Regulatory T cells in children with allergy and asthma: It is time to act

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Regulatory T cells in children with allergy and asthma: It is time to act夽 Anna Stelmaszczyk-Emmel ∗ Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland

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Article history: Accepted 13 November 2014 Available online xxx Keywords: Regulatory T cells Allergy Asthma Children Immunotherapy

a b s t r a c t Nowadays allergy and asthma are a huge medical problem. Despite deeper and more precise knowledge concerning their pathogenesis and the role of the immune system in these processes, so far immunotherapy is the only treatment which can modify the course of these diseases. Considering that regulatory T cells (Treg cells) have a great significance in pathogenesis of both diseases it seems appropriate to pay attention to their role in the treatment process. This work summarizes the Treg cells characteristics, the influence of allergen specific immunotherapy and other treatment modalities on Treg cells, and the possibility of using Treg cells in therapy. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

2. Allergy and asthma

Allergy and asthma are one of the most important issues for the public health. This is mainly because their occurrence is high and growing each year. Over the last few decades the prevalence of allergic diseases has dramatically increased. Allergy can be even described as an epidemic of the 21st century. At the moment, according to European Federation of Allergy & Airways Diseases Patients’ Association (EFA) around 113 million people in Europe suffer from allergic rhinitis and around 68 million from allergic asthma (EFA Book on Respiratory Allergies, 2011). WHO states that 235 million people suffer from asthma (WHO, Asthma, Fact sheet N◦ 307, 2013). Even though the prevention and control of allergy and asthma are the leading priority for many organizations and governments, it is estimated that next year 50% of Europeans will suffer ´ from allergy (Samolinski et al., 2012). Respiratory allergies affect around 20–30% of European population (EFA Book on Respiratory Allergies, 2011). Impaired immune responses to allergens are one of the most important factors in the development of allergy. It is already known that Treg cells play the key role in this situation. This work summarizes the classification, nomenclature, and characteristics of Treg cells, and their role in treatment.

Allergy is one of the immune tolerance-related disorders resulting from a failure of the regulatory network. It is caused by complex, both innate and adaptive, immune responses to natural environmental allergens with T helper type 2 (Th2) cells and allergen specific IgE predominance and are characterized by an inflammatory reaction associated with increased production of Th2 cytokines (Palomares et al., 2010; Ring et al., 2012). Asthma is a chronic inflammatory disease of the airways wall. There are many hypotheses on the pathogenesis of asthma; all of them involve the role of immune system and its components, such as dendritic cells (DCs), Th cells, mast cells, granulocytes, and NKT cells. Treg cells also have their part in it (Jutel, 2014; Lambrecht and Hammad, 2013). Asthma is not only associated with allergy, although more than half of asthmatic patients are allergic (Agache et al., 2012). The EFA states that allergies contribute to around 90% of asthma cases (EFA Book on Respiratory Allergies, 2011). Allergen specific immunotherapy (ASIT) is so far, along with allergen avoidance, the only specific treatment of allergic disorders, with the potential to modify the course of the disease. ASIT can be divided according to the way of delivery into subcutaneous immunotherapy (SCIT), sublingual immunotherapy (SLIT), and recently oral immunotherapy OIT. ASIT has already been used over a 100 years. Treatment of asthma is mainly symptomatic and is based on pharmacological therapies, which do not influence dysregulated immune responses. However, ASIT can be effectively applied and has immunomodulatory effects in allergic asthma (Burks et al., 2013).

夽 This paper is part of a special issue entitled “Molecular basis of ventilatory disorders” guest-edited by Dr. Mietek Pokorski. ∗ Tel.: +48 226296517; fax: +48 226296517. E-mail address: [email protected] http://dx.doi.org/10.1016/j.resp.2014.11.010 1569-9048/© 2014 Elsevier B.V. All rights reserved.

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3. Regulatory T cells 3.1. Characteristics of Treg cells The studies performed during the last two decades have shown that regulatory T cells are one of the most important players in the acquired immunity. Regulatory mechanisms are necessary to maintain peripheral tolerance of the immune system. The population of regulatory T cells is still not fully described, and new facts and evidence are published continuously (Romagnani, 2014). This can be a reason why there is no clear classification of Treg cells subsets and confusing nomenclature is still in use. Many studies distinguish four or five Treg cells subsets. They include, ‘natural’, Treg cells (nTreg), and, ‘inducible’, Treg cells (iTreg) which are subdivided into: induced Treg cells, type 1 regulatory T cells (Tr1), TGF-␤ expressing Th3, IL-17 producing FoxP3 Treg cells, CD8+Treg cells, double negative CD4 CD8 TCR␣␤+Treg cells, and TCR␥␦ Treg cells (Palomares et al., 2014; Siegmund et al., 2009). These subpopulations are distinguished due to conditions in which they are produced, specific markers, secreted cytokines, mode of action, and localization. To facilitate and clarify the classification after Third International Conference on Regulatory T Cells and Th Subsets and Clinical Application in Human Disease (October 2012), Abbas et al. (2013) proposed a recommendation for a new Treg cells nomenclature. According to their suggestion, anatomical location of Treg cells should be indicated in the subset name, because it is the most informative. It is recommended to use, ‘thymus derived Treg cells’ instead of ‘natural FoxP3 Treg cells’ and ‘peripherally derived Treg cells’ instead of ‘induced’ or ‘adaptive’. For those Treg populations which are of unknown origin the most appropriate term is, ‘FoxP3 Treg cells’. It should be clearly specified if Treg cells were generated ex vivo, and the suggested term for such cells is, ‘in vitro-induced Treg cells’. The discussion on the classification of Treg cells might not seem a crucial problem. However, the term, ‘Treg cells’, is often misused in the literature. That is why, it is recommended to use the term, ‘Treg cells’, only when there is definitive evidence justifying its use, when authors are sure that the population considered to be ‘Treg cells’ has (or had) suppressive ability or characteristic transcriptional, epigenetic, and protein marker of this cell population. There are suggestions that Tr1 cells, which do not have any specific markers, probably do not constitute a specific lineage, but rather have a transient status of different effector T cells (Romagnani, 2014). In this review the term ‘Treg cells’ will be used in accordance with the new recommendations. The largest population of regulatory T cells are thymus derived regulatory T cells. They were first described by Sakaguchi et al. (1995). Transcription factor FoxP3 is essential for their function. In addition, they express high levels of interleukin-2 (IL-2) receptor ␣chain (CD25). Low expression of the IL-7 receptor CD127 is helpful in their identification (Liu et al., 2006). They have naïve phenotype (CD45RA), but some antigen experienced memory tTreg cells expressing CD45RO can also be found (Miyara et al., 2009). Other markers, unexpressed in the general population, but supportive in describing the maturation status of the cells, suppressive abilities and subpopulation status include: coinhibitory receptor cytotoxic T lymphocyte antigen 4 (CTLA-4), CD39, HLA-DR, GITR, absence of CD49d, ICOS, Helios, OX40, GARP, CD73, CD147 (Baecher-Allan et al., 2006; Ito et al., 2008; Kaczmarek et al., 1996; Landskron and Taskén, 2013; Romagnani, 2014; Stockis et al., 2009). Another characteristic feature enabling to distinguish Treg cells from other T cells, especially T effector cells is Treg specific demethylation region (TSDR). TSDR is fully demethylated in tTreg cells, partially demethylated in pTreg cells and, what is important, methylated in T effector cells. Demethylation of TSDR is essential for stable FoxP3 expression. Analysis of TSDR demethylation is

possible using both blood and tissue samples, but it requires more effort than the assessment of surface markers expression (Baron et al., 2007; Floess et al., 2007; Wieczorek et al., 2009). Treg cells the control immune response by suppressing target cells by different mechanisms. Depending on the origin or subpopulation status they can act directly (cell-to-cell contact) or by cytokine production (TGF-␤, IL-10). In addition, they can also fulfil their function through cytolysis (perforin and granzyme B), and modulation of dendritic cells maturation and function (Cao et al., 2007; Loebbermann et al., 2012; Sakaguchi et al., 2009; Zhao et al., 2006). The division into Treg cells subpopulations is not clearly explained and understood. Many authors observed the Treg lineage plasticity. Generally, tTreg cells are considered to be involved in the prevention of autoimmune processes, while pTreg cells are responsible for the induction of oral and gut tolerance (Povoleri et al., 2013). Tr1 cells are classified as a distinct population of tTregs. They are induced in the periphery under specific conditions and they are antigen-specific. Chronic exposure to antigen and the presence of IL-10 are essential to their development (Levings et al., 2005; Vieira et al., 2004). Characteristic features of this subpopulation are: production of high levels of suppressive cytokines (IL-10 and TGF-␤) and lack of FoxP3 expression. They may express ICOS, CD18, LAG3 and CD49b. A specific marker for this population is still unknown. Their suppressive activity is based on secretion of IL-10. They are able to directly inhibit the T effector cells proliferation and indirectly suppress the T effector cells activation. They also produce perforin and granzyme B and use the cell-contact-dependent mechanism (Gregori et al., 2012; Mandapathil et al., 2010). 3.2. Regulatory T cells in allergy and asthma Treg cells play a major role in the regulation of allergic reactions. They induce and maintain immune tolerance to allergens. Physiologically, Treg cells should keep a state of tolerance to innocuous substances and limit incorrect or excessive immune responses. Several pathways allow Treg cells to control and modify the development of allergic reactions. Treg cells directly inhibit the activation of Th2 cells (suppress the production of IL-4, IL-5, IL-9 and IL-13), block the migration of effector T cells into inflamed tissue, suppress the production of IgE, induce IgG4 in B cells, and limit Th17-mediated inflammation as recently demonstrated in mice (Baecher-Allan et al., 2004; Kleer et al., 2004; Palomares et al., 2010). It is known that in allergic individuals the number of regulatory T cells is often decreased and their function is impaired (Akdis et al., 2004; Lee et al., 2007; Stelmaszczyk-Emmel et al., 2013; Xu et al., 2007). One of the hypotheses on asthma pathogenesis considers the role of deficiency in regulatory T cells. It can concern not only frequency but also functional deficiency, can be caused by genetic predisposition, environmental factors, any other trigger that may disturb fragile immunological balance (Lambrecht and Hammad, 2013). The number of pulmonary FoxP3 Treg cells is decreased in asthmatic children (Hartl et al., 2007) and their function can be inhibited by overproduction of TNF-␣, IL-6 and TSLP (Nguyen et al., 2010). 3.3. Influence of treatment on regulatory T cells 3.3.1. Allergen specific immunotherapy (ASIT) Changes in the balance between allergen-specific Treg cells and Th2, Th17, Th22 and/or Th1 cells is very crucial in the development and treatment of allergic diseases. ASIT induces several cellular and molecular events involving Treg cells. According to many authors, ASIT should generate the of allergen-specific regulatory T cells, down-regulate Th2 response, and induce a shift in the regulatory or Th1 phenotype (Akdis and Akdis, 2014; Scadding and Durham,

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2009). Clinical observations revealed that the effects of ASIT are ambiguous and not all patients show the same changes in immunological responses. In many studies, clinically successful ASIT goes together with increased frequency of Treg cells, increased production of IL-10, and also hypomethylation of FoxP3 (Fujimura et al., 2011; Scadding et al., 2010; Suárez-Fueyo et al., 2014; Swamy et al., 2012; Syed et al., 2014). Other authors show that actually there are changes in Treg cells (Kim et al., 2011; Lou et al., 2012; Månsson et al., 2010; Moed et al., 2013). According to Nieminen et al. (2009), induction of Treg cells and Th1 response persists over 3 years after ASIT. However, laboratory evaluation of immunological responses to treatment is difficult. In the studies above mentioned, the authors used different markers to assess the Treg cells population, so their results cannot be easily compared. There is still no convenient biomarker for the immunological assessment of the efficacy of ASIT. 3.3.2. Vitamin D3 Recently, great attention is focused on vitamin D3. As it is known from many epidemiological studies, apart from the well-described function in calcium homeostasis and bone health, it plays an important role in immunomodulation. Deficiency or insufficiency of vitamin D3 is connected with increased risk of autoimmune diseases (including allergy and asthma). Vitamin D3 influences cells involved in both innate and adaptive immune response. It also affects regulatory T cells. Vitamin D3 directly promotes FoxP3+ and IL-10+ Tregs and secretion of immunomodulatory cytokines (IL-10 and TGF-␤) acting on T cells, and upregulates the inhibitory cytotoxic T-lymphocyte antigen 4 (CTLA-4). However, its effect on Th2 and IgE production is still unclear. Because of the importance of Tregs cells in the pathophysiology of allergy and asthma, many approaches (together with patientsbased studies) have been evaluated to demonstrate vitamin D3’s ability to enhance the number and function of Treg cells in the immune-mediated diseases. The main question is, if therapeutic application of vitamin D3 in patients with autoimmune disorders can affect Tregs and can influence the course of disease (Baris et al., 2014; Chambers and Hawrylowicz, 2011; Chambers et al., 2014; Maalmi et al., 2012). 3.3.3. Other drugs – methimazole, rapamycin Other drugs which are also capable of influencing regulatory T cells are: rapamycin and methimazole. Methimazole is an antithyroid drug, used in the treatment of Graves’ disease (Sato et al., 2012). Its main mechanism of action is to lower the level of thyroid autoantibodies (Antonelli et al., 2006; Molnár, 2007). Regulatory T cells, like in other autoimmune diseases, play a role in the development of Graves’ disease. Currently, there are studies which help to understand their role in the immune response. An in vitro study published by Klatka et al. (2014) shown that methimazole is able to correct dysfunctionality in the suppressive function of regulatory T cells in Graves’ disease. Moreover, for the suppressive capacity of Treg cells, the ration Treg/T effector cells seems even more important. Rapamycin is a well known immunosuppressive drug used to prevent acute graft rejection. In vitro studies conducted by Battaglia et al. (2006) have shown that rapamycin promotes the expansion of fully functional FoxP3 Treg cells. Importantly from the clinical standpoint, these results were applicable not only for healthy subjects, but also for patients with autoimmune diseases (T1D). There are many other studies showing the use of rapamycin in these settings (Akimova et al., 2012; Gu et al., 2014; Lu et al., 2014; Moreira-Teixeira et al., 2012; Scottà et al., 2013). 4. Regulatory T cells in therapy Physiological importance and capability of changing the course of disease by decreasing unnecessary immune responses make Treg

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cells very promising agents for cell-based therapy. Several studies showed that antigen-specific Treg cells are more effective than polyclonal Treg cells. They are capable to move to, and act directly at, the site of disease (Albert et al., 2005; Sagoo et al., 2011; Tarbell et al., 2004). Successful transfers of purified Treg cells, able to inhibit or prevent a disease, have been documented in many animal models (Lapierre et al., 2013; Mukherjee et al., 2003; Prinz and Koenecke, 2012). There are several ongoing or completed clinical trials in which infusion of Treg cells is applied. There are clinical trials concerning the use of Treg cells in GvHD prevention, and hematologic cancers and also in T1D and organ transplantation. In the majority of studies, modified Treg cells are used. The cells were expanded in vitro; however, in one trial injected T cells were genetically modified. Differences between unmodified and genetically modified cells have been discussed in a recent review (Jethwa et al., 2014). The authors demonstrate theoretical advantages and limitations of immunotherapy using gene-modified vs. non-modified Treg cells. So far, in children there are no ongoing or documented studies utilizing genetically modified Treg cells. Satisfactory results have already been obtained in T1D in children treated with autologous expanded ex vivo Treg cells (Marek-Trzonkowska et al., 2013, 2014). The treatment was safe and well-tolerated and it seems fully justified to say that antigen-specific Treg cells are the right direction to explore. Many important issues such as: the dose of Treg cells, treatment schedule, etc. are still waiting to be worked out. Induction of Tregs-based therapies into clinical practice is difficult because Treg cells are present in a low number in the peripheral blood, their expansion is difficult and requires proper conditions in accordance with good manufacturing practice (GMP). Attention should be drawn not only to therapy with Treg cells infusion but also to combination therapy (ASIT and drugs which influence Treg cells) to improve impaired Treg cells function. References Abbas, A.K., Benoist, C., Bluestone, J.A., Campbell, D.J., Ghosh, S., Hori, S., Jiang, S., Kuchroo, V.K., Mathis, D., Roncarolo, M.G., Rudensky, A., Sakaguchi, S., Shevach, E.M., Vignali, D.A., Ziegler, S.F., 2013. Regulatory T cells: recommendations to simplify the nomenclature. Nat. Immunol. 14, 307–308, http://dx.doi.org/10.1038/ni.2554. Agache, I., Akdis, C., Jutel, M., Virchow, J.C., 2012. Untangling asthma phenotypes and endotypes. Allergy 67, 835–846. Akdis, M., Akdis, C.A., 2014. Mechanisms of allergen-specific immunotherapy: multiple suppressor factors at work in immune tolerance to allergens. J. Allergy Clin. Immunol. 133, 621–631, http://dx.doi.org/10.1016/j.jaci.2013.12.1088. Akdis, M., Verhagen, J., Taylor, A., 2004. Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J. Exp. Med. 199, 1567–1575. Akimova, T., Kamath, B.M., Goebel, J.W., Meyers, K.E., Rand, E.B., Hawkins, A., Levine, M.H., Bucuvalas, J.C., Hancock, W.W., 2012. Differing effects of rapamycin or calcineurin inhibitor on T-regulatory cells in pediatric liver and kidney transplant recipients. Am. J. Transplant. 12, 3449–3461, http://dx.doi.org/10.1111/j.1600-6143.2012.04269.x. Albert, M.H., Liu, Y., Anasetti, C., Yu, X.Z., 2005. Antigen-dependent suppression of alloresponses by Foxp3-induced regulatory T cells in transplantation. Eur. J. Immunol. 35, 2598–2607. Antonelli, A., Rotondi, M., Fallahi, P., Romagnani, P., Ferrari, S.M., Barani, L., Ferrannini, E., Serio, M., 2006. Increase of interferon-gamma-inducible CXC chemokine CXCL10 serum levels in patients with active Graves’ disease, and modulation by methimazole therapy. Clin. Endocrinol. (Oxf) 64, 189–195. Baecher-Allan, C., Viglietta, V., Hafler, D.A., 2004. Human CD4+ CD25+ regulatory T cells. Semin. Immunol. 16, 89–97. Baecher-Allan, C., Wolf, E., Hafler, D.A., 2006. MHC class II expression identifies functionally distinct human regulatory T cells. J. Immunol. 176, 4622–4631. Baris, S., Kiykim, A., Ozen, A., Tulunay, A., Karakoc-Aydiner, E., Barlan, I.B., 2014. Vitamin D as an adjunct to subcutaneous allergen immunotherapy in asthmatic children sensitized to house dust mite. Allergy 69, 246–253, http://dx.doi.org/10.1111/all.12278. Baron, U., Floess, S., Wieczorek, G., Baumann, K., Grützkau, A., Dong, J., Thiel, A., Boeld, T.J., Hoffmann, P., Edinger, M., Türbachova, I., Hamann, A., Olek, S., Huehn, J., 2007. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Eur. J. Immunol. 37, 2378–2389. Battaglia, M., Stabilini, A., Migliavacca, B., Horejs-Hoeck, J., Kaupper, T., Roncarolo, M.G., 2006. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+

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