Regulatory T cell heterogeneity and therapy in autoimmune diseases

Journal Pre-proof Regulatory T cell heterogeneity and therapy in autoimmune diseases

Rui Zhang, Jinlin Miao, Ping Zhu PII:

S1568-9972(20)30294-9

DOI:

https://doi.org/10.1016/j.autrev.2020.102715

Reference:

AUTREV 102715

To appear in:

Autoimmunity Reviews

Received date:

20 June 2020

Accepted date:

28 June 2020

Please cite this article as: R. Zhang, J. Miao and P. Zhu, Regulatory T cell heterogeneity and therapy in autoimmune diseases, Autoimmunity Reviews (2020), https://doi.org/ 10.1016/j.autrev.2020.102715

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© 2020 Published by Elsevier.

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Regulatory T Cell Heterogeneity and Therapy in Autoimmune Diseases Rui Zhanga,1, Jinlin Miaoa,b,1, Ping Zhua,* a

Department of Clinical Immunology, PLA Specialized Research Institute of Rheumatoid &

Immunology, Xijing Hospital, Fourth Military Medical University, Xi’an, China b

National Translational Science Center for Molecular Medicine, Fourth Military Medical

University, Xi’an, China *

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Corresponding author: Ping Zhu

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Department of Clinical Immunology, PLA Specialized Research Institute of Rheumatoid & Immunology, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road,

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Xi'an 710032, Shaanxi Province, PR China.

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Telephone: +86-29-84773951; Fax: +86-29-83240610

Rui Zhang and Jinlin Miao contributed equally to this work.

Highlights

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E-mail: [email protected]

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Regulatory T cells are heterogeneous and can be divided into different subsets.

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Non-lymphoid tissue resident Treg cells exert special tissue-related functions which go far beyond their immunosuppressive capability.

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Treg-targeted therapy, including Treg-based cellular transfer, IL-2 therapy and Treg targeted drugs, will become a promising strategy for autoimmune diseases treatment.

Abstract

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Journal Pre-proof Regulatory T (Treg) cells are a group of CD4+ T cell with high expression of CD25 and cell linage specific transcription factor forkhead box P3 (Foxp3) and play a vital role in maintaining immune homeostasis. In the last two decades, researchers have shown that Treg cells involved in the occurrence, development and prognosis of many diseases, especially in autoimmune diseases. Treg targeted therapies, such as low-dose interleukin-2 (IL-2) treatment and Treg infusion therapy,

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which are aimed at restoring the number or function of Treg cells, have become a hot topic in

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clinical trials of these diseases. It is believed that Treg cells are heterogeneous. Different subsets of Treg cells have various functions and play different parts in immunomodulatory. Gaining

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insights into Treg heterogeneity will help us further understand the function of Treg cells and

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provide news ideas for the selective therapeutic manipulation of Treg cells. In this review, we

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Keywords

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autoimmune diseases.

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mainly summarize the heterogeneity of Treg cells and their potential therapeutic value in

T-Lymphocytes, Regulatory; Subset; Immunotherapy

1. Introduction In 1995, Sakaguchi and his colleagues found that CD4+CD25+ cells contribute to maintaining self-tolerance by down-regulating immune response to self and non-self antigens in mice, which revealed potential involvement of these cells in autoimmune diseases [1]. Then in 2003, the close relationship between the transcription factor Foxp3 and development of CD4+CD25+ Treg cells

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was found [2-4]. These pillar researches gave us a general knowledge of Treg cells and initiated new lines of research into pathogenesis and possible treatments of immune-related diseases. Over the past two decades, researchers revealed the important role of Treg cells in the pathogenesis of various diseases. In many autoimmune diseases, including rheumatoid arthritis (RA) [5-10], systemic lupus erythematosus (SLE) [11-13], ankylosing spondylitis (AS) [14],

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systemic sclerosis [15,16] and so on, the qualitative or quantitative defects of Treg cells have been

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widely reported. Several risk factors, including genetics [17] and environmental factors [18], could also involve in the occurrence and development of these diseases by regulating the number

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and function of Treg cells. These findings indicate that targeting Treg cells is a promising new

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therapeutic approach in autoimmune diseases. And several clinical trials of Treg cells infusion

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have already obtained affirmative effects on alleviating disease activity [19,20].

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Since Treg cells are heterogeneous and show functional and phenotypic differentiation within different subsets, in this review, we mainly discuss different subsets of CD4+CD25+Foxp3+ Treg

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cells, including tissue Treg. We also summarize Treg-targeted therapies in autoimmune diseases which may provide us new insights into understanding and treatment of these diseases. 2. Function of Treg cells

CD4+CD25+Foxp3+ Treg cells are a component of the immune system with an indispensable role in maintaining immune tolerance and homeostasis. Recent researches reveal that they can also control non-immunological processes. Treg cells resident in tissue have unique tissue-related functions which are critical to safeguarding organismal homeostasis [21,22]. Treg cells exert immunosuppressive function through multiple mechanisms, including secreting inhibitory cytokines such as IL-10, IL-35 and TGF-β [23-25], metabolic regulation via

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IL-2 or CD 73 and CD39 [26-29], cytolysis [30] and modulation of antigen presenting cells (APCs) [31,32]. Aside from these classical mechanisms of how Treg cells exert their inhibition function, recent advances uncover other mechanisms of Treg function. Schmidleithner et al. report that Treg cells

highly express

enzyme

hydroxyprostaglandin

dehydrogenase,

which

catabolizes

prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2. And in visceral adipose tissue,

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Treg-cell-mediated generation of 15-keto PGE2 increase suppressive activity of Treg and impact

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conventional T cell proliferation [33]. Akkaya B et al. indicate a novel pathway for Treg-mediated suppression by depleting the complex of cognate peptide and major histocompatibility complex

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class II from the dendritic cell (DC) surface in an antigen-specific manner which reduces the

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capacity of DCs to present antigen and results in the failure to activate naive T cells [34]. These

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novel mechanisms give clearer understanding of Treg function and molecules involved may

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become new targets in Treg-associated treatment. 3. Treg subsets

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There are many controversies related to Treg cells in autoimmune diseases, especially the issue of decreased numbers versus dysfunction of Treg cells in the pathogenesis of autoimmune diseases. These controversies may partly be explained by Treg heterogeneity in their phenotype and function. Unfortunately, there is still no systematic and comprehensive classification method for Treg subsets up to now. For all this, making the acquaintance of the existing classification methods will pave the way for a better understanding of Treg heterogeneity as well as the pathogenesis of autoimmune diseases. At present, the following subpopulations of Treg cells are widely investigated by researchers. 3.1 tTreg and pTreg

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According to the origin, Treg cells can be divided into two groups: thymus-derived Treg (tTreg) cells or natural Treg (nTreg) cells developing from immature thymocytes and peripheral Treg (pTreg) cells or induced Treg (iTreg) cells developing in periphery from mature CD4+ T cells in certain conditions. It was once believed that transcription factor Helios [35] and cellular surface glycoprotein Neuropilin-1 (Nrp-1) [36,37] which were highly expressed by tTreg cells can be used

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to distinguish tTreg cells from pTreg cells. However later studies revealed that pTreg cells can

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upregulate these two factors under certain conditions [38,39]. So far there is no reliable maker to distinguish tTreg cells from pTreg cells.

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3.2 aTreg, rTreg and non Treg

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Another classification of Treg cells subsets in human is based on the expression of CD45RA and ﹣

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Foxp3 [40]: CD45RA+Foxp3lo resting Treg (rTreg) cells, CD45RA Foxp3hi activated Treg (aTreg) ﹣

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cells and cytokine-secreting CD45RA Foxp3lo non-suppressive T (non-Treg) cells, the majority of which are not Treg cells (Table 1). Further, rTreg cells could acquire an aTreg phenotype and exert

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suppression after proliferation upon TCR simulation. These three different subpopulations of Treg cells have been studied in many autoimmune diseases. In RA and Behcet’s disease, researchers reveal that the proportion of total Treg cells and aTreg cells, but not rTreg cells is significantly decreased compared to healthy controls (HCs) [41]. In multiple sclerosis, aTreg cells show a significant reduction and express higher levels of PD-1 compared to HCs which denotes an exhausted and dysfunctional status of aTreg cells [42]. Several studies demonstrate a significant increase in rTreg and non-Treg subsets in patients with active SLE [43,44]. And rTreg cells sorted from peripheral blood mononuclear cells of SLE patients fail to inhibit the responder T cell proliferation. These results indicate that different Treg subsets may

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serve diverse functions in autoimmune diseases and further study in those Treg subsets is likely to lead us to an in-depth understanding of the pathogenesis of these diseases. Table 1 Key properties of rTreg cells, aTreg cells and non-Treg cells rTreg cells

aTreg cells ﹣

non-Treg cells ﹣

CD25++CD45RA+Foxp3lo ﹣ CTLA-4loKi-67

CD25+++CD45RA Foxp3hi CTLA-4hiKi-67+

CD25++CD45RA Foxp3lo CTLA-4int

cytokine production

Poorly produces IL-2 or IFN-γ

Scarcely produces IL-2 or IFN-γ

Produces high amounts of IL-2, IFN-γ and IL-17

methylation status of the FOXP3 gene

CpG methylation sites in 5’ flanking region and the STAT5-responsive region were completely demethylated

The 5’ flanking region was also highly demethylated, the STAT5-responsive region was less demethylated

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Potently suppressed the proliferation of responder cells

Didn’t have suppression function

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3.3 Th-like Treg

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In vitro suppressive activity

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cell surface molecules

In recent years, Treg cells that phenotypically mirror effector T helper (Th) cells and are known as

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Th-like Treg cells have been widely studied in rodent and human. These Treg cells express specific Th-associated transcription factors and play a regulatory or proinflammatory role during different types of immune responses (Table 2). Th1-like Treg cells upregulate Th1 specific transcription factor T-box transcription factor 21 (T-bet) and are thought as a unique Treg subset specific in inhibiting type 1 inflammation [45-47]. Th1-like Treg cells also express C-X-C motif chemokine receptor 3 (CXCR3) in a T-bet-dependent manner which enables them to migrate to sites of type 1 inflammation and inhibit Th1 cell responses. Th17- and Th2-like Treg cells acquire expression of the transcription factor RAR-related orphan receptor γt (RORγt) [48] and GATA-3 [49] and restrain Th17 cells responses and Th2 cells responses, respectively. However, in some

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autoimmune diseases and tumors, Th-like Treg cells secrete pro-inflammatory cytokines and display diminished suppressive function [50-56]. Therefore, Th-like Treg cells may exert suppressive capacity or proinflammatory effect in a context-dependent manner and contribute to different prognosis outcomes of varying diseases. And studies in different subsets of Th-like Treg cells will give us further understanding of pathogenic mechanism of various immune-related

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disorders and potential strategies in the treatments of these diseases.

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Table 2 Characters of Th-like Treg cells

Th2-like Treg cells

Th17-like Treg cells

transcription factors

T-bet

GATA-3 and IRF-4

RORγt

cytokine production

IFN-γ

stimulating factors

IL-12, IFN-γ, IL-27

chemokine receptor

CXCR3, CCR5

4. Tissue Treg

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tissue specificity

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Th1-like Treg cells

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IL-4, IL-13

IL-17

IL-4, IL-5

IL-1β, IL-6, IL-12

CCR4

CCR4, CCR6

skin

gastrointestinal tract

Aside from different populations of Treg cells in peripheral blood and lymphoid organ, there are also various subsets of Treg cells in non-lymphoid tissue. Non-lymphoid tissue resident Treg cells show unique phenotype, tissue specific transcription factors and distinct T cell receptors (TCR) repertoire. And their special tissue-related functions go far beyond their immunosuppressive capability [21,22,57]. 4.1 Treg cells in adipose tissue

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The most widely demonstrated tissue-resident Treg cells are Treg cells in adipose tissue, especially in visceral adipose tissue (VAT) [58]. VAT Treg cells account for more than half of CD4+ cells [59] in adipose. These cells appear to be mostly of thymic origin for they highly express Helios and Nrp1 [60] and have a distinct TCR repertoire [59]. VAT Treg cells express majority of signatures as canonical Treg cells do but display a distinct chemokine/chemokine receptor pattern, produce abundant IL-10 and upregulate genes downstream of IL-10R [59]. They also express peroxisome

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proliferator-activated receptor γ (PPARγ), an important transcription factor for adipocyte

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differentiation [61]. And loss-of-function study reveals that PPARγ is an important factor

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controlling Treg cells accumulation and phenotype in adipose tissue [61]. VAT Treg cells play a

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vital role in the maintenance of immune and metabolic homeostasis of adipose tissue. They not

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only act as important negative regulators of VAT inflammation and insulin resistance [62,63], but

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also involve in adipose tissue function including efficient thermogenesis and lipolysis [64,65]. 4.2 Treg cells in skeletal muscle

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In skeletal muscle, Treg cells also show a transcriptome distinct from that of lymphoid-organ Treg cells. Genes encoding IL-10, amphiregulin (Areg) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) are highly expressed [66]. And just like VAT Treg cells, skeletal muscle Treg cells highly express Helios and Nrp1 and have a unique repertoire of TCRs which indicated a thymic origin [67]. Treg cells in skeletal muscle exert important function in tissue regeneration. They start to accumulate in impaired muscles within a few days after injury [67] and IL-33 receptor ST2 [68] and TCR specificity [69] play a critical role in Treg accumulation in injured muscles. Muscle Treg cells exert their tissue repair function by regulating co-infiltrating conventional T cells, containing the number of myeloid cells and promoting their transition from a

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pro- to an anti-inflammatory phenotype and enhancing satellite cells differentiation to promote muscle regeneration [67]. In mouse model of myopathy such as myositis and muscular dystrophy, adoptive transfer of Treg cells or low dose IL-2/anti–IL-2 complexes treatment significantly alleviated muscle inflammation and reduced muscular injury [70,71]. These results indicate a therapeutic potential

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of targeting Treg cells in myopathy. However, in Toxoplasma gondii infection model, skeletal

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muscle Treg cells exert a deleterious effect by promoting proinflammatory macrophages accumulation in muscle, leading to prolonged tissue damage [72]. Therefore, it is essential to

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conduct more studies on the consequences of inflammatory environment on Treg physiology.

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4.3 Treg cells in tumor

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In 1999, two Japanese research teams found CD4+CD25+ Treg cells could inhibit tumor rejection

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and promote tumor growth, revealing that Treg cells were also involved in tumor immune regulation [73,74]. Treg cells may play different roles in different stage of tumorigenesis. A latest

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study of pancreatic cancer shows that depletion of Treg cells result in deteriorative pancreatitis and pancreatic intraepithelial neoplasia, and lead to accelerated tumor progression. This study reveals a protective role of Treg cells in precancerous lesion [75]. Nevertheless, tumor-infiltrating Treg cells hinder tumor immune surveillance and inhibit effective host-anti-tumor immune response. In breast cancer [76], melanoma [77] and lung cancer [78], increased frequencies of circulating or tumor-infiltrating Treg cells usually indicate poor prognosis. While in some cancers, especially colon cancer, large infiltrations of Foxp3+ cells suggest a better prognosis [79,80]. However, further study shows that this group of Foxp3+ cells, which indicate a good prognosis, are Foxp3lowCD45RA+ non-Treg cells and increased frequencies of Foxp3hiCD45RA- Treg cells also

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lead to a poor prognosis [81]. Tumor infiltrated Treg cells also have different metabolic characteristics from their counterpart in periphery. Intratumoral Treg cells increase their lipid metabolism and have a higher lipid content. Compared with their circulating counterpart, these Treg cells express higher amounts of CD36 which is known to mediate long-chain fatty acid uptake and lipid metabolism. Genetic

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ablation of Cd36 in Treg cells selectively decrease lipid uptake and content in intratumoral Treg

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cells. And loss of CD36 expression perturbs accumulation of Treg cells in tumor [82]. Thus, targeting CD36 and thereby manipulating tumor infiltrating Treg cells may be a promising

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anti-tumor therapeutic strategy.

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4.4 Treg cells in other organs

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Treg cells in other nonlymphoid tissue also show their distinct tissue specific functions. Treg cells

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resident in skin involve in restraining skin inflammation [83,84], regulating fibroblast activation [85], repairing cutaneous injuries [86] and establishing tolerance to commensal microbes [87].

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Scientists also uncover that cutaneous Treg cells modulate hair follicle stem cells and promote hair follicle regeneration through Jag1-Notch pathway [88]. Similar to skin Treg cells, Treg cells in intestine play an important role in tissue repair [89,90] and microbiota tolerance [91]. In addition, intestinal Treg cells involve in establishing tolerance towards food antigens [92]. In myocardia, Treg recruitment can inhibit post-myocardial infarction inflammation, excessive matrix degradation and poor remodeling [93]. In bones, Treg cells protect against TNF-α induced bone destruction [94] as well as bone loss caused by ovariectomy [95]. In central nervous system, Treg cells have been proven to promote myelin regeneration [96]. These discoveries about unique functions of Treg cells in different organs demonstrate their ability to adopt to environment and

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lay the foundation for optimal apply of Treg cells in the treatment of related diseases. 5. Treg-targeted therapy Alone with the deep studies in the role of Treg cells in autoimmune diseases, Treg-targeted therapy becomes a promising avenue for the treatment of autoimmunity. Among various strategies, Treg-based cellular transfer and low-dose IL-2 modulation have become subjects of intense

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research currently (Figure). And these strategies have been applied in many clinical trials of a

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variety of immune-related disorders [97-104]. 5.1 Treg-based cellular transfer

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Treg cells used for adoptive cell therapy usually come from autologous peripheral blood [97-102]

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or umbilical cord blood [103,104]. To obtain clinically meaningful Treg numbers, Treg cells

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derive from autologous peripheral blood or cord blood need to be expanded ex vivo or induced in

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vitro before transfusion. And several protocols have been developed. Some researches show that cell-based artificial antigen-presenting cells (aAPCs) could massively increase nTreg yield

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[105,106]. In the meantime, these aAPCs could maintain Foxp3 expression and suppressive function of nTreg cells. In other researches, addition of compounds such as rapamycin, retinoic acid, transforming growth factor-β (TGF-β) or combination of IL-2, DNA methyltransferase and histone deacetylase inhibitors could provide us sufficient Treg cells with phenotypic and functional stability [107-109]. Lu Y and his colleagues suggest that tTreg cells treated with miR-146b-5p antagomir show higher expansion and enhanced suppressive function in vitro [110]. Zhang D et al. showed D-mannose can specifically induced Foxp3 expression and Treg cell fate in naive T cells in vitro [111]. In addition, probiotics and prebiotics also exert profound effect on Treg induction and expansion via different mechanisms in animal models or human cells cultured

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in vitro [112]. And these discoveries may provide new protocols to obtain sufficient Treg cells for adoptive cell therapy or directly regulate Treg cells in vivo for the treatment of autoimmune diseases. Treg adoptive therapy has been applied in many autoimmune diseases. In SLE, Maria et al. found that autologous adoptive Treg therapy augment activated Treg cells in inflamed skin [97].

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And this phenomenon may promote the resolution of skin inflammation in SLE. In type 1 diabetes

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(T1D), patients treated with autologous expanded ex vivo Treg cells obtain certainty degree of remission with no severe adverse effects observed [98,99]. In these studies, researchers also found

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that treatment with Treg cells can prolong survival of β-cells in T1D. Another disease widely

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treated with Treg transfer is Graft-Versus-Host Disease (GVHD) [102,103]. In these clinical trials

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of GVHD, Treg cell therapy has also been proven to be safe and effective.

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Aside from polyclonal Treg cells infusion, antigen-specific Treg cells have been proven to be vastly superior for therapeutic use in animal models [113,114]. There are various ways to obtain

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antigen-specific Treg cells, including antigen-stimulated expansion [115,116], T cell receptor (TCR) overexpression [117,118] and the use of chimeric antigen receptors (CARs) [119]. And antigen-specific Treg cells therapy has already been proved to be a promising clinical application for treating autoimmune diseases [115,120-123]. Regarding to particular Treg subset involved in Treg therapy, there are few studies to date. In Crohn’s disease, in vitro expanded CD4+CD25+CD127loCD45RA+ naïve Treg cells from patients’ blood home to inflammation sites in mouse model and suppress the activation of T cells [124]. In GVHD, researchers uncover that a higher proportion of aTreg cells in hematopoietic stem cells harvested from allogeneic donors correlated with no subsequent development of acute GVHD in

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corresponding transplant recipients [125]. And the therapeutic use of Th-like Treg subsets or tissue Treg cells in autoimmune disorders needs further delineate. It remains to be verified next whether using specific subpopulation of Treg cells will be an optimum proposal in Treg adoptive therapy. Treg-based cellular transfer may be a hopeful treatment for various immune-related diseases in the near further. The major bottleneck of the Treg adoptive therapy is how to maintain the

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phenotypic and functional stability of induced or expanded Treg cells in vivo and how to make

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better use specific subset of these fascinating cells in therapy. And further studies need to be conducted with more participates in longer follow-up period.

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5.2 Low-dose IL-2 treatment

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Treg cells constitutively express CD25 (IL-2 receptor α chain) at high level, which, together with

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the β and γ chains, forms the high-affinity IL-2 receptor. And IL-2 has been considered an

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essential growth factor for lymphocytes and is also a requisite for the proliferation and function of Treg cells [126]. Since researchers have shown that low-dose IL-2 (at single doses from 0.33 to

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4.5 million IU) could selectively promote the development and maintenance of Treg cells without activation of Teff cells [127,128], utilization of low-dose IL-2 has become a new remedy of autoimmune diseases.

In early studies of lupus mice, researchers showed exogenous recombinant IL-2 (rIL-2) treatment could partially restore Treg : Tconv balance and effectively delay disease progression and decrease mortality [129]. In NOD mice, Johnson MC et al. suggested that β-cell–specific IL-2 therapy not only increase the number of Treg cells in islet but also enhance fitness of islet Treg and suppress T1D in diabetes-free NOD mice [130]. In human T1D, IL-2 is well tolerated at low dose with no serious adverse events, but adverse

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events such as injection-site reaction and influenza-like syndrome which are all transient and of mild to moderate grades often occur [131-133]. Although low-dose IL-2 therapy is thought to be safe and can effectively augment Treg cells in T1D, but the numbers of other cells expressing IL-2 receptor – NK cells and eosinophils – are also increased [133], and efficacy of low-does IL-2 treatment needs further studies. As for GVHD, several studies have proven that low-dose IL-2

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therapy is an effective regimen which obtain well tolerance and may reduce the incidence of

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GVHD and infection [134,135]. However, selective expansion of NK cells also occurred during low-dose IL-2 therapy [136]. In SLE, administration of low-dose rIL-2 to active SLE patients

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resulted in extensive expansion of Treg cells, enhanced Treg function in peripheral blood and

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evidently reduced disease activity [137-139]. And a study in primary Sjogren's syndrome come to

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the same conclusion and further confirmed the unique therapeutic effect of low-dose IL-2

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treatment on immune-related disorders [140]. Another recent clinical trial in 11 autoimmune diseases including RA, AS, SLE, psoriasis, Behcet's disease, granulomatosis with polyangiitis,

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Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis and sclerosing cholangitis using low-dose IL-2 therapy in 46 patients also confirmed that this treatment selectively activate Treg cells and is safe across different diseases [141]. Furthermore, many clinical trials in different diseases such as RA (NCT02467504, NCT01988506) are still under way. These on-going studies may provide us with stronger evidence that IL-2 maybe the next revolution in immunotherapy. In addition, IL-2 complexed with several different monoclonal antibodies (IL-2/mAb complexes) emerged in recent year for their more selective proliferation of Treg cells and prolonged half-life of IL-2 in vivo. IL-2/mAb complexes, such as JES6–1 and F5111.2, bind to

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IL-2 and alter its conformation and binding interaction to IL-2R subunits，result in selective expansion of Treg cells [142]. Injection of IL-2/JES6–1 complexes sufficiently increases Treg proportion and alleviates diseases in animal models such as collagen-induced arthritis (CIA) [143], lupus nephritis [144] and congestive heart failure [145]. F5111.2, a human anti-IL-2 antibody, complexed with IL-2 also plays a protective role in mouse model of NOD, experimental

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autoimmune encephalomyelitis and GVHD [142]. However, the safety and efficacy of IL-2/mAb

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complexes in human need further verification.

In conclusion, there is still a long way to go before this potential therapy can be used in

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clinical practice. What is the safe and effective dose range including cumulative dose and whether

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there will be long-time complication require further researches. And double-blind,

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indispensable.

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placebo-controlled randomized trials and studies of deeper mechanism of IL-2 treatment are also

5.3 Pharmacologic targeting of Treg

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As scientists uncovered several signals promoting Treg survival and function, drugs targeting these signaling pathways could preserve Treg numbers and function, becoming potential therapeutic approaches of autoimmune diseases. mTOR kinase signaling plays an important role in T cell differentiation and function. Treg cells inhibited mTOR by rapamycin show improved lineage and functional stability in ex-vivo expansion experiment [109]. And mTOR-deficient T cells fail to differentiate to Th1, Th2 and Th17 cells but divert to Treg cells [146]. In animal model of many autoimmune diseases, such as autoimmune pancreatitis [147], T1D [148] and SLE [149], treatment with rapamycin lead to significant expansion of Treg cells and remission of diseases. In human SLE, rapamycin also

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exerts safety and efficiency in treatment of this disease [150]. Immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome is a fatal autoimmune disorder caused by loss-of-function mutations in Foxp3, leading to impaired Treg function and autoimmunity [151,152]. A lasted research demonstrates that rapamycin partial restores Treg function in IPEX patients

by

upregulating

immunoregulatory

protein

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Glucocorticoid-Induced-TNFR-Related-Protein (GITR) and Epstein-Barr-virus-Induced-3 (Ebi3)

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and results in a beneficial effect on clinical and histological evolution of IPEX pathology [153]. Histone deacetylases (HDACs) are portent epigenetic regulators of T cell-mediated immunity.

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Researches show that HDACs inhibitors act as an anti-inflammatory agent and become potential

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therapeutic approaches to autoimmune diseases [154]. HDACs inhibitors promote Treg function

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both in vivo and in vitro and lead to long-term allograft survival in mice [155]. In human,

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administration of vorinostat, an HDACs inhibitors, significantly increase the number of Treg cells and expression of Foxp3 in patients with hematologic malignances who have received allogeneic

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hematopoietic stem cell transplantation [156]. And there is a significant reduction in the incidence of severe GVHD. HDACs inhibitors could also enhance Treg function [157] or inhibit Th17 cell differentiation in CIA mouse model [158]. Peripheral blood mononuclear cells from RA patients cultured with lipopolysaccharide and HDACs inhibitor in vitro show increased proportion of iTreg cells and higher expression of IL-10 [157]. These results suggest HDACs inhibitors may have a beneficial effect in RA treatment. 6. Conclusion In autoimmune diseases, Treg cells have become currently a subject of intense research in both pathogenesis and treatment. Quantity or quality defects in Treg were found in many diseases and

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part of the Treg relevant etiology and mechanisms have been revealed. As a consequence, Treg targeted therapy emerged and became a promising protocol curing these diseases. However, because of Treg heterogenity and plasticity as well as complexity of the immune system, there are still various challenges. How Treg cells regulate immune response in different conditions or in different tissue and how Treg and conventional T cell in different subsets interact with each other

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Compliance with Ethical Standards

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comprehensive and thorough understanding of Treg cells.

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in diverse disease status need further researches. There is still a long way to go for us to get a

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Funding This work was supported by grants from the National Key Research and Development

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Program of China [grant number 2017YFC0909002] and the National Natural Science Foundation

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of China [grant number 81801599].

Conflict of Interest The authors declare that they have no conflict of interest.

Human and Animal Right and Informed Consent This article does not contain any studies with human and animal subjects performed by any of the authors.

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Figure. Treg-targeted therapy Treg cells used for adoptive cell therapy usually come from autologous peripheral blood or umbilical cord blood. In order to obtain clinically meaningful Treg

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numbers, Treg cells need to be expanded ex vivo or induced in vitro with artificial

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antigen-presenting cells or Treg inducers (such as rapamycin, retinoic acid, transforming growth factor-β, DNA methyltransferase and histone deacetylase inhibitors) before transfusion. Artificial antigen-specific Treg cells are generated by introducing synthetic receptors (CARs or engineered TCR). Treg-targeted drugs, such as rapamycin and HDACs inhibitors, promote Treg survival and function by targeting specific signal pathways which are of crucial importance for Treg development. Low-dose IL-2 modulation is another well-studied treatment which could selectively promote the development and maintenance of Treg cells without activation of Teff cells as well as correct the imbalance of Treg and Teff cells. aAPC, artificial antigen-presenting cells; CAR, chimeric antigen receptor; HDACs, Histone deacetylases; RA, retinoic acid; TCR, T cell receptor; UCB, umbilical cord blood. 39

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Outline Part 1：This part is a brief introduction of the function and subsets of regulatory T (Treg) cells, including some recent advances in this area. Part 2：This part mainly elaborates non-lymphoid tissue subsets of Treg cells, including visceral adipose tissue Treg (VAT Treg) cells, skeletal muscle Treg cells, intratumoral Treg cells and Treg

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cells in other organs. The characteristics and special tissue-related functions of different tissue

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Treg subsets as well as their status in different diseases are also been expounded.

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Part 3：This part is mainly about Treg-targeted therapy, including Treg-based cellular transfer,

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IL-2 therapy and Treg-targeted drugs in autoimmune diseases.

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