally, Sköldberg latter diagnosed by a growth hormone deficiency. The diagno- et al. (2003) identified also autoantibodies against histidine decar- sis and management of these conditions does not differ from boxylase expressed by EEC – like cells in the gastric mucosa. It the standard guidelines for each disorder separately (Perheentupa, is noteworthy, that it is not a routine procedure to perform EECs 2002; Betterle and Zanchetta, 2003; Husebye et al., 2009). staining on intestinal biopsies in case of diarrhea or malabsorption, as stressed by Ohsie et al. (2009). Besides, several studies showed OTHER NON-ENDOCRINE DISORDERS repeatedly that EECs were lacking in the intestinal mucosa and Enamel hypoplasia affect mainly permanent teeth (Perheentupa, were related to chronic diarrhea (Padeh et al., 1997; Ward et al., 2006), but also deciduous teeth (Pavlic and Waltimo-Sirén, 2009). 1999; Oliva-Hemker et al., 2006; Posovszky et al., 2012). Pavlic and Waltimo-Sirén (2009) recently suggested that an inad- Tubulo-interstitial nephritis, life-threatening autoimmune equate process of enamel formation might affect all ameloblasts and other rare manifestations have also been in phase. Ameloblasts have an epithelial origin with parenchy- reported in APECED (Perheentupa, 2002, 2006; Betterle and mal cells of endocrine origin. It is speculated that ameloblasts Zanchetta, 2003; Husebye et al., 2009). The main identified or secreted protein in the extracellular matrix may be the tar- autoantibodies are summarized in Table 2. get of autoantibodies leading to hypoplasia. Thereby, APECED would be the first model of dental hard tissue autoimmune disease TREATMENT (Pavlic and Waltimo-Sirén, 2009). Management of APECED relies in education of the patients to Ocular manifestations affect 25% of the patients and include know his disease, education of the local physician, and the mainly keratitis that can lead to blindness. It is assumed that the knowledge that new components of the disease may develop origin of keratitis is the result of autoimmunity against corneal during life. Psychological support is strongly recommended as epithelium (Merenmies and Tarkkanen, 2000; Perheentupa, 2006). this disease impairs greatly the quality of life of the patients However, to our knowledge no specific antibodies have been iden- (Perheentupa, 2006). Except candidiasis treatment that has been tified in APECED patients. Only antibodies against OBP1 have explained previously, treatment of APECED relies mostly on hor- been found in the AIRE mouse model against lacrimal glands mone replacement therapy according to affected organs (thyroid, (DeVoss et al., 2010). parathyroid, pancreas, etc.). In some rare and potentially lethal Hyposplenism or asplenia is often diagnosed upon the develop- situations, however, patients may require treatment ment of thrombocytosis, circulating Howell–Jolly bodies, abdomi- in association with immunosuppressive therapies. These rare sit- nal ultrasound imaging or in case of severe S. pneumoniae infection uations include autoimmune hepatitis, especially its fulminant (Pollak et al.,2009). Destruction of the spleen in APECED has been form, which may be lethal and therefore prompt immunosup- related to an autoimmune attack against the spleen (Perheentupa, pressive therapy is needed (Obermayer-Straub et al., 2001). The 2002, 2006; Betterle and Zanchetta, 2003; Husebye et al., 2009) same is true for interstitial nephritis and bronchiolitis in associ- although the exact mechanism remains obscure. Again, the mecha- ation to APECED. Immunosuppressive therapies have been also nisms proposed by Schaller et al. (2008) and by Matsumoto (2011) proposed in case of severe intestinal malabsorption with efficacy are of interest, as AIRE expression has also been described in lym- (Padeh et al., 1997; Ward et al., 1999). Very recently, Rituximab, phoid tissue and skin. A speculative hypothesis to the evolution of a chimeric monoclonal antibody targeting B cell lymphocytes

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Table 2 | Main identified target of autoimmune antibodies in APECED et al. (2006), describing high-titer antibodies to several type I patients. interferons in practically all APECED patients studied. This anti- interferon response was exceptionally strong, since serum titers Diagnosis Main identified circulating autoantibodies up to 1:1,000,000, and clearly exceeding the titers seen against organ-specific antigens, were found. α Addison’s disease 21 hydroxylase, 17 hydroxylase Furthermore, high-titer antibodies were seen against the Side-chain cleavage enzyme antibodies two main mediators secreted by Th17 cells, interleukin-17 and (or steroid cell antibodies) interleukin-22 (IL-17 and IL-22). Responses with lower titers were Hypoparathyroidism NALP5, Ca2+ sensing receptor occasionally seen against other , too. In our own as Hypothyroidism Thyroperoxydase yet unpublished observations we have detected occasional high- titer responses against several other interleukins and chemokines, Thyroglobuline as well, but in contrast to the aforementioned responses, these α Hypogonadism 17 hydroxylase responses are not characteristic to all APECED patients but rather Side-chain cleavage enzyme antibodies occur occasionally in only a few patients. (or steroid cell antibodies) The significance of these novel findings are still unclear, but Diabetes type I Glutamic acid decarboxylase 65-kDa isoform some information concerning the role of IL-17/IL-22 antibod- (GAD65) ies in the chronic candida infections, characteristic for APECED, Insulin has been obtained. Th17 cells secrete IL-17 and IL-22, which are cytokines with potent antifungal properties (Engelhardt and Tyrosine phosphatase (IA2) Grimbacher, 2012) and the occurrence of autoantibodies against Pituitary insufficiency Tudor Domain containing protein 6 (TDRD6) IL-17/IL-22 were reported to closely correlate to the presence of Atrophic gastritis/ Intrinsic factor, gastric parietal cell candida infection (Kisand et al.,2011; Engelhardt and Grimbacher, Biermer’s disease 2012). However, recent evidence points to a new interaction Intestine Glutamic acid decarboxylase 65-kDa isoform between AIRE and dectin-1, a pattern-recognition receptor that (GAD65) is important in antifungal innate immunity. Pedroza et al. (2012) recently showed that AIRE participates in the dectin-1 signaling Histidine decarboxylase pathway, and thus, missing AIRE activity could contribute to fun- Tryptophan hydroxylase gal susceptibility through this pathway. Dectin-1 is expressed on Autoimmune hepatitis Aromatic L-amino acid decarboxylase (AADC) and was recently shown to induce a non-canonical Cytochrome P450 1A2 caspase-8 inflammasome in response to fungal and mycobac- Cytochrome P450 2A6 terial infection (Gringhuis et al., 2012). The activation of the Cytochrome P450 1A1 dectin signaling pathway also leads to expression of IL-17 and Cytochrome P450 2B6 22 and defensins, however. Besides, other mechanisms such Dominant-negative mutations in STAT3, gain of mutation of Vitiligo Transcription factors: SOX 9, SOX 10, aromatic STAT1, mutations in IL-17F and IL-17R may be alternate causes L-amino acid decarboxylase (AADC) of CMC (Engelhardt and Grimbacher, 2012). Alopecia areata Tyrosine hydroxylase The significance of the antibody response toward interferons Nephropathy Antibody against tubular basement membrane and other cytokines is presently also unclear. One could speculate (Hannigan et al., 1996) that some of these antibodies against type I interferons as well as Pulmonary disease Potassium channel regulatory protein (KCNRG) reacting with IL-17 and IL-22 might have a protective function. As pointed out by Waterfield and Anderson (2011), antibodies to Eye OBP1* type I interferons do not seem to lead to increased susceptibility Non-tissue specific** IFN-α, IFN-β, IFN-ω, IL-22, IL-17F,IL-17A to viral infections. This resistance might be due to redundancy

−/− and it has to be seen whether this anti-interferon response is *Identified in a mouse model AIRE . directed only toward certain members of the interferon family. **Main non-tissue-specific antibodies according to Kisand et al. (2011). While Th17 cell response and the release of soluble IL-17 and IL-22 are evidently necessary for the defense against mucocuta- expressive CD20 has been successfully used in a young patient with neous candida infection, the same cytokines have a role in the bronchiolitis (Popler et al., 2012). The rationale for Rituximab use development of psoriasis. Similarly, interferons are known to be in APECED is supported by the presence of B cell infiltrates in the involved in the pathogenesis of several conditions, and one such affected organs (Gavanescu et al., 2008). chronic ailment is the autoimmune diseases belonging to the sys- temic lupus erythematosus (SLE) complex. Anti-interferon alpha AUTOANTIBODIES TOWARD INTERFERONS AND CYTOKINES antibodies are currently being tested as a therapeutic mean against At the beginning of this millennium, the antibody responses to SLE (Merrill et al., 2011). In order to be able to find out if the the main target organs affected in APECED, and the responsi- antibodies against interferons and other cytokines could have a ble target antigens were fairly well characterized. A new period protective role in APECED, large APECED patient cohorts have to in APECED studies started along the publication by Meager be studied in order to find out whether, e.g., psoriasis and SLE are

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significantly less common in APECED patients than in the general that in APECED, the primary defect outside thymus, where the population. autoreactive T cells are not destroyed, would be the cell destruction The reason for the antibody response toward soluble immune of the endocrine organs by cell-mediated immune response, fol- mediators is still unclear, and we do not yet know what exactly lowed by release of cellular components taken up by professional elicits them and thus, only speculative scenarios can be presented. antigen presenting cells and further stimulating the activation of It is conceivable to hypothesize, however, that the tissue destruc- CD4+ Th-cells and finally resulting in an autoantibody response tion preceding the failure of the endocrine organs may have a to these organ-specific antigens. However, simultaneous overex- role. Tissue destruction, be it caused by trauma, viral infec- pression of TRIMs and subsequent up-regulation of a variety of tion or autoimmune attack, would probably lead not only to soluble mediators of immune response and inflammation, such as the release of potential tissue-specific autoantigens and thus, to interferons and members of the IL-1 family would lead to autoan- autoantibody formation against these proteins, but could also tibody formation also against these cytokines. Lastly, one reason lead to an inflammatory response and production of several for the break of tolerance to immune mediators, and subsequent mediators of inflammation. One key group of molecules in this production of autoantibodies could be related the fact that AIRE respect is the acute phase proteins, notably those belonging to the expression seems to occur, in addition to thymic epithelial cells also IL-1 group. outside thymus, notably in dendritic cells, that normally express It is generally believed that the destruction of the endocrine also such mediators (Heath and Carbone, 2009) organs in APECED is caused by the autoreactive CD8+ cyto- The consequences of such cytokine-directed antibody response toxic T cells, although definitive evidence for this mechanism is are still an open question. In case of the Th17 type interleukins still lacking (Betterle and Zanchetta, 2003; Moraes-Vasconcelos (IL-17 and IL-22) there is convincing evidence that such antibod- et al., 2008). This hypothesis is reinforced by the examination of ies are linked to the CMC. However, at least in some cases, the microscopic examinations of samples, sometimes obtained post- antibodies may have a balancing, down-regulating effect on the mortem. Indeed, parathyroid, adrenal glands, or pathology expression of the corresponding biologically active molecules but disclosed also atrophy and lymphocytic infiltration that suggest also, by regulating the immune response to target organs. Thus, lymphocytic aggression of the organs leading to atrophy and it is possible to presume, that especially the antibodies to type I dysfunction (Betterle and Zanchetta, 2003). This is also stressed, interferons might have a protective effect, as some chronic immune indirectly, by the analysis of the AIRE-deficient mouse model, who diseases, such as psoriasis and SLE, are rare or non-existing among develop also a lymphocytic infiltration in some inner organs along APECED patients. with atrophy (Ramsey et al., 2002). However, cell destruction caused by an immune response CELL-MEDIATED IMMUNE RESPONSES against the endocrine organ would in fact lead to a similar sit- Although it is now a generally accepted view that the consequence uation that is thought to happen in viral infections. In fact, several of the AIRE defect in APECED will lead to the escape of the poten- autoimmune diseases, such as diabetes type I or chronic autoim- tially autoreactive T cells, there is in fact rather little direct evidence mune liver diseases are thought to be a consequence of preceding to show that the tissue destruction in the endocrine organs affected viral infection: enterovirus infection in the case of diabetes type in APECED is caused by cytotoxic CD8+ T cells. Furthermore, I and hepatitis B in the case of chronic active hepatitis. In viral most studies describing the phenotype of the lymphocytes infil- infections, a specific group of intracellular regulatory molecules, trating affected organs is not from APECED patients directly, but TRIMs (tripartite motif-containing proteins), have been shown to from patients suffering of solitary lesions that are similar to the have a key role in eliciting an autoimmune or auto inflammatory ones seen in APECED, such as solitary AD or diabetes. However, consequence (Jefferies et al., 2011). the solitary endocrine diseases, such as isolated thyroid disease The TRIM protein family is a form of RING domain containing or AD are remarkably similar in their clinical picture as well as E3 ligases and they exert a variety of biological functions, related immunological findings as those of APECED. Thus, in solitary to immunity and inflammation (Jefferies et al., 2011). Specifically, AD and in APECED with adrenocortical failure, autoantibodies of the more than 20 different TRIM proteins, some seem to up- recognize the p450c21 steroidogenic enzyme. Interestingly, in this regulate the expression of type I interferons and proinflammatory disease complex CD8+ T cells that reach against specific T cell cytokines, notable interleukin-1beta (IL-1beta). Furthermore, the epitopes in p450c21 has been demonstrated (Bratland et al., 2009; same mediators of immune response and inflammation are in Rottembourg et al., 2010) Likewise, in thyroid diseases, thyroglob- some cases known to up-regulate the expression of TRIMs. Thus, ulin and thyroid peroxidase are recognized by the autoantibodies, a vicious circle can theoretically occur and this in turn could irrespective if the condition is occurring alone, in association of lead to autoimmunity. So far, overexpression of TRIMs, or an APS-2 or as part of the APECED complex. The similar syner- autoimmune response toward them, has been shown to be linked gism in terms of the nature of autoantigens occurs in chronic to autoimmune and autoinflammatory processes in Sjögren’s immunological liver diseases, too. syndrome or rheumatoid arthritis (Jefferies et al., 2011). In chronic aggressive hepatitis the lymphocytic infiltrating cell Presently, we have no information how the occurrence of population has been shown to be of the CD8+ lineage (Si et al., autoantibodies toward the interferons and other mediators of 1984). In a murine model of Graves’ disease, the CD8+ cell immune response might affect the aforementioned vicious circle, population contains also the recently identified CD8+CD122+ but it is conceivable to speculate that such an antibody response T cells that are functionally similar to the CD4+CD25+ regulatory could have an balancing effect. One could thus form a hypothesis, T cells (Ryan et al., 2005). Furthermore, studies in thyroid and

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other affected organs show that one of the main cell population therefore impaired. Data concerning the CD8+ regulatory T cells in the lymphocytic infiltrate are in fact the CD4+ Th17 cells that in APECED patients is missing, however. secrete as effector molecules, the cytokines IL-17 and IL-22. In The finding that the regulatory T cell population in APECED is experimental autoimmune diseases, the balance between the Th17 functionally defective and that the expression of the key molecule effector cells and the two regulatory T cells, CD8+CD122+ and for Treg function, the FOXP3 is impaired, is consistent with clini- CD4+CD25+, seems to regulate both the occurrence and severity cal findings in IPEX syndrome, caused by a defect in the function of tissue destruction and functional failure. of the FOXP3 gene. However, it should be noted that the effect of There could thus be two distinct mechanisms operating in the FOXP3 mutations in Treg population also in the IPEX patients is pathogenesis of autoimmunity in the endocrinopathies: one medi- highly variable. Also, in contrast to APECED there seems to be a ated by soluble effector molecules, such as IL-17 and IL-22 as genotype–phenotype correlation in IPEX, as different mutations well as type I interferons, and an other one mediated by effector are associated in variable clinical picture, that show differences T cells, which are either of the CD8+ CTL cell or of the Th17 effec- in severity as well in the types of clinical components that are tor cell lineage. To counteract these, again two distinct biological present (Torgerson et al., 2007; d’Hennezel et al., 2009) A consis- processes would occur: the production of autoantibodies and sec- tent finding in IPEX is however the inability of the CD4+CD25 ondly, the emergency of the regulatory T cells. As to the regulatory high Tregs to suppress the function of autologous effector T cells T cell response, it is to note that one key immunological failure in (Bacchetta et al., 2006). There are, thus, several differences in the APECED, is the dysregulation of the Treg cell maturation (Ryan clinical picture of APECED and IPEX, but both conditions show et al., 2005; Kekäläinen et al., 2007; Saitoh et al., 2007; Laakso et al., clear immune destruction of at least some endocrine organs. Both 2010, 2011; Wolff et al., 2010). conditions also share some similarities in the GI symptoms. In normal thymus, Treg maturation follows a prepro- The proposed hypothesis that in APECED both tissue destruc- grammed scheme, and the immature CD8+CD4+FOXP3+ tive mechanisms and controlling regulatory mechanisms exist seems to be prone to apoptosis, whereas the more mature raises a question whether APECED could be treated or even form CD4+CD8−FOXP3+ cells form the active Treg popula- cured by immunological manipulations. To find an answer for tion (Lehtoviita et al., 2009). According to Endharti et al. (2011) this question is one of the further challenges for APECED research the CD8+CXCR3+ Tregs in humans are functionally similar to murine CD8+CD122+ Tregs. Furthermore, in APECED patients ACKNOWLEDGMENTS the recent thymic emigrant (RTE) pool of Treg cells shift to the acti- The study was financially supported by the European Science vated pool and the RTE reservoir is depleted. Most importantly, Foundation (ESF) and the Sigrid Juselius foundation (to Nicolas in APECED patients these cells express less FOXP3 than in the Kluger). We thank Professors Jaakko Perheentupa, Adrian Hayday, healthy controls (Laakso et al., 2010). Thus, in APECED the newly and Pärt Peterson, as well as Drs Kai Kisand, Annalisa Macagno, formed Treg cells have a developmental defect and their function is and Edward Stuart for stimulating discussions.

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Utility of KCNRG autoantibodies as Saitoh, O., Abiru, N., Nakahara, M., and Uibo, R., Aavik, E., Peterson, P., Per- J. Med. Genet. 29, 824–826. a marker of active pulmonary dis- Nagayama,Y.(2007). CD8+CD122+ heentupa, J., Aranko, S., Pelkonen, R., ease and successful treatment with T cells, a newly identified regulatory and Krohn, K. J. (1994a). Autoanti- rituximab. Pediatr. Pulmonol. 47, T subset, negatively regulate Graves’ bodies to cytochrome P450 enzymes Conflict of Interest Statement: The 84–87. hyperthyroidism in a murine model. P450scc, P450c17, and P450c21 in authors declare that the research was Posovszky, C., Lahr, G., von Schnurbein, 148, 6040–6046. autoimmune polyglandular disease conducted in the absence of any com- J., Buderus, S., Findeisen, A., Schaller, C. E., Wang, C. L., Beck- types I and II and in isolated Addi- mercial or financial relationships that Schröder, C., Schütz, C., Schulz, Engeser, G., Goss, L., Scott, H. son’s disease. J. Clin. Endocrinol. could be construed as a potential con- A, Debatin, K. M., Wabitsch, M., and S., Anderson, M. S., and Wabl, Metab. 78, 323–328. flict of interest.

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Received: 24 April 2012; accepted: 15 July Immun. 3:232. doi: 10.3389/fimmu. Copyright © 2012 Kluger, Ranki and reproduction in other forums, pro- 2012; published online: 02 August 2012. 2012.00232 Krohn. This is an open-access arti- vided the original authors and source Citation: Kluger N, Ranki A and Krohn K This article was submitted to Frontiers in cle distributed under the terms of the are credited and subject to any copy- (2012) APECED: is this a model for fail- Primary Immunodeficiencies, a specialty Creative Commons Attribution License, right notices concerning any third-party ure of T cell and B cell tolerance? Front. of Frontiers in Immunology. which permits use, distribution and graphics etc.

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“fimmu-03-00232” — 2012/7/31 — 17:51 — page 12 — #12 PERSPECTIVE ARTICLE published: 18 July 2012 doi: 10.3389/fimmu.2012.00210 Pathogenesis of autoimmunity in common variable immunodeficiency

Klaus Warnatz1,2* and Reinhard E. Voll 1,2

1 Centre of Chronic Immunodeficiency, University Medical Center Freiburg, University of Freiburg, Freiburg, Germany 2 Division of and Clinical Immunology, University Medical Center Freiburg, Freiburg, Germany

Edited by: Common variable immunodeficiency (CVID) presents in up to 25% of patients with autoim- Luigi Daniele Notarangelo, Harvard mune (AI) manifestations. Given the frequency and early onset in some patients with CVID, Medical School, USA AI dysregulation seems to be an integral part of the immunodeficiency. Antibody-mediated Reviewed by: AI cytopenias, most often affecting erythrocytes and platelets make up over 50% of these Mario Abinun, Newcastle upon Tyne Hospitals NHS Foundation Trust, UK patients.This seems to be distinct from mainly cell-mediated organ-specific autoimmunity. Rupali Das, Children’s Hospital of Some patients present like patients with AI lymphoproliferative syndrome. Interestingly, in Philadelphia, USA the majority of patients with AI cytopenias the immunological examination reveals a dys- *Correspondence: regulated B andT cell homeostasis. These phenotypic changes are associated with altered Klaus Warnatz, Centre of Chronic signaling through the antigen receptor which may well be a potential risk factor for disturbed Immunodeficiency, University Medical Center Freiburg, University immune tolerance as has been seen in STIM1 deficiency. In addition, elevated B cell- of Freiburg, Breisacher Str. 117, activating factor serum levels in CVID patients may contribute to survival of autoreactive 79106 Freiburg, Germany. B cells. Of all genetic defects associated with CVID certain alterations in TACI, CD19, and e-mail: klaus.warnatz@uniklinik- freiburg.de CD81 deficiency have most often been associated with AI manifestations. In conclusion, autoimmunity in CVID offers opportunities to gain insights into general mechanisms of human autoimmunity.

Keywords: autoimmune cytopenia, autoimmunity, CD21low B cells, common variable immunodeficiency, hypogammaglobulinemia

Autoimmunity is an integral part of immune dysregulation in together in most of the CVID patients remains obscure. Other a quarter of patients with common variable immunodeficiency causes of ALPS and ALPS-related disorders have not been excluded (CVID), often presenting as the first manifestation of the dis- systematically in AI-CVID. ease (Agarwal and Cunningham-Rundles, 2009). In recent years Other immunodeficiencies strongly associated with AI mani- analyses of the immune disturbances have revealed complex dys- festations comprise immune dysregulation, polyendocrinopathy, regulations of the immune system. In parallel, progress in our enteropathy X-linked (IPEX) syndrome, autoimmune polyen- comprehension of the pathogenesis of disorders docrine syndrome type 1, combined immunodeficiencies (CID) like systemic lupus erythematosus (SLE) allows for comparison of including hypomorphic severe (S)CID variants (Liston et al., common roots of human autoimmune (AI) disorders. 2008), both calcium channelopathies, Wiskott–Aldrich syndrome This perspective article is an attempt to summarize the factors (WAS), DiGeorge syndrome, Good syndrome, activation-induced which contribute to autoimmunity in CVID. deaminase (AID) deficiency, CD25 deficiency, Stat5b deficiency, Autoimmune cytopenias are the most common AI manifesta- and hair dysplasia (Al-Herz et al., 2011). tions in CVID and the focus of this article. In the context of distinct Most of these immunodeficiencies are associated with (i) dis- associated alterations of the cellular immune system AI cytope- turbed T cell homeostasis, (ii) altered antigen receptor, or (iii) nias appear to be a separate manifestation from organ-specific altered cytokine signaling. Aspects relevant in patients with CVID autoimmunity in CVID (Boileau et al.,2011; Cheng and Anderson, shall be discussed in the following sections. 2012). The presentation of AI-CVID patients resembles patients with autoimmune lymphoproliferative syndrome (ALPS) with the DISTURBED T CELL HOMEOSTASIS IN AI-CVID coincidence of lymphoproliferation and AI cytopenias (Seve et al., Disturbed T cell homeostasis is a common contributing fac- 2008; Wehr et al., 2008; Boileau et al., 2011). While none of the cel- tor to the development of autoimmunity in different forms of lular markers, such as increased double negative T cells or reduced monogenic primary immunodeficiency disorders (PIDs). Several switched memory B cells, helped to distinguish AI-CVID from features of disturbed cell homeostasis are also present in CVID. FAS-ALPS,increased serum levels of soluble Fas ligand, interleukin Lymphopenia affects mostly CD4 T cells and especially naïve CD4 (IL) 10, and vitamin B12 allowed a distinction between FAS-ALPS T cells, while CD8 T cells become relatively expanded (Giovannetti patients and AI-CVID to be made (Rensing-Ehl et al., 2010). None et al., 2007). Both CD4 and CD8 T cells are activated as determined of the tested CVID patients carried a genomic or somatic muta- by the expression of activation markers and Ki67. Thymic out- tion in FAS, rendering FAS-ALPS a differential diagnosis. Thus, put was decreased, but Ki67 expression was particularly strong the reason that lymphoproliferation and autoimmunity are seen in naïve and central memory T cells, suggesting homeostatic

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proliferation as described for other immunodeficiency models with or without a disturbed TCR signal (Picard et al., 2009; (Cassani et al., 2010). In addition, the Vβ repertoire of CD4 T cells Sauer et al., 2012), cytokine disturbance (Setoguchi et al., 2005), had contracted. These changes are well known to be associated and even persistent CD4 lymphopenia itself (Matsuoka et al., with an increased risk of autoimmunity as previously demon- 2010) might contribute to the reduction in regulatory T cells. strated in murine models and human AI disease (Datta and Interestingly, even ICOS deficiency disturbs maintenance and Sarvetnick, 2009). function of regulatory T cells (Kornete et al., 2012), thus poten- The severe reduction in naïve CD4 T cells in CVID has been sug- tially rendering regulatory T cell deficiency a crucial element in gested as a criterion for the diagnosis of late-onset CID (LOCID; AI dysregulation which is also common to different forms of Malphettes et al., 2009) for resembling the immunological and immunodeficiency. clinical phenotype of patients with hypomorphic SCID muta- tions (Liston et al., 2008; Cassani et al., 2010; De Ravin et al., DISTURBED B CELL HOMEOSTASIS IN AI-CVID 2010). Interestingly, the association of CD4 lymphopenia in pri- B cell homeostasis is also disturbed in CVID patients. Therefore, mary immunodeficiency seems to be stronger with granulomatous reduced switched development and the expansion inflammatory disease than AI cytopenias (Schuetz et al., 2008; of activated CD21low B cells are associated with the manifestation Mouillot et al., 2010). IL-7, which has a key role in the expansion of AI-CVID (Warnatz et al., 2002; Sanchez-Ramon et al., 2008; of autoreactive T cell clones in the lymphopenic host, was also Isnardi et al., 2010; Boileau et al., 2011). CD21low B cells con- found to be elevated in a subgroup of CVID patients (Holm et al., tain a high proportion of autoreactive clones (Rakhmanov et al., 2005). Though increased IL-7 levels were not associated with T cell 2009; Isnardi et al., 2010) suggesting a disturbed selection of the lymphopenia, they nevertheless correlated with a more frequent B cell repertoire. This may involve defects in central selection incidence of autoimmunity. The regular feedback mechanism of for some (Isnardi et al., 2010), but not all patients (Rakhmanov IL-7 regulation seemed to fail in the small group of AI-CVID et al., 2010). Several factors have been identified as interfering patients examined. The production of several other cytokines with B cell selection. Firstly, the signal strength of the BCR itself including IL-2, interferon (IFN)-γ, IL-4, and TNFα is altered in determines the outcome during selection (Khan, 2009). Several some CVID patients, but none of the reported alterations have mouse models have demonstrated that alterations in the signal- been examined for their role in eliciting autoimmunity (Fischer ing machinery (Cornall and Goodnow, 1998; Wang and Clark, et al., 1994; Fritsch et al., 1994; Mullighan et al., 1997). Testing the 2003) and the balance between co-stimulatory (Tedder et al., 1997) role of specific cytokines in this setting will be of great interest as and inhibitory co-receptors (Cornall et al., 1998) determine the it is likely to reveal potential therapeutic targets. counter-selection of AI B cell clones. In CVID patients dis- Skewing of CD8 T cells is often more prominent than that turbed antigen receptor signaling was described and is discussed of CD4 T cells (Giovannetti et al., 2007). Cytomegalovirus (CMV) below. causes associated with terminal differentiation Given the negative feedback loop of immune complexes on of CD8 effector T cells which results in a skewing of the repertoire. B cells and plasma cells via the inhibitory receptors (Seite et al., In CVID this phenomenon was exaggerated (Kuntz et al., 2011). 2010; Baerenwaldt et al., 2011) it is intriguing to speculate as to A chronic viral infection is therefore a potential trigger for the whether low serum IgG by itself may contribute to antibody- clinical manifestation of AI disease in a disturbed immune system mediated AI cytopenias as one of the first manifestations in (Marashi et al., 2011). AI-CVID. Signaling by FcγRIIB inhibits B cell activation and Selection, activation, and differentiation of T cells in CVID can even induce apoptosis in plasma cells (Xiang et al., 2007). may also be affected by an impaired response of the T cell recep- Additionally, a lack of inhibition of monocytes/macrophages tor after stimulation (Fischer et al., 1994; Boncristiano et al., 2000; by FcγRIIB may foster overwhelming inflammatory responses Paccani et al., 2005). However, to date, the published investiga- and granuloma formation, a serious clinical problem seen in tions neither report an underlying genetic defect nor a correlation a subset of AI-CVID patients. Lupus-like disease in FcγRIIB- between altered T cell receptor signaling and a higher preva- deficient C57BL/6 mice (Bolland and Ravetch, 2000)aswellas lence of autoimmunity. Currently, the only intrinsic T cell defect the increased risk of SLE in homozygous carriers of the dysfunc- which causes CVID was found in a total of 11 patients with defi- tional FcγRIIB I232T variant (Floto et al., 2005) clearly indicate ciency of the inducible costimulator (ICOS; Warnatz et al., 2006; a crucial role for this inhibitory receptor in the maintenance Takahashi et al., 2009). Only one of the original nine European of humoral tolerance. This hypothesis is supported by the fact patients presented with AI neutropenia, whereas AI manifestations that in most CVID patients the initiation of immunoglobulin were more prominent in the two Japanese patients presenting with replacement leads to an amelioration of the bouts of AI-mediated (rheumatoid) arthritis, inflammatory bowel disease, interstitial cytopenias. pneumonitis, and psoriasis. The other major factors, which contribute to B cell-mediated Finally, many reports have described reduced numbers of cir- autoimmunity, are related to survival signals during selection culating regulatory T cells in CVID, especially affecting Freiburg (Cancro, 2004). For B cells, overexpression of B cell-activating Ia patients with reduced switched memory B cells and expan- factor (BAFF) causes increased survival of autoreactive B cells and sion of CD21low B cells (see below; Fevang et al., 2007; Genre overt autoimmunity (Mackay et al., 1999; Thien et al., 2004). It is et al., 2009; Horn et al., 2009; Melo et al., 2009; Yu et al., 2009; noteworthy that most CVID patients present with elevated BAFF Arumugakani et al., 2010; Mouillot et al., 2010). Several of levels (Kreuzaler et al., 2012). Currently it is unknown whether the factors mentioned above, such as a CID-like phenotype elevated BAFF levels sustain the expansion of CD21low B cells in

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CVID. The number of circulating CD21low B cells increases in to mutations in the WAS protein (Zhang et al., 1999). Interestingly, other AI diseases, such as SLE (Wehr et al., 2004), rheumatoid WASP deficiency also leads to increased AI disease associated with arthritis (Isnardi et al., 2010), and cryoglobulinemia (Terrier et al., decreased CD27+ memory B cells and increased CD21low Bcells 2011), supporting an association with autoimmunity. In contrast (Park et al., 2005). Although WASP deficiency affects both T and to SLE, where switched memory B cells are relatively expanded B cell receptor signaling, B cell-intrinsic defects clearly contribute and active disease is associated with expansion of circulating plas- to autoimmunity in WAS (Recher et al., 2012). As indicated above, mablasts (Dorner and Lipsky, 2004), AI-CVID has a more severe previous reports have found disturbed TCR-induced calcium sig- reduction in the number of switched memory B cells when com- nals (Fischer et al., 1996) in 40–50% of CVID patients but a link pared to other CVID patients. This could represent a disturbed to immune dysregulation in the identified patients has not been peripheral differentiation and selection. Increased autoimmu- established. nity associated with poor germinal center function has also been observed in deficiency of the AID (Hase et al., 2008), but no abnor- ALTERED TYPE I INTERFERON SIGNAL malities of AID expression or function have been described in IN AUTOIMMUNE CVID CVID at this point. Cytokines have been implicated in AI dysregulation. Type I IFNs Of all the genetic mutations which are associated with CVID, are thought to be particularly important as (i) AI reactions are AI manifestations are most common in TACI-deficiency [18/50 induced in patients after treatment with type I IFNs, (ii) the (36%) vs 112/490 (23%) in wt TACI CVID; Salzer et al., 2009]. In IFN signature is increased in patients with SLE, and (iii) some particular, heterozygous C104R mutations seem to effect a pre- chronic viral infections are associated with autoimmunity (Hall disposition for autoimmunity (11/20 patients, 55%; Salzer et al., and Rosen, 2010). The mechanisms are manifold and include 2009). While partial TACI signals in a heterozygous state may con- induction of dendritic cell (DC) maturation and increased BAFF tribute to the survival of autoreactive B cells, a formal proof of this production, a positive feed back loop in toll-like-receptors (TLR) hypothesis is still missing. AI manifestations including glomeru- 7 and 9 signaling leading to class switched antibody production lonephritis and vasculitis (interestingly with deposits of IgA) as (Hall and Rosen, 2010). well as AI thrombocytopenia (AI-TP) have also been described for Type I IFNs have not been well examined in CVID patients. CD19 and CD81 deficiency, and are possibly related to the dis- There exists only a single report of increased type I IFN production turbed antigen receptor signal in these patients (see also below; in CVID patients (Strannegard et al., 1987); others have detected van Zelm et al., 2006, 2010; Vince et al., 2011). The other B cell- increased MxA expression as a marker of IFN exposure in leuko- intrinsic genetic defects associated with CVID (BAFF-R, CD20, cytes of only 2/13 CVID patients (Rump et al., 1995). So far no CD21) have not been reported with AI manifestations (Warnatz attempt to correlate in CVID IFN expression to AI manifestations et al., 2009; Kuijpers et al., 2010; Thiel et al., 2011, but to date only has been made. single patients have been described for each defect, thus precluding Type I IFN expression and the induction of AI reactions is definite conclusions. closely linked to the activation of TLRs on plasmacytoid DCs In recent years, a B cell population producing IL10 has (pDCs) and B cells (Green and Marshak-Rothstein, 2011). Dif- been described as regulatory B cells (Mauri and Bosma, 2012). ferent strains of AI prone mice rendered deficient in TLR7/9 or Currently, nothing is known about their existence and function MyD88 expression produce dramatically fewer autoantibodies and in CVID. develop less severe disease (Green and Marshak-Rothstein, 2011). Surprisingly, however, TLR9 deficiency in the presence of normal DISTURBED ANTIGEN RECEPTOR SIGNAL TLR7 function reduces only anti double-strain-DNA autoanti- IN AUTOIMMUNE CVID body levels, but not other autoantibodies and is associated with Several mouse models of increased BCR signals demonstrate an a more severe AI disease, suggesting a regulatory role of TLR9 increased prevalence of AI manifestations (Dorner and Lipsky, for TLR7-mediated immune disease. In CVID patients, pDC and 2006). On the other hand, models of decreased TCR signaling can B cell responses to TLR7 and 9 ligands are impaired (Yu et al., also represent a risk factor for autoimmunity (summarized in Lis- 2012). Subanalysis of the reported data suggests that a subgroup ton et al., 2008). Decreased TCR signals are thought to interfere of patients is more seriously affected by reduced TLR signaling. with negative selection either through a selective or a stronger While the authors correlate the reduced function to increased impact on tolerogenic signals (Liston et al., 2008)thuspoten- infection susceptibility no correlation to autoimmunity is tially impairing the generation of regulatory T cells (Liston and mentioned. Rudensky, 2007). In humans, ORAI (Feske et al., 2006) and Stim1 In summary, autoimmunity is a prominent clinical feature in deficiency (Picard et al., 2009) need to be mentioned as proto- CVID. Associated factors include disturbed B and T cell homeosta- types of reduced antigen receptor signal strongly associated with sis and selection, altered antigen receptor signals, increased BAFF the coincidence of immunodeficiency and autoimmunity in the levels, and possibly altered TLR signaling. Pathogenic mechanisms, affected patients. Also in B cells of the subgroup of CVID patients however, have not been identified yet on a molecular level. Fur- with an increased risk of AI manifestations, calcium signaling is ther research needs to consider established mechanisms in other reduced compared to other CVID patients and healthy controls genetically defined immunodeficiency disorders to unravel the (Foerster et al.,2010; van deVenet al.,2011). The exact mechanism underlying immune dysregulation in CVID. Our improved knowl- of the signaling defect and its potential interference with selection edge will not only steer potential treatment strategies but also our are unknown. In WAS, antigen receptor signaling is impaired due concept of autoimmunity in general.

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ACKNOWLEDGMENTS by the German Federal Ministry of Education and Research This work was supported by Deutsche Forschungsgemein- (BMBF 01 EO0803, to Klaus Warnatz and Reinhard E. schaft Grant SFB 620 project C1 (to Klaus Warnatz) and Voll). The authors are responsible for the contents of this FOR 831 project 8 (VO673/31, to Reinhard E. Voll) and publication.

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“fimmu-03-00210” — 2012/7/17 — 20:14 — page6—#6 MINI REVIEW ARTICLE published: 24 July 2012 doi: 10.3389/fimmu.2012.00189 Autoimmune cytopenias in common variable immunodeficiency

Jenna C. Podjasek1 and Roshini S. Abraham2*

1 Division of Allergic Diseases, Department of Medicine, Mayo , Rochester, MN, USA 2 Cellular and Molecular Immunology Laboratory, Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

Edited by: Common variable immunodeficiency (CVID) is a humoral immunodeficiency whose primary Rosa Bacchetta, Fondazione Centro diagnostic features include hypogammaglobulinemia involving two or more immunoglob- San Raffaele Del Monte Tabor, Italy ulin isotypes and impaired functional antibody responses in the majority of patients. While Reviewed by: increased susceptibility to respiratory and other infections is a common thread that binds Rosa Bacchetta, Fondazione Centro San Raffaele Del Monte Tabor, Italy a large cross-section of CVID patients, the presence of autoimmune complications in this Antonio Condino-Neto, University of immunologically and clinically heterogeneous disorder is recognized in up to two-thirds São Paulo, Brazil of patients. Among the autoimmune manifestations reported in CVID (20–50%; Chapel *Correspondence: et al., 2008; Cunningham-Rundles, 2008), autoimmune cytopenias are by far the most Roshini S. Abraham, Cellular and common occurring variably in 4–20% (Michel et al., 2004; Chapel et al., 2008) of these Molecular Immunology Laboratory, Division of Clinical Biochemistry and patients who have some form of autoimmunity. Association of autoimmune cytopenias Immunology, Department of with granulomatous disease and splenomegaly has been reported.The spectrum of autoim- Laboratory Medicine and Pathology, mune cytopenias includes thrombocytopenia, anemia, and neutropenia. While it may seem Mayo Clinic, Hilton 210e, 200 First Street Southwest, Rochester, paradoxical “prima facie” that autoimmunity is present in patients with primary immune MN 55905, USA. deficiencies, in reality, it could be considered two sides of the same coin, each reflecting e-mail: [email protected] a different but inter-connected facet of immune dysregulation. The expansion of CD21 low B cells in CVID patients with autoimmune cytopenias and other autoimmune features has also been previously reported. It has been demonstrated that this unique subset of B cells is enriched for autoreactive germline antibodies. Further, a correlation has been observed between various B cell subsets, such as class-switched memory B cells and plasmablasts, and autoimmunity in CVID. This review attempts to explore the most recent concepts and highlights, along with treatment of autoimmune hematological manifestations of CVID.

Keywords: common variable immunodeficiency (CVID), autoimmune cytopenias, immune thrombocytopenia, autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome, Evans syndrome

INTRODUCTION phenotype (Warnatz et al., 2002; Piqueras et al., 2003; Wehr et al., Common variable immunodeficiency (CVID) is a highly het- 2008; Eibel et al., 2010). In the last 10 years, monogenic defects erogeneous immunodeficiency with varying complexity. The key associated with antibody deficiency have been described in a small diagnostic elements include low IgG (2 SD below mean of age) subset of CVID patients or patients with hypogammaglobuline- along with low IgA and/or IgM (Park et al., 2008; Resnick et al., mia, or single or few families with a history of consanguinity. 2011). CVID is considered the most commonly encountered and These genetic defects include disease-causing mutations or poly- clinically relevant primary immunodeficiency in adults (Chapel morphisms in the TNFRSF13B (TACI), CD19, ICOS, TNFRSF13C et al., 2008; Park et al., 2008) and though the majority of patients (BAFF-R), CD81, CD20, MSH5, and CD21 genes (Grimbacher are diagnosed between the age of 20 and 40 years, at least another et al., 2003; Salzer et al., 2004, 2005, 2009; Castigli et al., 2005, 20% are diagnosed during childhood (>2 years) or adolescence 2007; Warnatz et al., 2005; van Zelm et al., 2006, 2010; Kane- (Cunningham-Rundles, 2010). gane et al., 2007; Pan-Hammarstrom et al., 2007; Schaffer et al., While recurrent sinopulmonary infections are one of the hall- 2007; Sekine et al., 2007; Zhang et al., 2007; Kuijpers et al., 2010; marks of this disease, gastrointestinal, viral, and systemic bacterial Frank, 2012; Thiel et al., 2012). However, single-gene defects were infections have also been reported (Park et al., 2008; Resnick identified in only a relatively small subset of CVID patients rais- et al., 2011). Besides infections, CVID is associated with a vari- ing the possibility that the majority (>75%) of CVID patients ety of non-infectious complications including pulmonary disease, have oligogenic or polygenic defects. This was recently substanti- autoimmunity, granulomatous disease, , ated by a genome-wide association study of 363 CVID patients, and malignancy (Chapel et al., 2008; Resnick et al., 2011). which revealed that copy number variations (CNV), including The clinical heterogeneity and complexity of CVID has led to gene duplications and/or deletions were present and this analysis renewed efforts over the past decade to identify causal genetic led to the identification of several “novel” genes, which may play defects as well as correlate the “immuno-phenotype” with clinical an important role in the immune response, and genetic variations

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therein could lead to a disease phenotype associated with CVID cells with lymphadenopathy and autoimmune cytopenias with (Orange et al., 2011). reduced plasmablasts – pre-terminally differentiated plasma cells Paradoxical as it may seem, autoimmune manifestations are (Wehr et al., 2008). not uncommon in patients with primary immunodeficiencies Data from Sanchez-Ramon et al. (2008) and Vodjgani et al. (PIDDs) and at least 25% of all PIDDs described in the 2011 IUIS (2007) provide independent substantiation of the association classification may have some form of autoimmune phenomenon between low class-switched memory B cells and clinical features (Bussone and Mouthon, 2009; Notarangelo, 2009; Al-Herz et al., of autoimmunity and splenomegaly in CVID patients reported 2011). The autoimmunity observed in PIDDs may be related by the EUROclass and other classification studies (Warnatz et al., either to a direct or indirect genetic effect, and includes defects 2002; Piqueras et al., 2003; Wehr et al., 2008). in genes that regulate immunological self-tolerance as well as Martinez-Gamboa et al. (2009) showed that there was a numer- genetic variations that alter immune regulation. Not surpris- ical decrease in memory B cell numbers in ITP patients who ingly, therefore, autoimmune features are identified relatively underwent splenectomy and alluded to a potential role for the frequently in CVID patients (Brandt and Gershwin, 2006; Knight spleen in maintaining memory B cell homeostasis. However, a and Cunningham-Rundles, 2006; Cunningham-Rundles, 2008). different study suggests that the age at which splenectomy is performed is more relevant to maintenance of marginal zone AUTOIMMUNITY IN CVID (memory) B cells numbers than consideration of splenectomy Autoimmune hematological abnormalities, specifically cytope- in isolation, regardless of age at which the procedure is done nias, are the most common of all autoimmune manifestations (Wasserstrom et al., 2008). in CVID and may present as thrombocytopenia, anemia or neu- Besides the correlation of B cell subsets, specifically switched tropenia. In the longitudinal study mentioned above, immune memory B cells, with autoimmunity, there is evidence from thrombocytopenia (ITP) was reported in 14% of patients, while multiple human and mouse models on the significance and impor- autoimmune hemolytic anemia (AIHA) and neutropenia was less tance of regulatory T cells expressing FOXP3 in suppressing common with only 7 and <1%, respectively, of the cohort affected or controlling autoimmunity (Buckner, 2010; Long and Buck- (Resnick et al., 2011). It should also be kept in mind that autoim- ner, 2011). It has been shown in at least a subset of CVID mune cytopenias may in fact be the presenting symptom for a patients, particularly those with autoimmune features, that there small subset of CVID patients, especially in children, where Evans is a substantial decrease in relative frequency (%) but not abso- syndrome (ES) has been reported to precede the clinical and lute quantitation of FOXP3+ Tregs raising the possibility of immunological phenotype of CVID (Savasan et al., 2007). Other abnormal immune regulation in these patients (Arumugakani autoimmune presentations reported in CVID include rheumatoid et al., 2010), though the mechanism of immune dysregula- arthritis, anti-IgA antibodies, vitiligo, and alopecia (Horn et al., tion in this context may extend beyond numerical changes to 2007; Park et al., 2008; Resnick et al., 2011).Averyrecentlongitu- possible functional alterations as well (Jang et al., 2011; Long and dinal study assessing clinical complications that cause morbidity Buckner, 2011). and mortality in CVID patients identified autoimmune compli- Another recent study demonstrated B cell receptor recombi- cations in 29% of a cohort of 473 patients studied over 4 decades nation bias in a subset of CVID patients and postulated that this (Resnick et al., 2011). Interestingly, in the same study, the pres- may predispose to decreased secondary recombination with sub- ence of autoimmunity was not associated with an increase in sequent defective central tolerance leading ultimately to the escape mortality. of autoreactive clones (Romberg et al., 2011). Further, a biomarker (soluble BAFF/BLys) produced by monocytes and dendritic cells IMMUNOLOGICAL AND PHENOTYPIC MANIFESTATIONS (DCs), which is a critical B cell survival and proliferation factor, OF AUTOIMMUNE CYTOPENIAS IN CVID and known to be abnormally increased in contexts of autoim- As alluded to previously, several clinical and immunological clas- munity, especially in rheumatologic diseases (Becker-Merok et al., sifications have been posited in an attempt to stratify and may 2006) was also been shown to be elevated in CVID patients be even simplify the complex and heterogeneous phenotypes but there was no demonstrable correlation with the incidence of seen in CVID (Warnatz et al., 2002; Piqueras et al., 2003; Chapel autoimmunity (Knight et al., 2007). et al., 2008; Wehr et al., 2008). The relatively more recent EURO- class study attempted to cohesively link the earlier Freiburg and CVID: OVERLAP WITH AUTOIMMUNE Paris classifications by correlating B cell subset immunopheno- LYMPHOPROLIFERATIVE SYNDROME types with clinical presentation specifically providing correlation AND EVANS SYNDROME for autoimmunity, granulomatous disease, and splenomegaly Published data have demonstrated a clear immunologic and (Warnatz et al., 2002; Piqueras et al., 2003; Wehr et al., 2008). clinical overlap between CVID, ES, and autoimmune lymphopro- Of particular relevance was the correlation of an expansion of liferative syndrome (ALPS). ES is characterized by the presence CD21low/dim B cells with splenomegaly (Wehr et al., 2008). The of autoimmune cytopenias in two or more hematopoietic lin- CD21low/dim B cells have been previously reported to be a subset eages. A small study evaluating 12 pediatric patients with ES of anergic B cells with defective signaling that has the capac- determined that half (6/12) also had elevated αβ TCR+ DNT ity to home to sites of inflammation (Rakhmanov et al., 2009, T cells (CD3+CD4–8–) and defective Fas apoptosis characteris- 2010; Foerster et al., 2010; Charles et al., 2011). Additionally, cor- tic of ALPS patients (Teachey et al., 2005). A subsequent larger relations were identified between an expansion of transitional B study of 45 patients with ES substantiated the earlier finding

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by demonstrating diagnostic criteria for ALPS in 21/45 patients (Seif et al., 2010). The correlation between ES,ALPS,and CVID was made in a dif- ferent study, which though limited in sample size (n = 7), showed development of hypogammaglobulinemia, as seen in CVID in 5/7 patients with ES. These patients also had increased Fas expression (Savasan et al., 2007). A larger cohort study of 68 patients with ES showed that only a relatively small proportion, 4/68 had CVID (Michel et al., 2009). In a separate study of ALPS patients (n = 66), an equally small number, 5/66 had hypogammaglobulinemia, suggesting a poten- tial phenotypic overlap with CVID. The majority of the ALPS patients in this study had reduced class-switched memory B cells, similar to what has been reported in two-third or greater of CVID patients (Rensing-Ehl et al., 2010).

MECHANISMS OF DEVELOPMENT OF AUTOREACTIVITY The development of self-reactive B cells is regulated both centrally (bone marrow) and peripherally through at least two indepen- dent check-points. It has been suggested that there may be a failure of both central and peripheral tolerance mechanisms in CVID due to immune dysregulation resulting in a flawed neg- ative selection process. Logically, this would suggest that there would be an increased selection of autoreactive B cells prior to affinity maturation () or memory B cell/plasma cell commitment in the secondary lymphoid organs (Haymore et al., 2008). This is a topic that is discussed in depth elsewhere in this journal series, and therefore, not addressed herein.

DIAGNOSIS AND TREATMENT FIGURE 1 | Diagnostic algorithm for autoimmune cytopenias in CVID. DIAGNOSIS AITP,autoimmune thrombocytopenia purpura; AIHA, autoimmune The evaluation of CVID patients for autoimmune cytopenias hemolytic anemia; AIN, autoneutropenia; IVIG, intravenous should include appropriate diagnostic work-up (Figure 1), how- immunoglobulin;. *non-exclusionary. ever, in the case of ITP this may primarily be a diagnosis of exclusion. A presumptive diagnosis of ITP can be arrived at by ruling out alternative pathological mechanisms through clinical lack of detectable autoantibodies does not exclude a diagnosis of history, physical review, complete blood count (CBC) analysis, and AIN (Bope and Kellerman, 2012). Most cases of AIN are asso- peripheral blood smears (Provan et al., 2010). Confirmation of the ciated with normal marrow reserve and pathogenesis is related diagnosis is usually determined by response to appropriate treat- to antibody-mediated destruction and in some cases, sequestra- ment. As per the previous discussion that autoimmune cytopenias tion. The diagnosis can include a bone marrow biopsy, which may precede a diagnosis of CVID, it would be reasonable to eval- would reveal a hypercellular marrow and usually a late matura- uate both pediatric and adult patients for immunoglobulin levels tional arrest, though in some cases, an early arrest can also be seen. on diagnosing ITP to rule out a possible CVID or selective IgA AIN may be associated with ITP and/or AIHA in CVID patients. deficiency (Provan et al., 2010). Additionally, follow-up may be Besides, the possible presence of anti-neutrophil antibodies, cir- required with periodic evaluation and correlation with clinical culating immune complexes may also be present in a subset of history to document evolution of the disease process. patients with AIN (Dinauer and Coates, 2009). Likewise, the diagnosis of AIHA mandates evidence of hemol- ysis along with detection of an autoantibody. There are a number TREATMENT of laboratory markers for establishing hemolysis, including a CBC A treatment algorithm for autoimmune cytopenias in CVID is with peripheral smear, increased indirect bilirubin, increased lac- provided in Figure 2. The American Society of Hematology tate dehydrogenase (LDH), and decreased haptoglobin. Autoan- has provided guidelines for the treatment of patients with ITP tibodies can be detected by a direct antiglobulin test (DAT) or and these include initiation of treatment in adult patients if the Coomb’s test (Gehrs and Friedberg, 2002). platelets are below 30 × 109/L. However, in pediatric patients, The diagnosis of autoimmune neutropenia (AIN) is similar the current guidelines state that treatment is based on clinical to ITP in that it is a diagnosis of exclusion. In some cases, symptoms associated with thrombocytopenia regardless of the detection of anti-granulocyte antibodies may be useful but the platelet counts (Neunert et al., 2011). Pediatric patients are far

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FIGURE 2 |Treatment algorithm for autoimmune cytopenias in CVID. AITP,autoimmune thrombocytopenia; AIHA, autoimmune hemolytic anemia; AIN, autoneutropenia; IVIG, intravenous immunoglobulin; ANC, absolute neutrophil count; G-CSF,granulocyte colony stimulating factor. *Rh+, non-splenectomized individuals only. more likely to experience spontaneous remissions. The treatment The treatment of AIN is primarily dictated by the severity of of choice as first-line therapy for ITP is the use of steroids at neutropenia-associated clinical symptoms and the underlying dis- 1 mg/kg for a duration of at least three weeks with subsequent ease context. Treatment with high-dose IVIG or steroids may be dose reduction and eventual withdrawal. Alternative therapeutic used if there is very profound neutropenia (ANC < 500/mm3) options could include a single dose of intravenous immunoglob- in conjunction with recurrent or fulminant infections. G-CSF ulin (IVIG) at 1 g/kg. Further use of IVIG is dependent on therapy is only of value if bone marrow reserves are depleted. clinical response to the initial dose. A combination of the above Splenectomy has little value in reversing neutropenia, especially two therapies may be utilized if a rapid response is required. if it is isolated, since the effect is transient, and can ultimately Rho(D) immune globulin is an option for Rh-positive individ- increase overall infection risk (Dinauer and Coates, 2009). uals who have not undergone a splenectomy and are unable to A separate study of 19 adult patients with steroid-refractory tolerate steroid treatment (Neunert et al., 2011). Splenectomy is autoimmune cytopenias, reported a 100% initial response rate to recommended as a therapeutic option only for those patients that a combination of low-dose Rituximab and Alemtuzumab (anti- fail corticosteroid therapy. CVID patients undergoing splenec- CD52 humanized monoclonal antibody). Infection occurred in tomy or receiving immunosuppressive medication may be at 6/19 patients after a median period of 70 weeks (Gomez-Almaguer increased risk for infection given their intrinsic immunological et al., 2010). Other reports have documented an initial response defects. rate of 78–92% for refractory autoimmune cytopenias treated While AIHA is treated much like ITP,it may be more challeng- with mycophenolate mofetil with no significant adverse events ing to manage, particularly in patients with ES (Cunningham- reported (Kotb et al., 2005; Rao et al., 2005). Thus, the approach Rundles, 2002; Wang and Cunningham-Rundles, 2005). For to treating autoimmune cytopenias in CVID is not dissimilar to refractory cases of ITP,AIHA,or both, Rituximab, a chimeric mon- the treatment of immune competent patients (Wang and oclonal anti-CD20 B cell-depleting agent, has been effectively used. Cunningham-Rundles, 2005). In a modest-size cohort of CVID patients (n = 33) with refractory autoimmune cytopenias (failure of at least 2–6 treatments prior to SUMMARY initiation of Rituximab), the initial response rate was remarkably This minireview, which is limited in scope, provides an encapsu- high at 84% (Gobert et al., 2011). Severe infection was an unfortu- lated discussion on the incidence and presentation of autoimmu- nate consequence in almost a quarter of these patients (8/33) over nity in CVID, specifically autoimmune cytopenias, their overlap a mean follow-up period of 39 months. Of note, half the patients with other clinical entities, some notable immunological hall- (4/8) were not on replacement at the marks, laboratory diagnosis and an overview of standard and new time of infectious diagnosis. An earlier study reports similar rates therapies. As mentioned in the text, a more exhaustive treatment of infection in patients with ITP who received standard treatment of autoimmunity in CVID, focusing on mechanistic aspects, is (Michel et al., 2004). provided elsewhere.

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immunologic outcomes in heterozy- commercial or financial relationships in common variable immunodefi- distributed under the terms of the gotes. J. Allergy Clin. Immunol. 120, that could be construed as a potential ciency. Front. Immun. 3:189. doi: Creative Commons Attribution License, 1178–1185. conflict of interest. 10.3389/fimmu.2012.00189 which permits use, distribution and This article was submitted to Frontiers in reproduction in other forums, pro- Received: 22 February 2012; accepted: 18 Primary Immunodeficiencies, a specialty vided the original authors and source Conflict of Interest Statement: The June 2012; published online: 24 July 2012. of Frontiers in Immunology. are credited and subject to any copy- authors declare that the research was Citation: Podjasek JC and Abraham Copyright © 2012 Podjasek and Abra- right notices concerning any third-party conducted in the absence of any RS (2012) Autoimmune cytopenias ham. This is an open-access article graphics etc.

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“fimmu-03-00189” — 2012/7/24 — 12:31 — page7—#7 MINI REVIEW ARTICLE published: 04 June 2012 doi: 10.3389/fimmu.2012.00129 TH17 cells in autoimmunity and immunodeficiency: protective or pathogenic?

Ashish K. Marwaha1,2, Nicole J. Leung 1,2, Alicia N. McMurchy 2,3 and Megan K. Levings 2,3*

1 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 2 Child and Family Research Institute, Vancouver, BC, Canada 3 Department of Surgery, University of British Columbia, Vancouver, BC, Canada

Edited by: In 2005 a newly discovered subset that secreted interleukin (IL)-17 became Rosa Bacchetta, Fondazione Centro the center of attention in immunology. Initial studies painted Th17 cells as the culprit for San Raffaele Del Monte Tabor, Italy destruction in many different autoimmune and auto-inflammatory diseases. Subsequently, Reviewed by: Rosa Bacchetta, Fondazione Centro the discovery of patients with primary immunodeficiencies in the IL-17 pathway taught San Raffaele Del Monte Tabor, Italy us that Th17 cells have a critical role in defense against certain fungal and bacterial infec- Francesco Annunziato, University of tions. Moreover, the paradoxical exacerbation of Crohn’s disease in the clinical trials of a Florence, Italy Secukinumab (AIN457), a fully human to IL-17A, has cast into doubt *Correspondence: a universal pro-inflammatory and harmful role for Th17 cells. Evidence now suggests that Megan K. Levings, Department of Surgery, University of British depending on the environmentTh17 cells can alter their differentiation program, ultimately Columbia, A4-186, 950 West 28th giving rise to either protective or pro-inflammatory cells. In this review we will summarize Avenue, Vancouver, BC, Canada V5Z the evidence from patients with immunodeficiencies, autoimmune, or auto-inflammatory 4H4. diseases that teaches us how the pro-inflammatory versus protective function ofTh17 cells e-mail: [email protected] varies within the context of different human diseases.

Keywords:Th17 cells, autoimmunity,T regulatory cells, immunodeficiency, inflammatory bowel disease, psoriasis, type 1 diabetes, secukinumab

INTRODUCTION thought to only produce IL-17A and IL-17F, which are 55% iden- In rare cases, a mutation in an essential gene can disrupt immune tical (Kolls and Linden, 2004). IL-17A can combine with IL-17F homeostasis, leading to clinical immunodeficiency. More com- to form a heterodimer and both can form homodimers (Wright monly, when individuals with a genetic predisposition are exposed et al., 2007). to environmental triggers, a failure of immune homeostasis can Th17 cells have many phenotypic characteristics that distin- lead to autoimmunity. In this review, we will discuss how par- guish them from other Th cell lineages. In addition to IL-17A and allel studies of immunodeficiencies and autoimmune diseases IL-17F, Th17 cells secrete other signature cytokines including IL- have advanced our knowledge of a CD4+ T cell lineage first 21 and IL-22 (Bending et al., 2011). They have also been reported characterized by production of IL-17A, Th17 cells. to produce IFN-γ (Annunziato et al., 2007), IL-4 (Cosmi et al., Th17 cells were identified in 2005 (Harrington et al., 2005; 2010), IL-10 (McGeachy et al., 2007), IL-9 (Beriou et al., 2010), Langrish et al., 2005; Park et al., 2005) and, as for other CD4+ IL-26, CXCL8, and CCL20 (Boniface et al., 2008). They are poor T cell lineages, their development, is controlled by a combina- producers of IL-2, which may result in their poor proliferative tion of cytokines which initiate a program of transcription fac- potential in vitro (Santarlasci et al., 2012). They constitutively co- tor expression and epigenetic re-modeling (van der Gast et al., express CCR4 and CCR6, but not CXCR3 (Acosta-Rodriguez et al., 2011). In humans, the cytokines which instruct Th17 cell lin- 2007b), and are derived from CD161+ precursors (Cosmi et al., eage development likely include IL-6, IL-21, IL-23, and IL-1β 2008). The effects of Th17 cells on other cells have recently been (Acosta-Rodriguez et al., 2007a; Chen et al., 2007; Evans et al., highlighted in many reviews (Annunziato and Romagnani, 2011; 2007; Wilson et al., 2007; Liu and Rohowsky-Kochan, 2008), with Gaffen, 2011; Gaffen et al., 2011; Ghoreschi et al., 2011; Milner, a potential synergistic role for TGF-β (Manel et al., 2008; Volpe 2011; Pappu et al., 2011; Wilke et al., 2011). et al., 2008; Yang et al., 2008a) via its ability to suppress Th1 cell Th17 cells initially developed a reputation as a destructive lineage commitment (Santarlasci et al., 2009). Cytokine-driven element in several diseases including multiple sclerosis (MS), activation of the signal transducer and activator of transcription rheumatoid arthritis (RA), psoriasis, and inflammatory bowel dis- (STAT) 3 pathway is an essential step in Th17 cell differentiation ease (IBD). In animal models this reputation, came from evidence (Holland et al., 2007; Yang et al., 2007; Ma et al., 2008), ultimately that the lack of IL-17-producing cells ameliorates experimental leading to expression of their lineage-defining transcription fac- autoimmune (EAE) and collagen induced arthri- tor: retinoid orphan receptor (ROR)C2 (Acosta-Rodriguez et al., tis (CIA; Cua et al., 2003). In humans, the reputation was due 2007a; Annunziato et al., 2007; Wilson et al., 2007; Manel et al., to correlative data documenting an increase in IL-17-producing 2008; Crome et al., 2009). Although the IL-17 cytokine family cells, particularly at sites of tissue inflammation (Wilke et al., includes six members (Kolls and Linden, 2004), Th17 cells are 2011). However, these original conclusions were over-simplified

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and as discussed below in some diseases Th17 cells clearly have a may not only relate to Treg dysfunction, but also to changes protective role. in Th17 cells. Indeed IPEX patients possessed an increased fre- quency of cells with a surface profile characteristic of Th17 cells THE ROLE OF Th17 CELLS IN PRIMARY (CD4+CCR6+CD161+), expressing RORC2, and producing IL- IMMUNODEFICIENCIES 17 (Passerini et al., 2011). Interestingly, in patients with point Much of what we know about human Th17 cells comes from mutations that do not abrogate FOXP3 expression, there was an the study of a rare primary immunodeficiency called Hyper- increased frequency of FOXP3+ cells within the CD161+ Th17 (Job’s, syndrome). This disease is caused by cell gate. These data suggest that dysfunctional Tregs may prefer- mutations in STAT3 (Holland et al., 2007) but the underlying cel- entially differentiate into Th17 cells and that an expansion of this lular basis for the characteristic phenotype of severe pneumonias, subset may underlie some of the clinical phenotype of IPEX. An mucocutaneous candidiasis, and Staphylococcus aureus abscesses alternative interpretation is that the Th17 cells in IPEX patients (Buckley et al., 1972; Grimbacher et al., 1999) remained unknown are highly activated and that in this case FOXP3 expression is until several groups found that these patients lack Th17 cells in a consequence of T cell activation and not Treg lineage commit- their peripheral blood (de Beaucoudrey et al., 2008; Ma et al., ment (Ziegler,2006). Regardless,in this immunodeficiency there is 2008; Milner et al., 2008; Renner et al., 2008). In addition, naïve strong correlative evidence that Th17 cells may have a detrimental Th cells from Job’s syndrome patients have low levels of RORC2 pro-inflammatory effect. expression and cannot be differentiated into Th17 cells in vitro (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; EVIDENCE FOR THE PRO-INFLAMMATORY ROLE OF Th17 IN Renner et al., 2008). HUMAN AUTOIMMUNITY One complication when interpreting data from Job’s syndrome The first recognition of the importance of Th17 cells came from patients is that STAT3 is activated downstream of other cytokines, studies of EAE. The notion of EAE as a Th1-mediated disease was making it difficult to attribute a clinical phenotype to one pathway. challenged when mice deficient in the p40 subunit of IL-12 were Recently, other immunodeficiencies have been described which found to be resistant to EAE whereas mice deficient in the p35 sub- involve more specific defects in the IL-17 pathway. For example, unit were actually more susceptible to disease (Becher et al., 2002). two patients with chronic mucocutaneous candidiasis (CMC) dis- Cua et al. (2003) solved this paradox by using genetically deficient ease, characterized by chronic or persistent infection with Candida mice to show that IL-23p19 and IL-12p40, but not IL-12p35, were albicans and S. aureus, were found to have an IL-17RA autosomal essential for EAE development. IL-23, which shares the IL-12p40 recessive deficiency or an IL-17F autosomal dominant deficiency subunit with IL-12, was subsequently found to stabilize Th17 cells, (Puel et al., 2011). In addition, patients with a deficiency in and these cells were found to be the main contributing factor in the intracellular adaptor molecule CARD9, which is essential for EAE (Langrish et al., 2005). Subsequently, a correlation between dectin-1 signaling, also suffer from systemic Candidiasis infection Th17 cells and human autoimmunity was sought. Below we dis- (Glocker et al., 2009) and have low numbers of Th17 cells in their cuss the evidence for a pro-inflammatory role in autoimmunity peripheral blood. and describe attempts to target this axis therapeutically. Together, these data have led to the hypothesis that in humans Psoriasis is an auto-inflammatory skin disease characterized by Th17 cells have an essential role in protective immunity the spe- recurrent demarcated red and scaly skin plaques. These plaques cific pathogens C. albicans and S. aureus. In accordance with this include infiltrating T cells (mainly Th cells) and dendritic cells in conclusion, Sallusto et al. have characterized different subsets of the dermis as well as cytotoxic T cells and neutrophil in the epi- human Th17 cells that can be differentiated in vitro with antigen dermis (Lowes et al., 2007). The resulting inflammatory process specific stimulation by C. albicans and S. aureus (Zielinski et al., results in rapid keratinocyte proliferation, abnormal keratinocyte 2012). differentiation, and angiogenesis (Lowes et al., 2007). Initially, Th17 cells have an intriguing close developmental link with increased levels of IFN-γ, TNF-α, and IL-12 in the serum and FOXP3+CD4+ regulatory T cells (Tregs). FOXP3 and RORC2 can lesions of psoriasis patients labeled this as a Th1-mediated disease directly interact via a DNA-independent mechanism, and dur- (Di Cesare et al., 2009). However, RORC, IL-1β, IL-6, and IL-23 are ing Th17 cell development FOXP3 is transiently expressed (Zhou also increased in psoriatic skin lesions (Di Cesare et al., 2009) lead- et al., 2008). Moreover, upon activation fully differentiated human ing to the possibility that Th17 and Th1 act in synergy to produce Th17 cells preferentially express FOXP3 in comparison to Th1 psoriatic inflammation. cells (McMurchy and Levings, unpublished data). Indeed there is Th17 cells are thought to be recruited to the skin by expres- increasing evidence for the existence of cells that co-express IL-17 sion of CCL20, the ligand for CCR6, then locally stabilized by IL-1 and FOXP3 (Ayyoub et al., 2009; Beriou et al., 2009; Miyara et al., and IL-23. Since both IL-17 and IFN-γ cause keratinocytes and 2009; Voo et al., 2009; Kryczek et al., 2011; Ye et al., 2011). antigen presenting cells (APCs) to produce more IL-1, IL-23, and Immune dysregulation, polyendocrinopathy, enteropathy X- CCL20, a positive feedback loop causing keratinocyte proliferation linked syndrome (IPEX) is a triad of autoimmune syndromes is established (Kryczek et al., 2008; Zaba et al., 2009). Several mon- including enteropathy, type 1 diabetes, and hyper-IgE (McMurchy oclonal antibodies targeting TNF-α and the p40 subunit shared by et al., 2010), resulting from mutations in FOXP3. The cellular basis IL-12 and IL-23 (Ustekinumab) have been approved for clinical for this disease has been attributed to Treg dysfunction (Bacchetta use in psoriasis. Since IL-17 can act synergistically with TNF-α to et al., 2006; D’Hennezel et al., 2009), but recently Bacchetta et al. induce keratinocytes to express inflammatory proteins (Chiricozzi investigated whether part of the cellular defect in IPEX patients et al., 2011), it is possible that anti-TNF-α acts in part by inhibiting

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Th17 cell-driven inflammation. Targeting IL-17 alone with Secuk- the role of Th17 in the NOD mouse model of T1D (Vukkadapu inumab (AIN457) or Ixekizumab, both fully human neutralizing et al., 2005; Jain et al., 2008; Emamaullee et al., 2009; van den antibodies to IL-17A, is also effective in psoriasis (Hueber et al., Brandt et al., 2010; Lee et al., 2011; Liu et al., 2011; Joseph et al., 2010; Leonardi et al., 2012), confirming that this is likely a major 2012), including a potential ability to convert into Th1 cells in vivo pathogenic cytokine in this skin disease. Since Th17 cells are not (Bending et al., 2009; Martin-Orozco et al., 2009). However, more the sole producers of IL-17 [other possible sources of this cytokine recent data suggest some of this apparent plasticity could be related in psoriatic plaques include γδ T cells (Cai et al., 2011), mast cells to the study of in vitro polarized Th17 cells, which are not suf- (Lin et al., 2011), neutrophils (Lin et al., 2011), and Tregs (Boven- ficiently stabilized at the epigenetic level (Bending et al., 2011; schen et al., 2011)], whether or not Th17 cells are the major source Cohen et al., 2011). Patients with T1D, have an increase in circulat- of this cytokine in skin remains to be determined. ing IL-17-producing cells (Honkanen et al., 2010; Marwaha et al., Another disease with strong links to Th17 cells is RA, a chronic 2010; Hughson et al., 2011), including FOXP3 expressing Th17 autoimmune disease that leads to joint destruction. T cells infil- cells (Marwaha et al., 2010), monocytes that secrete Th17 polar- trating the synovial fluid of RA patients produce high amounts izing cytokines (Bradshaw et al., 2009), and islet-antigen specific of IL-17A, IL-1β, and IL-6 (Cascao et al., 2010), especially during Th17 cells (Arif et al., 2011). There is also evidence that pancreatic early disease and pre-treatment (Chabaud et al., 1999; Ziolkowska lymph nodes from T1D patients have an increase in Th17 cells et al., 2000; Hwang and Kim, 2005; Leipe et al., 2010). Notably, (Ferraro et al., 2011) and that islets from T1D patients, who died the levels of IL-17 in the synovium correlate with joint damage, close to diagnosis, express IL-17A, RORC, and IL-22 (Arif et al., whereas those of IFN-γ correlate with protection (Kirkham et al., 2011). Mechanistically IL-17 enhances IL-1β, IFN-γ, and TNF- 2006). Recent evidence supports a role for IL-17F as well as IL-17A α-induced apoptosis in human islets (Arif et al., 2011). We have in RA, with the two related cytokines acting in synergy to induce also found that significantly elevated levels of CD8+IL-17+ cells other pro-inflammatory cytokines and chemokines in synovio- are detectable in the peripheral blood of a large subset of patients cytes, myeloid cells, and synovial fibroblasts (Lundy et al., 2007; with T1D at disease onset (Marwaha et al., 2010). In the context Tran et al., 2007; Hot and Miossec, 2011; van Hamburg et al., of mounting correlative evidence that IL-17-producing cells may 2011). Hence, analogous to the process in psoriasis, a positive be pathogenic in the early stages of T1D onset, clinical trials to pro-inflammatory feedback loop encourages more Th17 differ- test the effects of therapy with agents such as Secukinumab or entiation and maintenance in the joint (reviewed in Sarkar and Ustekinumab are warranted. Fox, 2010). Direct clinical evidence for the role of IL-17 in RA comes from recent clinical trials which found that Secukinumab A PROTECTIVE ROLE FOR Th17 IN THE GUT and another anti-IL-17A therapeutic known as LY2439821 signifi- The success of Ustekinumab, a human IL-12/23 monoclonal anti- cantly benefit these patients (Genovese et al., 2010; Morrison et al., body, in patients with moderate to severe Crohn’s disease held 2011). promise for the targeting of the IL-17 pathway to modulate this Multiple sclerosis is a neurological disease that results from disease (Sandborn et al., 2008). However, the paradoxical exacer- auto-inflammatory damage to the sheaths surrounding bation of Crohn’s disease in the clinical trial of a Secukinumab, nerves in the brain and spinal cord. This disease has historically cast into doubt the pro-inflammatory role of Th17 cells in the gut. been associated with the discovery of Th17 cells since, as discussed Whilst IL-17 cell-producing cells are found in high numbers in above, they have a major pathogenic role in EAE (Bettelli et al., inflamed mucosa in Crohn’s disease and patients, 2008; Dong, 2008; Dubin and Kolls, 2008; Weaver and Hatton, more recent data demonstrate that characterization on the basis 2009). MS patients have increased IL-17 mRNA in their blood as of IL-17 alone is insufficient to classify these cells as pathogenic. well as cerebrospinal fluid (Matusevicius et al., 1999), and expres- As described above, Th17 can co-secrete IFN-γ (Annunziato et al., sion of miRNA326 in their peripheral blood mononuclear cells 2007; Lee et al., 2009; Cosmi et al., 2011; Hirota et al., 2011)or promotes Th17 cell differentiation and correlates with disease co-express FOXP3 (Ayyoub et al., 2009; Beriou et al., 2009; Miyara severity (Du et al., 2009). Blood-brain barrier endothelial cells et al., 2009; Voo et al., 2009; Kryczek et al., 2011; Ye et al., 2011), layers are more permeable to in vitro polarized Th17 cells, espe- indicating the existence of multiple subsets of Th17 cells with cially if the monolayer is pre-treated with IL-17 or IL-22 (Kebir functional specialization (Figure 1). et al., 2007). These data led to the hypothesis that in MS Th17 In retrospect, data from mouse models of colitis heeded a warn- cells weaken the blood-brain barrier and enable the migration of ing as to the protective role for Th17 cells in Crohn’s disease. In immune cells into the normally immune privileged sites within the the dextran sulfate sodium (DSS)-induced colitis model, admin- central nervous system. If this is the case, then Th17 cells may have istration of a neutralizing IL-17A antibody (Ogawa et al., 2004), more of a facilitative than directly pathogenic role in the nervous deletion of IL-17A (Yang et al., 2008b), or of IL-22 (Zenewicz system, distinct from their clear role in the positive feedback loop et al., 2008) all resulted in a worsening of the colitis. In con- of inflammation in psoriasis and RA. Recruitment has begun for a trast, IL-17F-deficient (Yang et al., 2008b) and IL-21-deficient phase II clinical trials of Secukinumab in patients with relapsing- mice (Fina et al., 2008) were protected against DSS induced remitting MS. This trial will provide significant insight into the colitis. These data suggest there are non-redundant roles of IL- question of whether IL-17 blockade in MS can induce a clinically 17A versus F, and that, at least in the gut, IL-21 rather than relevant protective function. IL-17A or IL-22 may be a primary Th17-derived pathogenic Type 1 diabetes (T1D) is characterized by autoimmune destruc- cytokine. tion of pancreatic islet cells resulting in the loss of insulin How could Th17-derived cytokines exert a protective func- production. Murine studies have yielded conflicting results on tional role in the intestine? First, IL-17A improves barrier function www.frontiersin.org June 2012 | Volume 3 | Article 129 | 99 Marwaha et al. Th17 in autoimmunity and immunodeficiency

FIGURE 1 |The protective and pathogenic functions ofTh17 cells. pathway of tissue damage such as that seen in psoriasis or RA. In Crohn’s Depending on the local cytokine environment, different subsets of Th17 disease, Th17 cells can differentiate in the gut into protective or pathogenic cells arise and mediate distinct effector function. In the presence of IL-23, Th17 cells. In the presence of TGF-β and IL-21,Th17 are re-programmed into Th17 cells seem to be pro-inflammatory and can either make IFN-γ “regulatory” Th17 cells which seem to protect from intestinal themselves or work in concert with Th1 cells to drive a positive feedback inflammation. by strengthening tight junctions after inducing claudin and mucin under certain conditions γδ T cells (Stark et al.,2005),CD8+ Tcells expression (Kinugasa et al., 2000; Chen et al., 2003). Second, IL-22 (Shin et al., 1998; He et al., 2006), T follicular helper cells (Cua and improves barrier function by inducing epithelial cell proliferation Tato, 2010), Lymphoid Tissue induced (LTi) cells (Cupedo et al., (Brand et al., 2006) and enhancing goblet cell restoration and 2009), and NKT cells (Michel et al., 2007; Lee et al., 2008; Rachit- mucus production (Sugimoto et al., 2008). Also, a novel suppres- skaya et al., 2008), can all secrete IL-17. We must therefore start sive Th17 subset dubbed regulatory Th17 (rTh17) cells has recently to redefine the partial role that Th17 cells play in IL-17-guided been described.When Esplugues et al. (2011) used a CD3-antibody immune response. An additional consideration is their potential strategy to induce mucosal tolerance, Th17 cells were recruited for plasticity and co-secretion of cytokines that define other Th cell to the gut but then re-programmed into suppressive, FOXP3- lineages (e.g.,IFN-γ),although this is more likely a transient rather negative, rTh17 cells. The function of rTh17 cells depends on than permanent change based on epigenetic analysis (Bending IL-10, TGF-β, and CTLA-4, and does not occur in CCR6-deficient et al., 2011; Cohen et al., 2011). In summary, the notion that Th17 mice where Th17 are not recruited to the gut. The latter data indi- cells are purely pro-inflammatory cells is mistaken, rather these cate that the mucosal immunity micro-environment is critical for cells mediate a diverse set of responses in infection, autoimmunity, the development of rTh17 cells. and immunodeficiency.

CONCLUSION ACKNOWLEDGMENTS Different flavors of Th17 differentiation, ranging from highly The authors’ own work is supported by grants from the Canadian pro-inflammatory to suppressive, result from different cytokine Institutes for Health Research and the Juvenile Diabetes Research micro-environments in various diseases. Th17 cells can no longer Foundation. Megan K. Levings holds a Canada Research Chair in be identified solely on the production of IL-17A since the com- Transplantation. Alicia N. McMurchy is a Canada Vanier Scholar. bination of co-secreted cytokines is key to defining their effector Ashish K. Marwaha holds a Michael Smith Research Trainee award function. Moreover, IL-17 is not only produced by Th17 cells, and and a Radcliffe Travelling Fellowship at the University of Oxford.

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Frontiers in Immunology | Primary Immunodeficiencies June 2012 | Volume 3 | Article 129 | 104 REVIEW ARTICLE published: 02 August 2012 doi: 10.3389/fimmu.2012.00233 Dendritic cells: a double-edge sword in autoimmune responses

Giada Amodio and Silvia Gregori*

San Raffaele Telethon Institute for Gene Therapy (OSR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy

Edited by: Dendritic cells (DC) are antigen-presenting cells that play a pivotal role in regulating innate Rosa Bacchetta, Fondazione San and adaptive immune responses. In autoimmunity, DC act as a double-edged sword since Raffaele, Italy on one hand they initiate adaptive self-reactive responses and on the other they play a Reviewed by: pivotal role in promoting and maintaining tolerance. Thus, DC are the most important Rosa Bacchetta, Fondazione San Raffaele, Italy cells in either triggering self-specific responses or in negatively regulating auto-reactive Elena Perez, University of South responses. The latter function is mediated by DC in the steady-state or specialized subsets Florida, USA of DC, named tolerogenic DC. Clinical and experimental evidence indicate that prolonged *Correspondence: presentation of self-antigens by DC is crucial for the development of destructive autoim- Silvia Gregori, San Raffaele Telethon mune diseases, and defects in tolerogenic DC functions contribute to eradication of Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells self-tolerance. In recent years, DC have emerged as therapeutic targets for limiting their and Gene Therapy, San Raffaele immunogenicity against self-antigens, while tolerogenic DC have been conceived as ther- Scientific Institute, Via Olgettina 58, apeutic tools to restore tolerance. The purpose of this review is to give a general overview 20132 Milan, Italy. e-mail: [email protected] of the current knowledge on the pathogenic role of DC in patients affected by autoimmune diseases. In addition, the protective role of tolerogenic DC will be addressed.The currently applied strategies to block immune activation or to exploit the tolerogenic potential of DC will be discussed.

Keywords: dendritic cells, autoimmune diseases, tolerance

INTRODUCTION cellular interactions or through secretion of pro-inflammatory Dendritic cells (DC) are professional antigen-presenting cells and immuno-regulatory cytokines (Banchereau and Steinman, (APC) specialized in capturing and processing antigens (Ags) to 1998; Larregina and Falo, 2005; Merad et al., 2008; Rescigno present to T cells. DC constitute a front-line defense against patho- and Di Sabatino, 2009; Lambrecht and Hammad, 2010; gens, are located throughout the body, and form complex networks Thomson, 2010). that allow them to communicate with different cells. Therefore, In the bloodstream, DC circulate as immature cells charac- DC are critically involved in the initiation of adaptive immune terized by a low expression of human leukocyte antigen (HLA) responses and, as such, are defined immunogenic DC. These DC class II and co-stimulatory molecules, high endocytic activity, and might be implicated in the induction of autoimmune responses low T cell activation potential. Circulating DC constantly patrol via the activation of auto-reactive T cells and the consequent the surrounding environment for pathogens, such as viruses and eradication of self-tolerance. Conversely, DC in the steady-state, bacteria. Upon Ag encounter, DC undergo a complex process of or specialized subsets of DC, termed tolerogenic DC, promote maturation meanwhile they travel to the lymph nodes, where they and maintain tolerance through several non-overlapping mech- activate helper and cytotoxic T cells as well as B cells. Imma- anisms. Tolerogenic DC can induce apoptosis of effector T cells, ture DC in the steady-state migrate at low ratio to the lymph skew T cell phenotype, and promote anergy and/or regulatory T nodes without undergoing activation, can present Ags to T cells cells (Tregs; Morelli and Thomson, 2007; Gregori, 2011). Thus, in the absence of co-stimulation and induce clonal T cell anergy defects in the activities of tolerogenic DC may also contribute to (Schwartz et al., 1989), deletion of auto-reactive T cells (Hawiger break self-tolerance and to induce autoimmune responses. et al., 2001; Steinman and Nussenzweig, 2002), and promote Tregs An optimal balance between immunogenic and tolerogenic (Dhodapkar et al., 2001). Tolerogenic DC, both circulating and DC is therefore fundamental to prevent self-reactive immune tissue resident, contribute to the induction and maintenance of responses and to maintain immune self-specific homeostasis. In self-specific tolerance. this review, we will give an overview of the different role of both In humans, two major and intrinsically different subpopula- immunogenic and tolerogenic DC in promoting autoimmune tions of DC have been described: myeloid DC (myDC), called also disease onset and/or progression, focusing primarily on human conventional DC, and plasmacytoid DC (pDC), which differ in pathological conditions. their transcriptional program, development, phenotypic markers, and immunological functions (Belz and Nutt, 2012). myDC pick HUMAN DENDRITIC CELL SUBSETS up Ags in the periphery and move to T cell areas of peripheral lym- Dendritic cells are present in all tissues and they function as phoid organs to initiate immunity through a number of different an important bridge between innate and adaptive immunity, by events including maturation and cytokine secretion, all of which

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are regulated by recognition of pathogens via Toll-like receptors 2004). Similar to myDC, immature pDC as well as alternatively (TLR; Watts et al., 2010). activated pDC are involved in promoting tolerance (Hanabuchi Myeloid DC are present in the peripheral blood and in several et al., 2010; Martin-Gayo et al., 2010). pDC are characterized by tissues where they acquire specialized functions. In the blood- the expression of BDCA-2, BDCA-4 (Dzionek et al., 2001), IL-3R stream, several subpopulations of immunogenic myDC, all of (CD123; Jahnsen et al., 2000), and ILT7 (Cao and Bover, 2010). them expressing CD11c, and the myeloid markers CD13 and pDC are found in the peripheral blood, lymph nodes, and the CD33, are present (Table 1). These cells include CD16+ (they thymus, and they are recruited to sites of inflammation under are also characterized by the expression of M-DC8; Schakel et al., pathological conditions (Swiecki and Colonna, 2010). 1999), BDCA-1+, and BDCA-3+ (Dzionek et al., 2001) that have different ability to stimulate allogeneic T cells (MacDonald et al., DENDRITIC CELLS IN CENTRAL AND PERIPHERAL 2002). Distinct phenotypical and functional characteristics are dis- TOLERANCE played by myDC resident in peripheral tissues. These myDC can be To avoid autoimmune reactions, self-reactive lymphocytes have to distinguished according to the expression of specific markers: lan- be deleted or rendered tolerant. Several mechanisms are operating gerin (CD207) expressing cells (Geissmann et al., 2002; Larregina in the central and peripheral compartments to induce and main- and Falo, 2005) are Langerhans cells (LC) and interstitial dermal tain tolerance. Defects in these mechanisms are associated with the DC localized in the skin; CD103+ DC reside in the lamina propria activation of immune responses against self-Ags (Goodnow et al., (LP) of the small intestine (Jaensson et al., 2008; Rescigno and Di 2005). Central tolerance occurs in the thymus and leads to the Sabatino, 2009); C-type lectin+ (DC-SIGN) DC are present in the deletion of self-reactive T cells through the positive and negative decidua (Laskarin et al., 2007); BDCA-1+ and BDCA-3+ DC have selection (Hogquist et al., 2005). The role of DC in central toler- been described in the lung (Demedts et al., 2005; Table 1). ance has become evident in the last decades. Thymic myDC are In addition to immunogenic myDC, other subsets of myDC very efficient in mediating negative selection of developing thy- with tolerogenic properties have been described such as DC mocytes (Brocker et al., 1997; Ohnmacht et al., 2009). In addition, expressing the scavenger receptor CD163 and immunoglobulin- peripheral myDC can migrate to the thymus and contribute to like transcript 3 (ILT3; Maniecki et al., 2006). We recently negative selection (Bonasio et al., 2006; Proietto et al., 2008). Both identified DC-10, which are tolerogenic DC characterized by the thymic myDC and pDC play an important role in promoting posi- expression of CD11c+, CD14+, CD16+, CD83+, and the tolero- tive selection of Tregs (Proietto et al., 2008; Hanabuchi et al., 2010; genic molecules HLA-G and ILT4 (Gregori et al., 2010). DC-10 Martin-Gayo et al., 2010). Thus, myDC and pDC cooperate in the display a mature phenotype since they express both HLA class thymus to promote on one hand negative selection of self-reactive II and co-stimulatory molecules. They have a unique cytokine T cells, and on the other positive selection of Tregs. secretion profile consisting of high levels of IL-10 in the absence To control immune responses to self-Ags that are not expressed of IL-12 (Gregori et al., 2010). Specialized subsets of tolerogenic in the thymus or may escape negative selection, different mech- DC have been described in each tissue where they maintain tissue anisms of tolerance are operational in the periphery during the homeostasis and tolerance (reviewed in Gregori, 2011). entire lifespan. Mechanisms of peripheral tolerance include cell Plasmacytoid DC are component of the innate immune system death with consequent clonal deletion, development of a state and are specialized in producing interferon-α (IFN-α)uponacti- of T cell unresponsiveness, and active suppression mediated vation via TLR7- and TLR9-mediated recognition of nucleic acids, by Tregs. DC, via the production of the immuno-modulatory and participate in T cell immunity (reviewed by Colonna et al., cytokines IL-10 and TGF-β or the expression of the tolerogenic

Table 1 | Different subsets of human dendritic cells.

Tissue distribution Markers Reference

Myeloid DC Immunogenic BDCA-1 Blood/tissues CD11c, BDCA-1 MacDonald et al. (2002), Dzionek et al. (2000, 2001) BDCA-3 Blood/tissues CD11c, BDCA-3 MacDonald et al. (2002), Dzionek et al. (2000, 2001) M-DC8 Blood/tissues M-DC8, CD16 Schakel et al. (1999) DC-SIGN Blood/decidua CD11c, DC-SIGN Laskarin et al. (2007) Langerhans cells Skin CD207, CD1a Larregina and Falo (2005), Geissmann et al. (2002) Myeloid DC tolerogenic CD163 Blood CD11c, ILT3 Maniecki et al. (2006) DC-10 Blood/tissues CD11c, CD14, CD16, CD83, HLA-G, ILT4 Gregori et al. (2010) CD103 Lamina propria CD11c, CD103 Jaensson et al. (2008), Rescigno and Di Sabatino (2009) Plasmacytoid DC pDC Blood/tissues BDCA-2, BDCA-4, CD123, ILT7 Dzionek et al. (2000, 2001), Jahnsen et al. (2000), Cao and Bover (2010)

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molecules indoleamine 2,3-dioxygenase (IDO) or ILTs (Morelli myDC in the CNS at early stages of the disease and their pres- and Thomson, 2007; Gregori, 2011), can regulate several of these ence within the demyelinating lesions indicate that myDC play a processes. role in re-activating T cell responses to myelin upon entry into the CNS (Wu and Laufer, 2007). In addition to their identifi- ROLE OF DENDRITIC CELLS IN PRIMING AND SUSTAINING cation in the CNS during the disease, analyses of myDC in the SELF-REACTIVE IMMUNE RESPONSES peripheral blood of MS patients revealed their ability to secrete In genetically susceptible individuals, autoimmune diseases may pro-inflammatory cytokines at higher levels than DC from nor- develop as a result of alterations in the expression of self-Ags by mal donors (Karni et al., 2006). These activated myDC, polarize DC, or access to immune privileged sites, or modification of the CD4+ T cells toward IFN-γ-producing effector cells (Karni et al., activation state of DC that became potent activators/inducers of 2006; Vaknin-Dembinskyet al.,2006). Thus, myDC in MS patients self-reactive effector T cells. Multiple evidences from pre-clinical are highly immunogenic and contribute to disease induction and models of autoimmune diseases indicate that DC loaded with progression. self-Ags acquired an activated phenotype and are able to trig- The role of pDC in the MS pathogenesis is less clear. No dif- ger autoimmune responses via the induction of T helper 1 (Th1) ferences in the absolute number of pDC have been found in the and Th17 responses (Torres-Aguilar et al., 2010). Priming of self- peripheral blood of MS patients. However, a reduced stimula- reactive T cells by activated DC that have taken up apoptotic cell tory activity of pDC and a limited expression of co-stimulatory debris may also lead to break-down self-tolerance and can result molecules upon in vitro activation were described, suggesting an in autoimmunity (Lleo et al., 2008). The pro-inflammatory envi- impairment in the maturation and an altered regulatory functions ronment generally observed in organs target of autoimmunity of pDC in MS patients (Stasiolek et al., 2006). can modify several tolerogenic DC functions, shifts the balance between tolerogenic and immunogenic DC toward the latter, and Systemic Lupus Erythematosus contributes to the development of autoimmune diseases. The induction of SLE and disease severity is associated with a Several factors in autoimmune patients indicate that the dys- defect in clearance of apoptotic cells by macrophages (Herrmann regulation in the immunogenic and tolerogenic DC is associated et al., 1998). This results in hyper-activation of DC and leads to with excessive self-reactive responses and inflammation. the chronic inflammation observed in SLE (Seitz and Matsushima, 2010). When apoptotic cells are not rapidly removed, they release ABERRANT ACTIVATION OF IMMUNOGENIC DENDRITIC CELLS blebs, in which SLE auto-Ags are clustered, and induce maturation IN HUMAN AUTOIMMUNE DISEASES of DC. These DC can stimulate the production of IL-2, IFN-γ, and, In the last decades, studies of DC in patients indicate that aberrant in particular, IL-17 by T cells that sustain autoimmune responses DC activation or functions are associated with different autoim- (Fransen et al., 2009). Although myDC are reduced in the periph- mune diseases as including Rheumatoid Arthritis (RA), Multiple eral blood of SLE patients (Robak et al., 2004; Migita et al., 2005), Sclerosis (MS), Systemic Lupus Erythematosus (SLE), Psoriasis, they contribute to effector T cell activation because of their acti- and Inflammatory Bowel Disease (IBD; Table 2). vated phenotype (Ding et al., 2006; Gerl et al., 2010). In line with Rheumatoid Arthritis this notion, monocytes from SLE patients undergo an accelerated In the peripheral blood of RA patients, but also in synovial fluids differentiation in vitro and express high levels of co-stimulatory and tissues, increased numbers of myDC and pDC are present molecules (Ding et al., 2006; Gerl et al., 2010). (Lebre and Tak, 2009). Studies on myDC from synovial fluid of Plasmacytoid DC are also reduced in the peripheral blood of RA patients show that these cells display an activated phenotype as SLE patients (Robak et al., 2004; Migita et al., 2005), but they accu- they express high levels of HLA-DR and co-stimulatory molecules. mulate in inflamed skin lesions (Mori et al., 1994) where they Interestingly, myDC in inflamed tissues are associated with T cells are selectively attracted by ChemR23, the chemokine receptor for in structures similar to germinal centers where they stimulate self- chemerin (Vermi et al., 2005). Moreover, circulating pDC from reactive T cells (Santiago-Schwarz, 2004). These myDC are also SLE patients migrate in response to CCL19 (Gerl et al.,2010). It has involved in promoting synovial inflammation due to their abil- been proposed that the increased responsiveness to CCL19 might ity to secrete pro-inflammatory cytokines (Jongbloed et al., 2006; lead to pDC accumulation in T cell area of lymph nodes where Lebre et al., 2008). they increase the priming of self-reactive T cells and contribute to The role of pDC in the RA pathogenesis is dual: on one hand SLE pathogenesis (Gerl et al., 2010). in synovial tissues pDC via the secretion of type I IFNs contribute to local inflammation, although at lower extend as compared to Psoriasis myDC (Pettit et al., 2000; Takakubo et al., 2008); on the other In psoriatic lesions, the frequency of myDC is 30-fold increased hand, pDC could play a role in activating B cells via the expression with respect to normal skin (Zaba et al., 2007). The large propor- α of B cell-activating factor (Lebre et al., 2008), leading to antibody tion of these cells secretes TNF- , IL-12, IL-23, and the inducible production, which sustain tissue damage. (iNOS; Lowes et al., 2005). These cytokines activate keratinocytes and fibroblasts to secrete pro-inflammatory Multiple Sclerosis cytokines (IL-6 and IL-1) that induce effector Th1 and Th17 The active participation of DC in the MS pathology is supported by cells, contributing to dermal inflammation and epidermal their presence and activation in the central nervous system (CNS) hyperplasia characteristic of psoriasis (Zheng et al., 2007; of MS patients (Pashenkov et al., 2002). Increased frequency of Pene et al., 2008).

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Table 2 | Role of dendritic cell subsets in human autoimmune diseases.

Disease DC subsets Localization Function Reference

Rheumatoid Arthritis myDC Blood/synovial tissues ↑ Effector T cell priming Santiago-Schwarz (2004), ↑ Pro-inflammatory cytokines Takakubo et al. (2008) ↑ DC activation pDC Synovial tissues ↑ IFN-α production Takakubo et al. (2008), ↑ B cell activation Lebre et al. (2008)

Multiple Sclerosis myDC Cerebrospinal fluid ↑ T cell activation Pashenkov et al. (2002), ↑ Pro-inflammatory cytokines Wu and Laufer (2007), Karni et al. (2006) pDC Blood Impairment in maturation Stasiolek et al. (2006) ↓ Ability to prime FOXP3+ Tregs

Systemic Lupus Erythematosus myDC Blood ↑ DC activation Seitz and Matsushima (2010), ↑ Effector Th1/Th17 cell priming Fransen et al. (2009) pDC Inflamed lesions/LN ↑ Effector T cell priming Robak et al. (2004), Migita et al. (2005), Gerl et al. (2010)

Psoriasis myDC Psoriatic lesions ↑ Pro-inflammatory cytokine Lowes et al. (2005), ↑ Effector T cell priming Zheng et al. (2007) pDC Psoriatic lesions ↑ IFN-α production Nestle et al. (2005)

Inflammatory Bowel Disease myDC Inflamed lesions ↑ DC activation te Velde et al. (2003), ↑ Pro-inflammatory cytokines Hart et al. (2005) ↑ T cell activation pDC Lamina propria ↑ DC activation Baumgart et al. (2011) ↑ IFN-α production ↓ TNF-α production

The frequency of IFN-α-secreting pDC is also increased in pDC from healthy subjects. However, these pDC were impaired in psoriatic lesions and participate to local inflammation (Nestle their ability to secrete IFN-α (Baumgart et al., 2011). Thus, these et al., 2005). results suggest that aberrant activation of pDC or alteration in their regulatory functions could play a role in the pathogenesis of IBD. Inflammatory Bowel Disease Several studies in Crohn’s disease (CD) and ulcerative colitis (UC) These examples clearly indicate that hyper-activation of myDC patients have demonstrated an abnormal intestinal accumulation is one of the key factors in promoting self-reactive T cell immunity. of DC expressing BDCA-1, which contribute to excessive T cell Moreover, an aberrant pDC distribution and function contribute activation (de Baey et al., 2003; te Velde et al., 2003; Silva et al., to the local inflammation in target organs of autoimmunity. In this 2004). DC from CD patients have an altered cytokine production scenario, activated DC are recruited to the inflamed tissues where profile since they produce higher levels of IL-12 and IL-6 than DC they secrete pro-inflammatory cytokines (i.e., IL-1, TNF-α, IFN- from healthy donors (Hart et al., 2005). Thus, myDC accumulate α, and IL-6) or express high levels of co-stimulatory molecules in the intestine of IBD patients where they activate pathogenic that induce an immune-stimulatory loop causing re-activation of T cells. self-reactive T cells and recruitment and/or the activation of other It has been recently reported that pDC might participate to immune cells, including additional DC. inflammation in the mucosa of CD and UC patients. Indeed high frequency of pDC was found in inflamed mucosa of CD and UC ALTERATION OF TOLEROGENIC DENDRITIC CELL FUNCTIONS patients. Studies on pDC from the peripheral blood of flaring CD AND AUTOIMMUNITY and UC patients demonstrated that they express higher levels of In homeostatic and resting conditions (in the absence of inflam- CD40 and CD86, and they secrete higher amounts of TNF-α than mation) DC preserve an immature or semi-mature phenotype,

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and actively participate in the maintenance of tolerance toward and it acts as an anti-inflammatory molecules, controlling apop- self-Ags. In these conditions, tissue resident tolerogenic DC tosis, T cell proliferation and activation (Otterbein et al., 2000; control self-reactive T cell responses by preventing excessive local Pae et al., 2004). In non-pathological conditions, Foxp3+ Tregs inflammation and autoimmune-mediated tissue damages. The promote IDO expression in myDC through the interaction of cyto- presence of high levels of pro-inflammatory mediators observed toxic T-lymphocyte antigen 4 (CTLA-4) with CD80 and CD86 in chronic inflamed tissues decreases the regulatory activity of (Fallarino et al., 2002, 2003; Grohmann et al., 2002). Resulting tolerogenic DC. myDC acquire the ability to generate Foxp3+ Tregs (Mellor and One of the most important features of tolerogenic DC is Munn, 2004). During inflammation, chronically activated myDC, their ability to secrete immuno-regulatory cytokines, such as although expressing high levels of CD80 and CD86, become refrac- IL-10 and TGF-β. IL-10 directly suppresses T cell responses by tory to the inhibitory signal induced by Foxp3+ Tregs and unable inhibiting the secretion of IL-2 and IFN-γ (Vieira et al., 1991) to express IDO. and by preventing T cell proliferation (Taga and Tosato, 1992). Indoleamine 2,3-dioxygenase can also be expressed by pDC Similarly, TGF-β potently inhibits T cell responses (Gorelik and alternatively activated with anti-CD40L and IL-3 (Martin-Gayo Flavell, 2002). IL-10 controls a number of different cells impli- et al., 2010) or with thymic stromal lymphopoietin (TSLP; cated in inflammatory responses, including APC (Mosser and Hanabuchi et al., 2010). These IDO expressing pDC have been Zhang, 2008). The expression of HLA class II, co-stimulatory shown to promote the induction of Foxp3+ Tregs. In the synovial molecules (de Waal Malefyt et al., 1991) and pro-inflammatory fluid of RA patients, IDO expressing pDC have been identified cytokines (Fiorentino et al., 1991a,b) is down-regulated by IL-10. (Takakubo et al., 2008), but their limited number and the presence On the other hand, IL-10 up-regulates the expression of tolero- of an increased frequency of activated myDC impair their ability to genic molecules such as ILT3 and ILT4 (as reviewed in Suciu-Foca counteract self-reactive effector T cell responses by the induction et al., 2005), and HLA-G (Moreau et al., 1999) on APC, render- of Tregs. ing them capable of dampening immune responses and inducing Immune cells and non-immune cells can play an important Tregs (Carosella et al., 2011). In the steady-state, DC secrete high role in driving the development of tolerogenic DC. It has been levels of IL-10, can modulate the activation of neighboring myDC, shown that human intestinal epithelial cells (IECs) through the and promote the de novo induction of tolerogenic DC. In vitro secretion of TSLP,TGF-β, and retinoic acid drive the development studies demonstrated that maturation of monocytes derived DC of CD103+ tolerogenic DC (Iliev et al., 2009). CD103+ DC pro- in the presence of exogenous IL-10 is inhibited, and resulting mote the de novo induction of Foxp3+ Tregs and inhibit Th1 and DC become able to induce anergic/suppressive T cells (Steinbrink Th17 responses (Iliev et al., 2009). In CD patients, IECs do not et al., 1997, 2002). Moreover, differentiation of monocytes derived express TSLP and fail to control DC-mediated pro-inflammatory DC in the presence of IL-10 results in a population of mature responses, resulting in abnormal release of IL-12 (Rimoldi et al., myDC, called DC-10, which secrete high levels of IL-10 and are 2005) and reduced ability to induce CD103+ DC (Rescigno and potent inducers of Ag-specific IL-10-producing type 1 regulatory Di Sabatino, 2009). This perturbation in the cross-talk between (Tr1) cells in vitro (Gregori et al., 2010; Pacciani et al., 2010). In IECs and DC disrupts the intestinal immune-homeostasis and addition to their ability to secrete high levels of IL-10, DC-10 promotes gut inflammation. strongly express ILT4 and HLA-G, which are necessary for effi- In conclusion, chronic inflammation and the presence of high cient Tr1 cell induction. In inflamed tissues, high amounts of levels of pro-inflammatory cytokines in target organs of autoim- pro-inflammatory cytokines lead to the down-regulation of IL-10 munity and in the periphery alters the regulatory activity of production that could impair the modulation of already differen- tolerogenic DC and generate an imbalance between tolerogenic tiated DC, and the de novo induction of tolerogenic DC, including and immunogenic DC, which sustains constant activation of DC-10. self-reactive T cells leading to tissue damage. It has been reported that mutations in IL-10 or in its receptor lead to the loss of IL-10 function and cause severe intractable infant STRATEGIES TO PROMOTE TOLERANCE BY and adult enterocolitis (Glocker et al., 2009, 2010), demonstrat- TARGETING DENDRITIC CELLS ing the critical role of IL-10 in maintaining intestinal tolerance. Autoimmune diseases are the result of a potent and de-regulated More recently, it has been shown that DC generated from periph- immuno-responses toward self-Ags mediated by a variety of eral monocytes of IBD children carrying a mutation in IL-10R immune cells, including B and T lymphocytes, and APC. The secrete significantly higher amounts of TNF-α, IL-12, and IL-23 critical role of DC in the initiation and in the progression of than DC from healthy controls (Begue et al., 2011). These data autoimmune diseases indicates that DC targeting therapies could indicate that impairment in the ability of DC to produce IL- represent a good alternative to current immuno-modulatory ther- 10 and to respond to it is critically involved in the pathogenesis apies already approved for the treatment of autoimmune diseases. of IBD. Two alternatives approaches can be foreseen to modulate DC: In addition to soluble factors, tolerogenic DC can express (i) therapies targeting immunogenic DC to lower their activation, immuno-regulatory enzymes such as IDO and heme oxygenase-1 (ii) therapies targeting tolerogenic DC to improve their function (HO-1), which suppress T cell responses and promote immune and induction. tolerance. IDO inhibits effector T cell proliferation by reducing Treatment with monoclonal antibodies (mAb) against pro- tryptophan that is necessary for cell division (Mellor and Munn, inflammatory cytokines or their receptors aiming to reduce the DC 2004). HO-1 is the rate-limiting enzyme in heme catabolism immunogenicity are currently under clinical investigation for the

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treatment of autoimmune diseases. Administration of Anakinra, a to generate human tolerogenic DC using IL-10, TGF-β, vitamin recombinant version of IL-1Rα, in combination with methotrex- D3, dexamethasone or rapamycin has been performed (Boks et al., ate (MTX), or of , a humanized mAb that competes 2012). Results from this study demonstrated that DC activated with IL-6 for receptor binding, provided good clinical benefit in the presence of IL-10 (IL-10 DC) showed the most powerful in RA patients (Smolen et al., 2008; Niu et al., 2011). Positive tolerogenic characteristics with high IL-10 production and low results were obtained also in patients with RA, CD, and psoria- T cell activation. Based on these results the authors suggested sis treated with anti-TNF-α mAb (Infliximab; Present et al., 1999; that IL-10 DC are the best suitable subset of tolerogenic DC for Cohen et al., 2000; Ricart et al., 2001). Two recent phase II clini- tolerance inducing therapies. We developed a protocol to gener- cal trials, proved the efficacy and safety of a two different mAbs ate human tolerogenic DC by differentiating monocyte derived against IL-17 (Ixekizumab; Leonardi et al., 2012)oritsreceptor DC in the presence of exogenous IL-10. Resulting cells, called (Brodalumab; Papp et al., 2012) for the treatment of Psoriasis. DC-10, represent a powerful subset of tolerogenic DC. DC-10 Despite these encouraging results, additional studies are needed are phenotypically and functionally stable and upon activation to evaluate the safety of long-term treatment with these mAbs they maintain their cytokine production profile (high IL-10/IL- and to define the optimal schedule for their efficacy. Notably, 12 ratio) and their ability to differentiate adaptive Ag-specific to obtain stable clinical benefit, chronic administration of these Tr1 cells (S. Gregori, personal communication). In alternative mAbs is required since clinical symptoms return after treatment to IL-10, a method to generate clinical grade tolerogenic DC withdrawal. from patients with RA using vitamin D3 and dexamethasone An alternative approach to block DC immuno-stimulatory has been also developed (Harry et al., 2010) and a clinical trial activity is the inhibition of co-stimulatory molecules (CD80 and for treating RA patients will be initiated soon (Moreau et al., CD86). In pre-clinical models of autoimmune diseases the efficacy 2009). Results from this first proof of principle clinical trial of CD28/B7 blockade by CTLA-4Ig has been shown (Salomon and will provide informations on the safety and efficacy of tolero- Bluestone, 2001). Interestingly, while the efficacy and tolerabil- genic DC-based cell therapy to restore tolerance in autoimmune ity of CTLA-4Ig () have been reported across multiple settings. international, randomized, double blind, placebo control trials in patients with active RA (Massarotti, 2008), its effect in other CONCLUSIONS AND PERSPECTIVES autoimmune diseases, such as Psoriasis and MS, is still not clear Over the past years significant progresses have been achieved (Sakthivel, 2009) and additional investigations are required. in understanding the pathological role of DC in autoimmune Results from these clinical trials indicate that therapies with diseases and how tolerogenic DC regulate and maintain tol- mAb aim at inhibiting pro-inflammatory cytokines or co- erance toward self-Ags. Although a number of questions still stimulatory signaling pathways are efficacious; however, they remain to be addressed, inhibition of the immunogenic branch required long-term administration with consequent long-term of DC function or induction of the tolerogenic one has become detrimental effects for patients. a feasible approach to restore tolerance in autoimmune diseases. Another alternative strategy to restore tolerance in autoim- Current approaches based on the administration of mAb against munity is to improve the induction and function of tolerogenic immunogenic proteins have been successful, however the lack of DC. The majority of the efforts have been focused on gener- information regarding long-term safety and the chronic infusion ating tolerogenic DC in vitro to be subsequently administered limited their broaden application. Alternatively, in vitro differenti- in vivo as cell therapy, rather than in promoting in vivo the expan- ated tolerogenic DC are of great potential interest as cell therapy for sion of tolerogenic DC. Different immune-modulatory agents there-establishment of immunological tolerance in autoimmune have been used in order to modify the phenotype, cytokine pro- diseases. Nevertheless, the optimal type of tolerogenic DC still files and activity of DC. Encouraging results have been obtained remains to be defined. It has to be taken into account that tolero- by treating DC with biological agents such as dexamethasone genic DC should be resistant to maturation either induced by in (Piemonti et al., 1999) or vitamin D3 (Penna and Adorini, 2000) vivo transfer or by inflammatory mediators. Moreover, the route or cytokines such as TNF-α (van Duivenvoorde et al., 2004, 2007) and dose of administration as well as the need of in vivo pharma- or IL-10 (Steinbrink et al., 1997, 2002; Sato et al., 2003; Gregori cological treatments for maintaining their tolerogenic functions et al., 2010). In pre-clinical models of arthritis (van Duivenvo- have to be still determined. Further studies in orde et al., 2004, 2007), EAE (Menges et al., 2002), and type 1 model as well as in large animals will elucidate these aspects diabetes (T1D; Feili-Hariri et al., 2002) the efficacy of in vitro and will allow the establishment of protocols with tolerogenic induced tolerogenic DC-based cell therapy has been demon- DC-based cell therapy for clinical application in autoimmune strated. In addition, repetitive injection of immature DC has been diseases. shown to protect mice from collagen-induced arthritis (Charbon- nier et al., 2006). To date, in the field of autoimmune diseases, ACKNOWLEDGMENTS no data have been published using immuno-modulatory pDC as This work was supported by Telethon Foundation “Comitato therapeutic tools. Telethon Fondazione Onlus” Core Grant OSR-TIGET project E2 Despite the fact that in vitro generated human tolerogenic DC (Rome) and by the Italian Ministry of Health. Dr. Giada Amodio have been studied in research settings, the described methods conducted this study as partial fulfillment of her PhD in Molec- have not been translated into clinical grade protocols. Recently ular Medicine, Program in Basic and Applied Immunology, San a comparative analysis of good manufacturing practice protocols Raffaele University, Milan, Italy.

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Role of ChemR23 nal authors and source are credited and myeloid dendritic cells act as regula- Accumulation of plasmacytoid DC: in directing the migration of myeloid subject to any copyright notices concern- tory dendritic cells to induce anergic roles in disease pathogenesis and and plasmacytoid dendritic cells to ing any third-party graphics etc.

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