Regulatory T Cells: Pathophysiological Roles and Clinical          Applications

Ryota Sakai; Kyoko Komai; Mana Iizuka-koga; Akihiko Yoshimura; Minako Ito

doi:10.2302/kjm.2019-0003-OA

Abstract

Inflammation and immune responses after tissue injury play pivotal roles in the resolution of inflammation, tissue recovery, fibrosis, and remodeling. Regulatory T cells (Tregs) are responsible for immune tolerance and are usually activated in secondary lymphatic tissues. Activated Tregs subsequently regulate effector T cell and dendritic cell activation. For clinical applications such as the suppression of both autoimmune diseases and the rejection of transplanted organs, methods to generate stabilized antigen-specific Tregs are required. For this purpose, transcriptional and epigenetic regulation of Foxp3 expression has been investigated. In addition to conventional Tregs, there are some Tregs that reside in tissues and are called tissue Tregs. Tissue Tregs exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Such tissue Tregs could also be useful for Treg-based cell therapy. We recently discovered brain Tregs that accumulate in the brain during the chronic phase of ischemic brain injury. Brain Tregs resemble other tissue Tregs, but are unique in expressing neural cell-specific genes such as the serotonin receptor (Htr7); consequently, brain Tregs respond to serotonin. Here, we describe our experiences in the use of Tregs to suppress graft-versus-host disease and to promote neural recovery after stroke.

Introduction

Regulatory T cells (Tregs) suppress unwanted immunity against a variety of antigens, including self-antigens, commensal bacteria-derived antigens, and environmental allergens,¹ thereby preventing the development of autoimmune diseases, colitis, and allergies.²^,³^,⁴ Tregs express Forkhead box P3 (Foxp3) as a major master transcription factor and suppress excessive immune responses by recognizing various self-antigens and foreign antigens.⁵ Tregs are generated mainly via two different pathways. The first is direct development from CD4 and CD8 double-positive T cells in the thymus. Tregs that develop via this route are called thymus-derived Tregs (tTregs), or naturally occurring Tregs (nTregs). tTregs are believed to develop from precursor thymic T cells by recognizing self-antigen-MHC complexes expressed on thymic antigen-presenting cells with relatively high avidity. Therefore, tTregs express a T cell receptor (TCR) repertoire with a bias for self, and are important for the prevention of autoimmunity.⁶^,⁷ The second pathway of Treg generation is differentiation from naïve CD4⁺ T cells in the periphery upon antigen stimulation with an appropriate combination of cytokines, including interleukin-2 (IL-2) and transforming growth factorTGF-β).⁸^,⁹ Tregs produced in this way are called induced Tregs (iTregs) or peripherally induced Tregs (pTregs); the term “iTreg” is often used for Tregs generated in vitro, whereas the term “pTreg” indicates Tregs generated from naïve T cells in vivo. It is estimated that tTregs compose most of the systemic Treg population, whereas pTregs are highly enriched in certain organs, including the gut and maternal placenta.⁸^,¹⁰ TGF-β is essential for the generation of both iTregs and pTregs,¹¹ and lamina propria CD103-positive dendritic cells (LPDCs) are major producers of TGF-β in the intestine.¹²^,¹³ Furthermore, several commensal bacteria reportedly stimulate CD103⁺ LPDCs and increase TGF-β production.¹²

Various mechanisms of immune suppression by Tregs have been proposed: IL-2 consumption by CD25 expression, suppression of co-stimulation by CTLA4 expression, and suppression of inflammation by anti-inflammatory cytokine IL-10 and TGF-β.¹¹^,¹⁴ In recent years, in addition to its immune suppression functions, Tregs localized in non-lymphoid tissues have attracted attention in various fields. Tregs are either present in various tissues at steady state or accumulate after tissue injury; they play important roles in tissue homeostasis and repair by interacting with tissue cells. These Tregs are called tissue Tregs; they exhibit common properties among tissues, but they also have characteristics specialized for each tissue.¹⁵^,¹⁶ In this review article, we describe the molecular mechanisms that govern Treg differentiation and maintenance, focusing on the roles of transcription factors and epigenetic modifications. We will describe an example application of Tregs in a murine graft-versus-host disease (GVHD) model.¹⁷ We will also review tissue Tregs and their application to treat diseases of the central nervous system.¹⁸^,¹⁹

Mechanism of Treg Development

Tregs are characterized by the expression of transcription factor Foxp3, which plays crucial roles in the differentiation, maintenance, and function of Tregs. The importance of Foxp3 is evident, because differentiation of tTregs and pTregs is severely impaired in humans and mice with Foxp3 loss-of-function mutations, leading to death as a result of severe inflammatory diseases.²^,³^,⁴ Given the critical roles of Foxp3 in Treg biology, the molecular mechanisms underlying the induction of this transcription factor have been extensively analyzed (Fig. 1).²⁰

Figure 1.

Signals and transcription factors involved in Foxp3 induction and stable expression in iTregs. A thematic view of the Foxp3 locus and the CNSs are shown, as described in previous reports.²²^,²³^,²⁴ Activation of transcription factors Smad2 and Smad3 is essential for Foxp3 induction in iTregs, and Smads are recruited to the conserved CNS1 region. The CNS2 region serves as an enhancer for Foxp3 transcription and is bound by transcription factors such as Foxp3, STAT5, and CREB. In tTregs, CpG islands of this region are hypomethylated, and are designated TSDRs. CNS2 is the major TSDR, and is heavily methylated in freshly generated iTregs. As a consequence, important transcription factors cannot be recruited to these cells, and Foxp3 expression is unstable. CNS0 is also hypomethylated in Tregs and is an important binding site for Satb1. PKA, protein kinase A; PKC, protein kianse C; JAK, Jauns kinase.

In addition to the promoter, previous studies identified several intronic enhancers at the Foxp3 gene locus that are important for Treg differentiation.²¹ Each enhancer was shown to differentially contribute to tTreg and pTreg differentiation. The enhancers were designated as conserved noncoding sequence (CNS) 0, 1, 2, and 3, with the numbers reflecting their distances from the transcriptional start site (Fig. 1).²²^,²³ CNS0 was identified as a Satb1-binding site.²³ Satb1 is a global genome organizer that induces both transcriptional regulation and epigenetic regulation via the formation of novel nuclear architectures.²⁴ Satb1 is believed to function as a pioneering element required for the subsequent activities of the other CNS elements that lead to the initiation of Foxp3 expression.²³

The CNS1 enhancer contains binding sites for transcription factors, including Smads, NFAT, AP-1, and retinoic acid receptor (RAR) (Fig. 1).²⁰ Retinoic acid (RA) contributes to high and stable Foxp3 transcription through RAR.²⁵ CD103⁺ dendritic cells (DCs) in the lamina propria and mesenteric lymph nodes express the enzyme retinal aldehyde dehydrogenase, which synthesizes RA.²⁶ TGF-β induces binding of Smad2/3 to CNS1, which is a critical step for iTreg/pTreg differentiation.⁷^,²³ Smad2 and Smad3 are redundantly essential for the induction of Foxp3 by TGF-β stimulation.⁹ TGF-β1-deficient mice and Smad2/3 double-deficient mice exhibited relatively normal tTreg development in the thymus, but showed significantly reduced numbers of pTregs.⁹^,²⁷ These reports confirmed the essential role of TGF-β–Smad2/3 signaling in pTreg development. Accelerated mucosal T helper type 2 (Th2) cell-type inflammation and abortion were observed in CNS1-deleted mice, suggesting the importance of pTregs for suppression of excessive immunity against commensal bacteria and fetuses.²⁸^,²⁹

The CNS2 enhancer contains binding sites for transcription factors, including Stat5, NFAT, Runx1/Cbfβ, CREB, and Foxp3 (Fig. 2).³⁰ For example, IL-2 signaling is critical for tTreg and pTreg differentiation, and Stat5-responsive elements exist in the Foxp3 CNS2 and promoter region.³¹^,³²^,³³^,³⁴^,³⁵^,³⁶ CNS2 enhancer is important for the maintenance of Foxp3 expression, particularly under inflammatory conditions in which Tregs are exposed to inflammatory cytokines and stronger TCR stimulation.³⁷^,³⁸ The CNS2 locus is highly enriched with CpG sites, and the methylation status is an especially important determinant of the activity of this enhancer. In tTregs, the CpG sites are fully demethylated, which contributes to sustained expression of Foxp3 in tTregs and the stability of the tTreg lineage phenotypes (Fig. 2).³⁷^,³⁸^,³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³ DNA demethylation at this locus is also observed in pTregs, albeit with slightly reduced penetrance compared with tTregs,³⁹^,⁴⁴^,⁴⁵ rendering pTregs a stable subset.³⁹^,⁴⁵ However, this region is rarely demethylated in in vitro-generated iTreg cells and, consequently, iTregs are highly unstable.³⁹^,⁴⁰ Full methylation of this locus was also reported to prevent abnormal Foxp3 induction in non-Tregs, including CD8⁺ T cells and natural killer cells.⁴¹^,⁴⁶^,⁴⁷

Figure 2.

The role of CNS2 demethylation in stable Foxp3 expression. In tTregs, the CpG sites are fully demethylated, which contributes to sustained Foxp3 induction. The CNS2 enhancer contains binding sites for transcription factors, including STAT5, NFAT, Runx1/Cbfβ, CREB, and Foxp3. In iTregs, however, CNS2 is highly methylated, and therefore cannot bind these transcription factors. As a consequence, Foxp3 expression in this cell subset is highly unstable. MBD, methyl-CpG-binding domain; Dnmt1, DNA methyltransferase 1.

The CNS3 enhancer reportedly contains binding sites for c-Rel and is important for the differentiation of tTregs and pTregs (Fig. 1).²¹^,⁴⁸ In CNS3-enhancer-deleted mice, Treg development was severely impaired, and c-Rel was critical for tTreg development at the Foxp3 promoter.⁴⁹ We found that TFAR6, an adaptor protein that activates NF-κB (including c-Rel), is essential for stable Foxp3 expression.⁵⁰

Recruitment of Nr4a Factors to the Promoter of Foxp3

The promoter at the Foxp3 locus is activated by various types of stimuli, including TCR stimulation and cytokine signaling. We recently found that members of the Nr4a nuclear receptor transcription factor family of nuclear orphan receptors have essential roles in Treg differentiation by directly acting on the Foxp3 promoter⁵¹^,⁵²^,⁵³ (Fig. 1). The Nr4a family is composed of the closely related molecules Nr4a1, Nr4a2, and Nr4a3, which belong to a nuclear receptor superfamily.²⁰ The expression of Nr4a factors is highly enriched in tTregs compared with other T-cell subsets.⁵⁴^,⁵⁵^,⁵⁶^,⁵⁷ Nr4a factors act directly on the Foxp3 promoter and strongly induce Foxp3 expression.⁵² Nr4a family members are redundantly important for tTreg development in the thymus, as revealed by the nearly complete loss of Tregs in mice with the specific deletion of all three Nr4a factors in T cells.⁵¹ Nr4a factors are also necessary for in vitro iTreg differentiation. Expression of all Nr4a factors is induced by TCR stimulation, suggesting that Nr4a factors are crucial mediators of TCR signaling in Foxp3 induction.⁵⁶^,⁵⁸^,⁵⁹ Forced activation of Nr4a in T cells is sufficient for Foxp3 expression without strong TCR stimulation.⁵¹ The whole promoter region also regulates Foxp3 expression by sensing both cytokine and TCR signaling, which is mediated by transcription factors including Nr4a factors.

After tTreg development, Nr4a factors are expressed at high levels in the periphery. A knockout mouse strain in which all Nr4a genes were specifically deleted in Tregs showed a global reduction of genes exclusively expressed in Tregs, including Foxp3, Il2r, and Ikzf4. These mice also developed Th2-type systemic autoimmune diseases. Nr4a-deficient Tregs easily lost Foxp3 expression and became Th2 and T follicular helper cells. These findings demonstrate that Nr4a controls a novel genetic program required for Treg cell maintenance and function.⁶⁰

In addition to Treg development, Nr4a factors were recently reported to be important for CD8⁺ T cell exhaustion⁶¹^,⁶²^,⁶³. “T cell exhaustion” is a phenomenon of dysfunction or physical elimination of antigen-specific T cells reported in chronic viral infections as well as in cancer. Unlike conventional T cells, exhausted T cells lose the ability to proliferate, to produce cytokines, and effector functions in response to antigens. Exhausted T cells express high levels of NR4a factors.⁶¹ The phenotypes of T cell exhaustion partly overlap with anergic phenotypes of Tregs, including a high expression of checkpoint receptors (e.g., PD-1, Tim-3, and CTLA4) and low expression of cytokines such as IL-2 and IFNγ. Inhibitors of Nr4a could potentiate anti-tumor immunity by reducing the number of Tregs and enhancing CD8⁺ T cell activity.⁶⁴ Therefore, NR4a factors seem to be important for immune tolerance by conferring anergic phenotypes to both CD4⁺ and CD8⁺ T cells (Fig. 3).

Figure 3.

The role of Nr4a nuclear receptor transcription factors in gene regulation of Tregs and CD8⁺ T cells. Strong or chronic TCR signals in response to autoantigens or tumor antigens induce the expression of Nr4a genes. In Tregs, Nr4a upregulates Foxp3 and transcription factor Eos expression, while suppressing cytokine production. In exhausted T cells, Nr4a upregulates inhibitory receptors, while suppressing cytokine expression. Tim3 is an immune checkpoint protein.

Treg-specific Epigenetic Modifications and Stability of Tregs

Ohkura et al. showed that the establishment of a Treg-specific CpG hypomethylation pattern [called Treg-specific demethylated regions (TSDRs)] is crucial for Treg development.⁴¹ TSDRs are distributed in genes that are important for Treg differentiation and function, including the CNS2 enhancer (Fig. 1) of Foxp3 and specific regions in Ctla4, Il2ra (encoding CD25), Ikzf4 (encoding Eos), and Tnfrs18 (encoding GITR). Demethylation of the upstream enhancer, which is close to CNS0, was also observed in tTregs.²³^,⁶⁵^,⁶⁶ Without the establishment of a TSDR hypomethylation pattern, even Foxp3-positive cells cannot acquire full suppressive activity. Instead, Foxp3-positive TSDR-methylated cells, which are similar to in vitro-generated iTregs, show highly unstable Treg-associated gene expression. Demethylation of CNS2 at the Foxp3 locus is believed to be maintained by stable binding of Foxp3 and the Cbf-β-Runx1 complex or CREB/ATF to demethylated CNS2.

Recent reports revealed that members of the ten-eleven translocation (Tet) family of demethylation factors have important roles in CpG demethylation at CNS2 in Tregs.⁶⁷^,⁶⁸^,⁶⁹ Mice in which both Tet1 and Tet2 were specifically deleted in T cells or Tregs developed autoimmune diseases as a result of reduced Treg differentiation and function.⁷⁰ Moreover, Stat5 interacts with Tet1 and Tet2 and recruits them to CNS2, as well as to the promoter and CNS1, thereby mediating CpG demethylation at those loci.⁷⁰ In Tet2/Tet3 double-deficient mice, Tet2 and Tet3 proteins were shown to mediate demethylation of CNS1 and CNS2 in the Foxp3 locus and other TSDRs in tTregs.⁶⁶^,⁷¹^,⁷²^,⁷³^, Hydrogen sulfide promoted the expression of Tet1 and Tet2, which were recruited to the Foxp3 locus by TGF-β and IL-2 signaling to maintain Foxp3 demethylation and Treg-associated immune homeostasis.⁷⁰ We found that reduced oxygen concentrations during iTreg induction resulted in the upregulation of Tet enzyme expression and the promotion of CNS2 demethylation.⁷⁴

Recent studies have demonstrated that demethylation of CNS2 by Tet enzymes is promoted by vitamin C in iTregs, leading to stabilized Foxp3 expression.¹⁷^,⁶⁵^,⁷²^,⁷⁴ Vitamin C was shown to potentiate Tet activity,⁷⁵^,⁷⁶ thereby facilitating demethylation of the Foxp3 CNS2 region and increasing the stability of Foxp3 expression in TGF-β-induced iTregs.⁷⁷ Compared with untreated iTregs, iTregs generated under low oxygen conditions in the presence of vitamin C retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model.

Use of iTregs to Prevent GVHD

Preparation of iTregs could be an effective strategy for Treg-mediated adoptive immunotherapy (Fig. 4).⁷⁴ Antigen-specific Tregs are expected to induce antigen-specific tolerance, rather than broad immunosuppression. Because iTregs can be efficiently expanded from naïve T cells, antigen-specific iTregs are a realistic choice for adaptive immune therapy of autoimmune diseases, suppression of rejection during organ transplantation, and prevention of GVHD in bone marrow transplantation. We showed that antigen-specific, polyclonally expanded iTregs induced stronger tolerance than polyclonal tTregs in a heart transplantation model.⁷⁸ However, iTregs generated by conventional methods did not effectively suppress GVHD in an animal model, because these iTregs were very unstable after adoptive transfer, probably as a result of the extreme inflammatory conditions. Consequently, we sought an optimal method to generate stable iTregs that can prevent GVHD in a murine model. Alloantigen-specific iTregs were generated by coculture of naïve T cells with allogenic DCs in the presence of TGF-β and RA. By examining various agents and genes, we found that a high concentration of vitamin C (100 µg/ml) stabilized Foxp3 expression most effectively in adoptively transferred iTregs under the GVHD environment (Fig. 4A). Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C treatment caused almost complete CNS2 DNA demethylation in alloantigen-specific iTregs; such treatment also reduced iTreg conversion into pathogenic Foxp3-negative cells (Fig. 4B).

Figure 4.

Vitamin C-treated iTregs ameliorate GVHD. A mouse GVHD model was induced by transferring bone marrow cells and conventional T cells from C57BL/6 mice into lethally irradiated BALB/c mouse recipients. Alloantigen-specific iTregs were generated by culturing naïve T cells from C57BL/6 mice and bone marrow-derived dendritic cells from BALB/c mice in the presence of TGF-β and retinoic acid with or without vitamin C, as described.¹⁷ (A) (left) DNA demethylation status of CNS2 in iTregs induced in the presence of vitamin C. Black circles represent DNA methylation of the CpG island. (Right) Vehicle or vitamin C-treated iTregs were transferred intravenously together with conventional T cells and bone marrow cells into lethally-irradiated BALB/c recipient mice. The mice were euthanized and analyzed by flow cytometry on day 7. Foxp3 levels in transferred iTregs (H-2 K^b+) are shown. (B) Survival time was estimated and plotted using the Kaplan–Meier method. Data from Kasahara et al.¹⁷

We observed stable, very high FOXP3 expression in human iTregs that underwent vitamin C-treatment.¹⁷ Vitamin C treatment of iTregs also shows promise for innovative clinical applications of adoptive Treg immunotherapy. Further understanding of the mechanisms that facilitate the establishment of a Treg-specific transcriptional program will allow the production of sufficient numbers of antigen-specific Tregs with appropriate stability.

Tregs Reside in Tissues, Including the Brain

Tregs usually represent about 10% of CD4⁺ T cells and are considered to exist in lymphoid tissues and sites of inflammation; however, it has recently been discovered that they accumulate in various tissues in addition to lymphoid tissues. Tissue Tregs recognize the self-antigen characteristic of the tissues and have a limited TCR repertoire. Such tissue-resident Tregs (e.g., in fat, muscle, skin, lung, and intestines) exhibit phenotypes quite different from those present in lymphoid tissue¹⁵^,⁷⁹^,⁸⁰ (Table 1). The following features are common to various tissue Tregs: high expression of genes such as Il10, Il1rl1 (encoding ST2, IL-33 receptor), Areg (amphiregulin), Klrg1, Ctla4, Tigit, Gata3, Batf, and Irf4 and low expression of Lef1, Tcf7, and Bcl2 compared with lymphatic tissue Tregs.⁷⁹^,⁸¹ Analysis of the A384T mutation of the Foxp3 gene has shown that BATF is an important regulator of tissue Tregs.⁸² However, tissue Tregs also have tissue-specific features that are induced by the localized microenvironment, and these features seem to play pivotal roles in the phenotypes of tissue Tregs. The features of various tissue Tregs are shown in Table 1.

Table 1. Features of various tissue Tregs

Tissue	Transcription factor	Function	Specific molecules	Localization receptor	References
Fat (VAT)	PPARγ	Suppression of fat inflammation, metabolic modifications, aging	IL-10	?	¹²³^,¹²⁴^,¹²⁵^,¹²⁶
Muscle	PPARγ	Proliferation of satellite cells	Areg	CCR2	¹²⁷^,¹²⁸^,¹²⁹^,¹³⁰
Heart		Suppression of activation, M2 induction	IL-10	CCR5	¹³¹^,¹³²^,¹³³^,¹³⁴
Lung		Suppression of pulmonary fibrosis Proliferation of alveolar type II cells	Areg	CD103	¹³⁵^,¹³⁶^,¹³⁷
Skin	GATA3	Regeneration of hair follicles, skin homeostasis, wound healing	Jag1-Notch	CCR4,6,8, CD103	¹³⁸^,¹³⁹^,¹⁴⁰^,¹⁴¹
Colon	Helios-RORgt+	Suppression of inflammation	IL-10, CTLA4	?	¹⁴²
	Helios+Gata3+	Tissue repair	Areg	?	¹⁴³
Brain	PPARγ	Suppression of astrogliosis	Areg, CCN3	CCR6,8	¹⁸^,⁸⁷

VAT, visceral adipose tissue.

The number of Tregs in the brain is extremely low under normal conditions. However, the involvement of Tregs in neuroinflammatory diseases such as multiple sclerosis (MS) has been intensively studied.⁸³^,⁸⁴ Tregs from MS patients have defects in their suppression ability owing to a decrease in the expression levels of CTLA-4, Foxp3, and some genes important for Treg function, and these defects may contribute to the onset of disease.⁸⁵ In a mouse model of experimental autoimmune encephalomyelitis, which mimics MS, symptoms worsen as a result of the depletion or deficiency of Tregs.⁸³ In mice lacking T cells, remyelination is delayed after lysolecithin injection, suggesting that T cells are required for remyelination of the central nervous system.⁸⁶^,⁸⁷ It has been reported that the secretory protein CCN3 produced by Tregs promotes the differentiation and myelination of oligodendrocyte precursor cells.⁸⁷ Previously, Tregs infiltrated into the brain were reported to suppress neuroinflammation and reduce the severity of experimental stroke at the acute phase.⁸⁸ However, the role of Tregs in stroke has become controversial,⁸⁹^,⁹⁰ mostly because the number of Tregs during the acute phase of ischemic brain injury is extremely low (less than 100 cells per brain), and antigen-specific proliferation and activation of Tregs may not occur in such a short period (i.e., within 3 days). Therefore, only a bystander effect, such as the secretion of IL-10, may be observed during the acute phase of ischemic stroke.

Tregs in Ischemic Brain Injury

We have been studying the immune responses after ischemic brain injury. The resulting brain damage causes neuronal cell death and destruction of neuronal circuits, resulting in impaired movement, sensation, and higher brain functions. Although thrombolytic therapy delivered as intravenous recombinant tissue-plasminogen activator alleviates ischemic brain damage, to be effective, it should be administered within 4.5 h after the onset. After this very early period, only rehabilitation is available as the main treatment for promoting functional recovery for most ischemic stroke patients.

Sterile inflammation is usually associated with tissue injury and cell death. In the case of ischemic stroke, inflammation occurs both in humans and in mouse models, and it is suggested that neuroinflammation is an attractive treatment target for reducing brain damage.⁹¹^,⁹²^,⁹³^,⁹⁴ Currently, innate immune cells, including microglia, macrophages, and γδ T cells, are thought to play major roles in neuroinflammation after stroke, because inflammation is apparent within a few days after stroke onset⁹⁵^,⁹⁶ (Fig. 5). By day 3 after disease onset, IL-17 secreted by γδ T cells contributes to damage in the ischemic penumbra region.⁹⁵^,⁹⁷ Then, after 3–4 days, infiltrated M1-type macrophages are converted to M2-type repairing macrophages, and few symptoms of inflammation are observed 1 week after stroke⁹⁸ This is mostly because M2-type macrophages facilitate the scavenging of necrotic cells and tissue debris and support neural repair by producing trophic cytokines such as IGF-I (Fig. 5). However, our group and others have identified a massive accumulation of lymphoid cells, including Tregs, in the chronic phase, more than 2 weeks after stroke onset.⁹⁹^,¹⁰⁰ Tregs account for approximately half of CD4⁺ T cells localized inside and around the cerebral infarction lesion in close proximity to scar-forming astrocytes and surviving neuronal cells (Fig. 5).

Figure 5.

Schematic showing the time-dependent recruitment of inflammatory cells into the brain following focal cerebral ischemia in mice. In this review, we call days 1–3 after stroke the onset acute phase, days 3–7 the sub-acute phase, and the period after 2 weeks the chronic phase. This figure shows conceptual changes of infiltration and accumulation of immune cells. The numbers of each immune cell type shown by the lines are indicative only. The inflammation processes occur both in the peri-ischemic region and the ischemic core. DAMPs, damage-associated molecular patterns; CTLs, cytotoxic T lymphocytes; Th, T helper.

Characterization of Brain Tregs

Like other tissue Tregs, brain Tregs are Helios⁺ KLRG1⁺ tTregs; they possess a unique TCR repertoire and express high levels of CTLA-4, PD-1, Areg, and ST2. Moreover, similar to other tissue Tregs, TCR signaling, IL-2, and IL-33 are essential for the proliferation of brain Tregs (Fig. 6). In the brain, both astrocytes and oligodendrocytes express IL-33. It is notable that IL-33 reportedly promotes tissue recovery after central nervous system injury⁸⁷ and induces M2 type macrophage-related genes.¹⁰¹ It is also highly possible that brain Tregs are involved in the repair function of IL-33 in the central nervous system (Fig. 6).

Figure 6.

Schematic view of the accumulation of brain Tregs and their nerve control mechanism. Brain Tregs are activated by recognizing self-antigens, infiltrate the brain in a chemokine-dependent manner, and then undergo interleukin (IL-2, IL-33)- and serotonin-dependent proliferation and produce amphiregulin (Areg). Areg suppresses excessive activation of astrocytes by inhibiting the production of IL-6 from microglia and astrocytes and thereby protects neuronal cells. There may be other neuroprotective mechanisms involving brain Tregs.

Moreover, brain Tregs express several unique genes, such as central nervous system-related genes, that are not present in other tissue Tregs. In particular, brain Tregs express serotonin receptor 7 (Htr 7), which raises cAMP levels (Fig. 6).¹⁰² cAMP promotes the proliferation of Tregs and potentiates their functionality.¹⁰³ The administration of serotonin or a selective serotonin reuptake inhibitor (SSRI) during the chronic phase after stroke increases the number of brain Tregs and improves neurological symptoms (Fig. 7). Compared with wild type Tregs, Htr7-deficient Tregs do not increase in number in the brain and cannot reduce neurological deficits after transfer into T cell-deficient mice. As expected, in vitro, Tregs isolated from the ischemic brain proliferate and are activated in an Htr7-dependent manner. Brain Tregs also express CCR6 and CCR8 and infiltrate the brain with the expression of CCL20 and CCL1 in the cerebral infarct area. Intraventricular injections of CCL1 and CCL20 increase the number of Tregs, leading to improved neurological recovery.

Figure 7.

Expression of serotonin receptor 7 (HTR7) in brain Tregs and the therapeutic effect of serotonin or selective serotonin reuptake inhibitor (SSRI). (a) Expression levels of Htr7 were measured using quantitative RT-PCR. Tregs were isolated from the brain of mice with focal cerebral ischemia, from the spleen of normal mice, and TGF-β-induced iTregs produced in vitro. Brain Tregs, but not other Tregs, expressed Htr7. (b) Ischemic mice were administered serotonin (5-HT) or phosphate buffered saline (control) intraventricularly on days 7, 9, 11, and 12 or were administered SSRI fluoxetine intraperitoneally on days 7–13 after stroke onset. Flow cytometric analysis of the frequency of Tregs among CD4⁺ T cells from ischemic brains (left) and neurological scores (right) were determined on day 14. Lower scores indicate better neurological behavior. Data are modified from Ito et al.¹⁸

The depletion or reduction of brain Tregs causes excessive activation of astrocytes and apoptosis of neurons in the motor cortex. Therefore, brain Tregs reduce the activation of astrocytes. Excess activation of astrocytes, so-called astrogliosis, is thought to lead to a delay in the recovery of motor function after brain or spinal cord injury.¹⁰⁴ Although astrogliosis is necessary for forming scar tissue to protect neuronal cells from necrotic areas, excessive activation of astrocytes increases neurotoxic factors, resulting in neural cell damage and inhibition of neuronal outgrowth.¹⁰⁵

Inflammatory cytokines such as IL-6 are important for the activation of astrocytes,¹⁰⁶ and brain Tregs suppress IL-6 levels in vivo and in vitro. Areg is known to suppress the production of inflammatory cytokines, including IL-6 and TNFα, in several inflammatory diseases.¹⁰⁷ We found that Areg suppresses IL-6 production from microglia and astrocytes in vivo (Fig. 6). Therefore, Areg is an important functional molecule of brain Tregs. Areg may also be directly involved in the proliferation of neural stem cells.¹⁰⁸

Clinical Implications of Tregs for Central Nervous System Diseases

It has not yet been demonstrated that Tregs accumulate in the brains of ischemic stroke patients. However, in human cerebral infarctions, correlations between the number of peripheral Tregs, the Treg/Th17 ratio, or IL-17/IL-10 levels and the severity of stroke have been reported.¹⁰⁹^,¹¹⁰^,¹¹¹^,¹¹² It is notable that stroke patients show greater immunoreactivity to brain self-antigens.¹¹³ SSRI is known to ameliorate neurological symptoms after stroke onset.¹¹⁴^,¹¹⁵ Therefore, it is highly likely that brain Tregs work on neural repair in human stroke patients. Consistently, a pilot study of FTY720 (fingolimod), an immuno-modulating drug, in stroke patients revealed that the administration of FTY720 was only effective within 3 days after stroke onset.¹¹⁶

Neural inflammation occurs not only in cerebral infarction but also in various types of damage to cerebrospinal tissues, such as spinal cord injury, autoimmune diseases such as multiple sclerosis, and in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.¹¹⁷^,¹¹⁸^,¹¹⁹ It is possible that these various types of neural inflammation also trigger acquired immunity and that brain Tregs infiltrate and accumulate in the central nervous system, thereby playing an important role in relieving neurological symptoms.¹²⁰ Tregs in such central nervous system diseases have not been well characterized; however, the Tregs of MS patients were found to proliferate as a result of serotonin stimulation.¹²¹ Therefore, Tregs present in the central nervous system may be similar to the brain Tregs that we have characterized.

Conclusion

In summary, our knowledge of Treg development and stability has been expanded by recent research. We have established methods for the generation of stable antigen-specific Tregs to suppress GVHD in mice. We also showed that the number of brain Tregs was increased by chemokines or serotonin and promoted neuronal recovery after ischemic brain injury. The molecular mechanisms whereby Tregs acquire brain-specific characteristics, including serotonin receptor expression, remain to be clarified. The identification of self-antigens and the induction of brain-specific Tregs may also be useful in therapies for relieving symptoms not only in cerebral infarction but also in other central nervous system diseases. In the future, it may be possible to administer autologous Tregs directly into the brain for the treatment of cerebral inflammation.¹²² More than 25 years have passed since Tregs were discovered. Now that elucidation of the mechanisms of Treg differentiation, maintenance, and function are almost complete, the era of therapeutic application has begun.

Acknowledgments

This work was supported by JSPS KAKENHI (S) JP17H06175, Challenging Research (P) JP18H05376, and AMED-CREST JP18gm0510019 and JP19gm1110009 grants to AY; JSPS KAKENHI 17K15667, 19H04817, and 19K16618 to MI; the Takeda Science Foundation; the Uehara Memorial Foundation; the Kanae Foundation; and the SENSHIN Medical Research Foundation, Keio Gijuku Academic Development Funds.

Conflict of Interest

The authors declare no competing financial interests.

References

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