CD4+CD25−LAG3+ regulatory T cells in humoral immunity

Regulatory T cells (Tregs) are necessary for the maintenance of peripheral immune tolerance. These Tregs are divided into two main subsets: thymus‐derived Tregs and peripherally‐derived Tregs. As a thymus‐derived Treg subset, CD4+CD25+ Tregs (CD25+ Tregs) that express the transcription factor, Forkhead box P3 (Foxp3), have been extensively studied. Type 1 regulatory T cells are representative Foxp3‐independent peripherally‐derived Tregs that produce a large amount of the inhibitory cytokine, interleukin (IL)‐10, and suppress immune responses through IL‐10 production. Accumulating evidence has shown that lymphocyte activation gene 3 (LAG3) is a reliable cell surface marker for type 1 regulatory T cells. Peripherally‐derived CD4+CD25−LAG3+ Tregs (LAG3+ Tregs) produce a large amount of IL‐10 and suppress colitis in a mouse model in an IL‐10‐dependent manner. LAG3+ Tregs characteristically express transcriptional factor early growth response gene 2 (Egr2), and ectopic expression of Egr2 conferred a suppressive function to CD4+ T cells. Intriguingly, polymorphism of the EGR2 gene is associated with susceptibility to systemic lupus erythematosus characterized by a wide spectrum of auto‐antibodies, and mice lacking Egr2 specifically in lymphocytes develop a lupus‐like autoimmune disease. We recently reported that Egr2‐expressing LAG3+ Tregs effectively suppress humoral immune responses in a transforming growth factor β3‐dependent manner. In addition, transforming growth factor β3 and IL‐10 synergistically regulate Toll‐like receptor‐mediated humoral immune responses. In the present review, we focus on the role of LAG3+ Tregs in humoral immunity, and also discuss its therapeutic potential for the treatment of autoimmune diseases.


Introduction
The immune system protects the body by eliminating various pathogens, such as bacteria, viruses and cancer cells. In contrast, excessive immune responses against self-antigens cause tissue damage, leading to the development of autoimmune diseases. Autoantibodies induce various autoimmune diseases, including systemic lupus erythematosus (SLE), which is characterized by severe inflammation in multiple organ systems. 1 Immune system homeostasis is maintained by self-non-self discrimination. Immune tolerance is maintained by many immune cell subsets, such as CD4 + regulatory T cells (Tregs), CD8 + Tregs, regulatory B cells, dendritic regulatory cells and regulatory macrophages. [2][3][4][5] Among these regulatory cells, the CD4 + Tregs play a pivotal role in maintaining immunological non-responsiveness to self-antigens. 6 CD4 + Tregs are divided into two main subsets depending on the location of their differentiation. One is naturally occurring, thymus-derived Tregs (tTregs) and the other is peripherally derived Tregs (pTregs). tTregs characteristically express the cell surface markers interleukin (IL)-2 receptor-a (CD25) and the master regulatory gene, Forkhead box P3 (Foxp3). tTregs differentiate primarily by strongly recognizing autoantigens in the thymus. On the contrary, pTregs differentiate from na€ ıve T cells in the periphery. Some of the CD25 + Tregs differentiate in the periphery and there are other Treg subsets that are induced only in the periphery. We previously reported on peripherally-induced CD4 + CD25 À LAG3 + regulatory T cells (LAG3 + Tregs) that characteristically express transcription factor early growth response gene 2 (Egr2) and produce a large amount of the immunosuppressive cytokines, IL-10 and transforming growth factor b3 (TGF-b3). 7,8 The present review focuses on the recent advances and potential clinical application of LAG3 + Tregs in autoimmune diseases.

Classification of Treg subsets
In 1995, thymus-derived Tregs were identified as a CD4 + CD25 + T-cell population, 9 and subsequent study showed that Foxp3 is a key transcription factor necessary for the development and function of the Tregs. 10 From the point of view of Foxp3 dependency, Abbas et al. proposed to classify these Foxp3dependent Tregs into three populations based on the place from which they derive. 11 One is thymusderived, naturally occurring Tregs (tTregs); another is Tregs that develop in the periphery from Foxp3 À conventional CD4 + T cells (pTregs); and the other is in vitro-induced Tregs.
Mutation of the Foxp3 gene in Scurfy mice results in extensive lymphoproliferation and severe inflammatory infiltration in some organs, such as the lung, skin and liver. 12 The autoimmune regulator (Aire) gene plays an important role in the negative selection of self-reactive thymocytes and the development of tTregs. 13 Intriguingly, crossing Scurfy mice with Airedeficient mice did not extend the range of the affected sites, and many organs remained unaffected, indicating that additional important mechanisms other than central tolerance and the Foxp3 system are required to maintain self-tolerance in the periphery. 14 Foxp3-independent Tregs are thought to develop in the periphery and consist of heterogenous subsets, such as type 1 Tregs (Tr1 cells) 15 and iTr35. 16 We have proposed that Tr1 cells present in vivo as peripherally derived Tr1 (pTr1), and Tr1 cells induced in vitro as in vitro-induced Tr1 (iTr1). 17 Both Foxp3-dependent Tregs and Foxp3-independent peripherally developed Tregs are thought to play important roles in the maintenance of immune tolerance.

Foxp3-dependent CD4 + CD25 + Tregs
In 1996, Sakaguchi et al. reported that thymusderived naturally occurring T cells that express CD25 function to suppress autoimmunity. 9 The transcription factor, Foxp3, was identified as a specific molecular marker in CD4 + CD25 + Tregs (CD25 + Tregs). Mutations of the human FOXP3 gene result in impaired development or dysfunction of Tregs and cause immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome. The clinical features of IPEX syndrome include chronic dermatitis, enteropathy characterized by severe diarrhea and autoimmune endocrinopathy. 18 Scurfy mice, which harbor a frame shift mutation in the Foxp3 gene, show extensive proliferation of CD4 + CD8 À T lymphocytes, multiorgan infiltration and elevation of cytokines. 12 In addition, forced expression of Foxp3 in na€ ıve conventional T cells confers Treg-like suppressive activity. 10,19 Therefore, Foxp3 is considered to be a master regulator of CD25 + Tregs.
Periphery differentiated Foxp3 + pTregs also participate in the control of immunity at sites of inflammation, especially at the mucosal surfaces. 20 An experiment involving the adoptive transfer of CD4 + Foxp3 À cells into non-lymphopenic mice suggested that peripheral conversion could account for the development of 4%-7% of peripheral Foxp3 + T cells. 21 However, it is not exactly clear what the ratio of tTregs and pTregs is in the peripherally existing Foxp3 + Treg. 22 CD25 + Tregs are thought to show their inhibitory ability through several mechanisms. 23 Cytotoxic T-lymphocyte protein 4 (CTLA4) on CD25 + Tregs prevent costimulation through CD28 on effector T cells. 24 Also, CD25 + Tregs express the high-affinity IL-2 receptor and absorb IL-2, thereby potentially preventing the activation of other T cells. 25 CD25 + Tregs play an important role in suppressing autoimmune diseases and maintaining immune homeostasis in humans. 26,27 The number or the function of tTreg cells in peripheral blood are defective in most autoimmune diseases, such as type 1 diabetes and multiple sclerosis. 28,29 Foxp3-independent CD4 + CD25 À LAG3 + Tregs Foxp3-independent Tregs are thought to be induced in the periphery and consist of several different subsets. Tr1 cells are representative Foxp3-independent pTregs defined by their characteristic of producing the inhibitory cytokine, IL-10, and suppressing immune responses through IL-10 production. 15 Tr1 cells also produce high levels of TGF-b, low levels of IL-2, and variable levels of IL-5 and interferon-c in the absence of IL-4 after stimulation. 15 As neutralizing anti-IL-10 and anti-TGF-b antibodies block the inhibitory ability of Tr1 cells, Tr1 cells regulate immune responses through the immunosuppressive cytokines, IL-10 and TGF-b. 30,31 Tr1 cells suppress antigen-specific effector T-cell responses through a cytokine-dependent mechanism, whereas CD25 + Tregs suppress the proliferation of other T cells in a cell contact-dependent manner. 32 Tr1 cells are the Treg subset identified by its cytokine profile, and the full biological characterization of Tr1 cells has been hampered by the lack of specific surface markers or transcriptional factors. In 2009, we identified peripherally differentiated IL-10-producing CD4 + CD25 À Tregs that express lymphocyte activation gene 3 (LAG3) on the cell surface and the transcription factor early growth response gene 2 (Egr2). 7 Approximately 2% of the CD4 + CD25 À T-cell population in the spleen are LAG3 + Tregs. LAG3 + Tregs produce a large amount of IL-10 in vitro on antigenic stimulation, and suppress the in vivo development of colitis induced in RAG-1 À/À recipients by the transfer of na€ ıve T cells in an IL-10-dependent manner. Unlike Foxp3 + tTregs, the development of LAG3 + Tregs does not require high-affinity interactions with selecting peptide/major histocompatibility complex (MHC) ligands expressed in the thymus. LAG3 + Tregs do not express Foxp3 protein, and LAG3 + Tregs from Scurfy mice still express Il10 mRNA and show in vitro suppressive activity. These findings revealed that LAG3 + Tregs are equivalent to pTr1 cells that exist in a steady state.
In 2013, Gagliani et al. subsequently reported that not only LAG3, but also CD49b, are reliable markers of mouse and human Tr1 cells in vivo. 33 Concomitant expression of LAG3 and CD49b is specific for Tr1 cells, because T helper cell 1 (Th1), Th2, Th17 and CD25 + Tregs do not co-express these markers. LAG3 and CD49b are found to correlate with IL-10 expression in CD4 + T cells from mice treated with a dose escalation strategy for efficient self-antigen-specific tolerance induction. 34 In HIV-infected individuals, the frequency of LAG3 + CD49b + Tr1 cells was increased, as well as serum levels of IL-10. 35 Tr1 cells are also shown to play an important role in the prevention and treatment of autoimmune diseases, organ transplantation and chronic inflammatory diseases. 36 Co-transfer of Tr1 cells prevented colitis induced in SCID mice by pathogenic CD4 + CD45RB high T cells. 30 Tr1 cells induced by intestinal bacteria alleviated the development of intestinal inflammation. 37 Intestinal Tr1 cells prevent autoimmune diabetes in non-obese diabetic mice. 38 These findings in widely varying model disease systems indicate that IL-10-producing Tr1 cells are associated with disease protection. In addition, clinical trials of cell therapy with Tr1 cells that are generated and expanded in vitro are underway. 39 Molecular properties of LAG3 LAG3 (CD233) is a type I membrane protein belonging to the immunoglobulin superfamily, and was first reported as a cell surface protein expressed on activated T cells and natural killer cells. 40 LAG3 expression is also detected in other immune cell subsets, including plasmacytoid dendritic cells, 41 B cells, 42 exhausted CD8 + T cells 43 and Tregs. 44 LAG3 has structural homology to CD4 and has a higher affinity to MHC class II than CD4. 45 Although CD4 molecules are mainly expressed on the cell surface, nearly half of the cellular content of LAG3 is retained in intracellular compartments, and LAG3 cell surface expression is tightly regulated in order to regulate its potent inhibitory activity. 46 Although initial analysis of LAG3-deficient mice did not show any defects in T cells, 47 subsequent examination showed that LAG3 negatively regulates T-cell expansion and development of the memory Tcell pool. 48 Ectopic expression of LAG3 conferred regulatory activity to na€ ıve T cells. 44 The crosslinking of the T-cell receptor (TCR) and LAG3 molecule results in less calcium release than TCR stimulation alone in activated T cells. 49 LAG3 on CD25 + Tregs is also reported to interact with MHC class II molecules on dendritic cells and induces an immunoreceptor tyrosine-based activation motif-mediated inhibitory signaling pathway that suppresses dendritic cell function. 45 However, LAG3 protein is hardly detected on the cell surface of CD25 + Tregs, 44 indicating that LAG3 expression in CD4 + T cells is not a marker for CD25 + Tregs. 7 On the contrary, Tr1 cells that produce IL-10 and show suppressive ability stably express LAG3 on their cell surface. 33 Intriguingly, in recent years, a plasma cell subset that expresses LAG3 and suppresses immunity through IL-10 production has been reported, indicating the importance of LAG3 as a marker of IL-10 producing cells. 50 LAG3-deficient mice do not develop autoimmunity, but the inhibitory function of LAG3 becomes evident only on susceptible backgrounds or active induction of disease. 51 LAG3 deficiency induces lethal myocarditis in mice deficient for the gene encoding the inhibitory co-receptor programmed cell death 1 (PD-1). In addition, LAG3 deficiency alone accelerated type 1 diabetes in non-obese diabetic mice. 52 From these results, LAG3 is considered to be an important molecule for the regulation of autoimmunity in relation to other molecules.

Role of the Egr2 gene in LAG3 + Tregs
Gene expression analysis shows that LAG3 + Tregs characteristically express Egr2. 7 Egr2 is a zinc fingertype transcription factor involved in peripheral nerve myelination, and mice deficient for Egr2 were embryonic lethal due to neuropathy, so research was mainly focused on neurology. 53 In 2005, Egr2 was identified as a factor related to the anergy induction of T cells, and exploration of the immunological role of Egr2 has proceeded since that time. 54 Mice lacking Egr2 specifically in lymphocytes show lupus-like autoimmune disease. 55 We previously reported that regulatory polymorphisms in EGR2 are associated with susceptibility to SLE. 56 Retroviral gene transfer of Egr2 converts na€ ıve CD4 + T cells into the IL-10-secreting and LAG3-expressing phenotype, and Egr2-transduced CD4 T cells show antigen-specific immunosuppressive capacity in vivo. 7 Furthermore, we have also identified EGR2-expressing CD4 + CD25 À CD45RA À LAG3 + T cells in peripheral blood mononuclear cells from healthy human donors. 8 Similar to murine LAG3 + Tregs, these human LAG3 + T cells produce large amounts of IL-10 in response to TCR stimulation and suppress humoral immunity. When the frequency of LAG3 + Tregs in the peripheral blood of rheumatoid arthritis patients was examined, it was lower in patients with rheumatoid arthritis, especially those with higher disease activity. 57 Further studies are required to evaluate the role of human LAG3 + Tregs in other autoimmune diseases.

LAG3 + Tregs control humoral immunity through TGF-b3
The humoral immune response is mediated by antibodies that are secreted by B cells. Although antibodies function to protect hosts from antigens, excessive antibody or autoantibody production results in the development of autoimmune diseases, such as SLE. In germinal centers, interaction with T follicular helper (Tfh) cells and germinal center B cells allows affinity maturation and isotype switching of antibodies. 58 Follicular regulatory T cells are a specific type of Treg that enter the B cell follicle, and specifically suppress Tfh cells and B cells. 59 Follicular regulatory T cells express FOXP3 and CTLA4, which are similar to conventional Tregs. 59 Intriguingly, follicular regulatory T cells also express CXCR5, Bcl-6, PD-1 and ICOS, the phenotype of which is similar to that of Tfh cells. 60 However, conflicting findings have been reported on the numbers and function of Treg in SLE patients. 61 The clinical manifestations of IPEX syndrome, caused by a mutation in the FOXP3 gene, are considerably different from those of SLE. 62 Thus, the precise roles of CD25 + Tregs in SLE remain elusive.
As described above, Egr2-deficient mice showed lupus-like pathology, so the involvement of LAG3 + Tregs in antibody production was also speculated. In 2015, we reported that transfer of LAG3 + Tregs suppressed anti-NP antibody production in NP-OVA immunized mice. 8 Also, adoptive transfer of LAG3 + Tregs from MRL/+ mice to lupus-prone MRL-FAS lpr / lpr (MRL/lpr) mice suppressed the formation of germinal center B and Tfh, proteinuria progression, and anti-dsDNA antibody titers. From these experiments, LAG3 + Tregs are considered to be the Treg subset possessing the ability to regulate humoral immune responses. Microarray analysis and quantitative real-time polymerase chain reaction of LAG3 + Tregs showed a significant increase of TGF-b3 expression. In addition, TCR stimulation of LAG3 + Tregs induced the production of a large amount of TGF-b3 protein, but not TGF-b1 or TGF-b2. TGF-b3 effectively suppressed anti-immunoglobulin M or anti-CD40 plus IL-4-stimulated B-cell activation and antibody production in vitro. Treatment with a TGF-b3 blocking antibody cancelled the LAG3 + Tregmediated suppression of antibody production in NP-OVA immunized mice, and also abrogated the therapeutic effects of LAG3 + Tregs in MRL/lpr mice. These results show that the TGF-b3 produced by LAG3 + Tregs plays a critical role in suppressing humoral immunity.

Function and immunosuppressive ability of TGF-b
TGF-b is a pleiotropic cytokine with potent regulatory and inflammatory activity. 63 There are three known isoforms of TGF-b (TGF-b1, TGF-b2 and TGF-b3), but most of the immunological studies were carried out on TGF-b1. As mice lacking TGF-b1 produce autoantibodies, including anti-dsDNA antibodies, and die as a result of excessive inflammatory responses with massive infiltration of lymphocytes, the importance of TGF-b1 in controlling autoimmune disease is well recognized. 64,65 TGF-b1 plays important roles in regulating the differentiation of T cells into effector and regulatory subsets. 66 TGF-b1 suppresses Th1 effector T-cell differentiation, 67 and promotes the conversion of na€ ıve CD4 + T cells to CD25 + pTregs by upregulating the expression of Foxp3. 68 In contrast, TGF-b1 induces IL-17-producing Th17 cells in the presence of IL-6, IL-1b and/or IL-21. 69 The effects of TGF-b1 on T-cell differentiation are highly contextdependent: TGF-b1 can drive either pro-or antiinflammatory responses, depending on the amount of TGF-b1 and on other factors present at the time of exposure to TGF-b1. 66 TGF-b2 is thought to be less involved in immune regulation because of its low expression in immune cells. 70 Also, TGF-b3 had not been considered to play an important role in immunity, because TGF-b3-deficient mice die within 20 h of birth with phenotypic features including delayed pulmonary development and defective palatogenesis. 71 However, a recent study has shown that TGF-b3, together with IL-6, induced highly pathogenic Th17 cells. 72 The production of TGF-b3 from immune cells has recently been recognized. 73,74 Although TGF-b3 mRNA is reported to be expressed in lymphocytes, such as CD4 + T cells, CD8 + T cells and B cells, TGF-b3 protein is highly produced by LAG3 + Tregs. 8,74 As has already been described, LAG3 + Tregs suppress humoral immune responses in a TGF-b3-dependent manner. 8 In addition, we have recently reported that TGF-b3 regulates humoral immunity in combination with IL-10 through modulating metabolic signals. 75 TGF-b3 and IL-10 synergistically regulate Toll-like receptor-mediated humoral immune responses in B cells by suppressing mammalian target of rapamycin signaling both in vivo and in vitro. Inhibition of mammalian target of rapamycin signaling by TGF-b3 and IL-10 further suppresses B-cell cellular metabolism, such as glycolysis and oxidative phosphorylation. The fact that LAG3 + Tregs produce both TGF-b3 and IL-10 implies the existence of a mechanism for maintaining humoral immune tolerance by the coproduction of these cytokines.

Conclusion
Breakdown of immune tolerance mechanisms causes autoimmune diseases and allergies. Treatment for these diseases is progressing daily, but there are still many diseases that have not yet reached the development of appropriate treatments with few sideeffects. Analysis of the regulatory mechanism of Tregs, which has been confirmed to exist in the steady state, is important for clarifying the mechanism of autoimmune diseases, and it will also lead to the development of new therapeutic strategies. To treat autoimmune diseases, many approaches aimed at increasing the number and function of Tregs have been studied. Currently, the most promising and aggressively explored therapy is adoptive transfer of Tregs. 76 Recent clinical trials have shown the safety of adoptive immunotherapy with Tregs and hinted at a possible therapeutic effect. 77 A phase 1/2a clinical study was carried out for refractory Crohn's disease patients, in which ova-specific Tr1 cells were isolated from patients' peripheral blood mononuclear cells, exposed to ovalbumin and administrated intravenously. This treatment was well tolerated and showed dose-related efficacy. 78 However, modifying the antigen specificity, stability and function of therapeutic Tregs is important to ensure the efficacy of Treg therapy. In order to solve these problems, a novel treatment combining Treg transfer and gene therapy has been developed. 79 To increase the therapeutic potential of Treg therapy, further investigation is required to clarify the function of various Treg subsets.