Regulatory T cells induced by B cells: a novel subpopulation of regulatory T cells

Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4+Foxp3+ regulatory T cells, several subsets of Foxp3- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4+CD25- T cells into CD25+Foxp3- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3+ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.


Background
Regulatory T cells are a therapeutic strategy for immune dysregulated diseases and a potential target for cancer immunotherapy. In addition to CD4 + Foxp3 + regulatory T (Treg) cells, studies have emphasized the roles of CD4 + Foxp3regulatory T cells, such as TGF-β-producing T helper 3 (Th3) cells, IL-10-producing type 1 regulatory T (Tr1) cells, and others. Accumulating evidence demonstrate that naïve B cells possess the ability to promote naïve CD4 + T cells into CD25 + Foxp3regulatory T cells with the expression of lymphocyte activation gene-3 (LAG3, CD223), inducible co-stimulator (ICOS, CD278), programmed cell death protein 1 (PD1, CD279), and glucocorticoid-induced TNFR family-related protein (GITR). B-cell-induced CD4 + Foxp3regulatory T cells exert the inhibition through both IL-10-independent and cell-cell contact-dependent mechanisms, although they also show IL-10-mediated suppression. Furthermore, these B cell-induced regulatory T cells protect mice from several immune disorders, including graft-versus-host disease, experimental allergic asthma, collagen-induced arthritis, and inflammatory bowel disease. Here, we review the phenotypes and functional mechanisms of thymus-derived and peripherally derived CD4 + Foxp3 + regulatory T cells, Th3 cells, Tr1 cells, B-cell-induced Foxp3regulatory T cells, and B-cell-induced Foxp3 + regulatory T cells. The present article focuses on B-cellinduced CD4 + Foxp3regulatory T cells, which we have named Treg-of-B cells.

Main text
CD4 + Foxp3 + regulatory T cells Sakaguchi et al. demonstrated that CD4 + CD25 + T cells contributed to maintaining self-tolerance in a nonantigen-specific manner [1]. Immune dysregulation, polyendocrinophathy, enteropathy X-linked (IPEX) syndrome is a recessive immune disorder. Reports showed that IPEX is caused by mutations of FOXP3 gene, which is orthologouse of the Foxp3 gene mutated in scurfy mouse [2][3][4]. Further studies demonstrated that Foxp3 expressed predominantly in CD4 + CD25 + T cells than CD4 + CD25 -T and CD19 + B cells. Moreover, retroviral transduction of Foxp3 in naïve CD4 + CD25 -T cells converted these cells toward Treg cells phenotype. Thus, Foxp3 has been identified as the master transcription factor of Treg cells [5].
Peripherally derived Foxp3 + regulatory T cells Foxp3 + regulatory T cells induced in vivo are called peripherally derived regulatory T (pTreg) cells and those generated in vitro are called in vitro-induced regulatory T (iTreg) cells [18]. Studies demonstrated that CD4 + Foxp3 -T cells differentiated into Foxp3 + CD25 + CD45RB low anergic T cells with suppressive functions in the presence of TGF-β1 in vitro as well as in vivo [19] and rescue Foxp3-deficient scurfy mice [20]. In the absence of tTreg cells, oral antigen administration induced the generation of CD4 + CD25 + Foxp3 + regulatory T cells in a TGF-β1-dependent manner [21]. Gut-associated lymphoid tissue CD103 + DCs played an important role in the de novo conversion of naïve T cells into pTreg cells, and retinoic acid facilitates that process [22]. Additionally, lung-resident tissue macrophages expressed retinal dehydrogenases, and TGF-β1 promoted pTreg cell induction under steady-state conditions [23]. Evidence has shown that the tumor environment induced pTreg cell generation to escape immune clearance [24]. One report demonstrated that tTreg and pTreg cells shared similar phenotypes, and neuropilin-1 serving as a surface marker to distinguish tTreg cells from pTreg cells [25].

CD4 + Foxp3regulatory T cells
The most well-defined Foxp3regulatory T cells are Th3 cells and Tr1 cells. Th3 cells have been identified as TGF-β-producing CD4 + LAP + T cells exhibiting TGF-βmediated suppression [26]. Tr1 cells have been characterized by the higher production of IL-10 and IL-10-mediated suppressive functions [27].

Type 1 regulatory T cells
The first study on Tr1 cells reported that naïve T cells repeated stimulation with peptide-pulsed splenocytes in the presence of IL-10 induced IL-10-producing CD4 + T cells with suppressive ability and hypoproliferative ability [33]. Akbari et al. demonstrated that bronchial DCs promoted Tr1 cells in vitro in an IL-10-and ICOS/ ICOS ligand (ICOSL)-dependent manner in the context of nasal tolerance [34]. By microarray analysis Tr1 and Th0 cell clones, CD49b, LAG3, and CD226 have been identified as the surface markers of Tr1 cells [35].
In addition to cytokines, reports have demonstrated that Tr1 cells could be induced by different proteins, different APCs, and different types of T cells. Galectin-1 promoted IL-10 expression in CD4 + T cells in an APCindependent pathway by binding to CD45 on T cells and inducing the expression of c-Maf and AhR [43]. In vitro activation of CD4 + CD44 hi Foxp3 -T cells through anti-CD3/CD28 antibodies and IL-2 generated CD49b-, LAG3-, c-Maf-, and AhR-expressing Tr1 cells [44]. Nie et al. found that long-term stimulation of lipopolysaccharide (LPS) conferred ICOSL expression in bone marrow-derived mast cells through NF-κB, subsequently promoting Tr1 cell development [45]. These reports suggest that the generation mechanisms for Tr1 cells consist of a fine-tuning program.

B cells in tolerance induction
B cells have been shown to have a role in the fine equilibrium for immune tolerance. Genetically B-celldeficient mice delayed recovery from experimental autoimmune encephalomyelitis and suggested B cells might contribute to immune modulation [46]. Collagen fragments expressed on B cell MHC class II sufficiently delayed the onset and decreased the severity of arthritis [47]. The role of B cells in oral tolerance has been investigated because B-cell-deficient mice exhibit a defective oral tolerogenic response characterized by lower levels of IL-10 and TGF-β in the spleen and gut-associated lymphoid tissues [48]. Gutgemann et al. showed that B cells interacted with T cells at the B-T border in the spleen after 4 h of oral administration of proteins [49]. Furthermore, orally antigen treated B cells have an enhanced ability to induce CD4 + regulatory T cells in vitro [50]. Anterior chamber-associated immune deviation was characterized by antigen-specific downregulation of the immune response to antigen occurs in the anterior chamber of the eye [51], and this phenomenon was abrogated in the absence of B cells [52]. Studies suggested that splenic B cells presented antigens derived from ocular APCs and induced CD4 + CD25 + regulatory T cells via IL-10 and MHC class II [52,53]. These evidence emphasize the role of B cells in the induction and maintenance of self-tolerance.
There is accumulating evidence demonstrating that specific B cell subsets modulate immune responses named as regulatory B (Breg) cells by Mizoguchi et al. [54]. Breg cells dampened immune responses though the secretion of IL-10, TGF-β, directly interact with activated CD4 + T cells, and the production of antibody that neutralized harmful soluble molecules [55]. Several Breg cells have been described in mice and IL-10-producing Breg cells are the most widely studied [56]. IL-10 produced by a variety of Breg cells suppressed inflammatory cytokines and promoted regulatory T cell differentiation [57,58]. These indicate that B cells contribute to the maintenance of tolerance.
In addition, naïve B cells functioned as antigenpresenting cells presented antigen and resulted in T cell tolerance to antigen [59]. Raimondi et al. demonstrated that adoptive transfer of antigen-presenting B cells four times in a week lead to antigen-specific CD4 + T cells tolerance independent of naïve or activated B cells [60,61]. Antigen-presenting follicular B, marginal zone B, and B-1a cells rendered antigen-specific T cells hyporesponsiveness without Foxp3 + Treg cells induction [62]. One study reported that B cells contributed to Treg cells homeostasis and cooperated with Treg cells to ameliorate inflammation [63]. These findings suggest that B cells play a role in immune modulation and might through the manipulation of CD4 + Treg cells.

B-cell-induced CD4 + Foxp3 -Treg-of-B cells
Naïve splenic B2 cells, peritoneal B-1a cells, and mucosal Peyer's patch B cells have been shown to induce CD4 + CD25 + Foxp3regulatory T cells, which named Treg-of-B cells by our group, without additional cytokines or molecules [50,64]. Naïve splenic B cells and naïve splenic CD4 + CD25 -T cells formed a stable immunological synapse and promoted CD62L hi CD25 + Foxp3regulatory T cell generation [65]. In our reports, transwell insertion during B-T coculture abrogated Treg-of-B cell induction suggesting that cell-cell contact between B and T cells was essential. By applying blocking antibodies during B-T coculture, both CD80 and CD86 on splenic B cells were required to induce functional Treg-of-B [64]. In consistent with above, Etemire et al. demonstrated that addition of anti-CD28 antibody to the B-T cell co-culture decreased the suppressive activity of Treg-of-B cells. Lower activity of the PI3K/AKT pathway was associated with Foxp3regulatory T cell generation [66]. IL-10-deficient Treg-of-B cells and Treg-of-B cells induced in the presence of anti-IL-10 neutralizing antibody remained their suppressive function suggesting that IL-10 was not critical for their induction [64,67,68]. These results suggest that the interaction between B-T cells is indispensable for the differentiation of Treg-of-B cells.

Treg-of-B cells differ from well-known Treg cells
To date, several molecules have been identified for their strong association with Treg-of-B cells that are conserved in single peptide-induced and anti-CD3/CD28 antibodies-induced methods. Treg-of-B cells expressed higher levels of LAG3, ICOS, PD1, GITR, OX40 (CD134), and CTLA4 compared to those on naïve CD4 + CD25 -T cells (Fig. 1). Another group demonstrated that antigen-presenting B cells facilitated naïve T cells to convert into CD4 + CD25 + CD62L + Foxp3 -IL-10-producing regulatory T cells [65]. Our published and unpublished data showed that Treg-of-B cells did not express Foxp3, Helios, or neuropilin-1 [67,69], and these also confirmed by using Foxp3-GFP reporter mice [64]. These evidence differentiates Treg-of-B cells from Foxp3-expressing Treg cells (Table 1).
Th3 cells are well-known that they exert TGF-βdependent inhibition and express LAP on surface [26]. Although Treg-of-B cells produced TGF-β compared with naïve CD4 + CD25 -T cells [68,69], TGF-β did not play a role in their suppressive mechanism [64]. In our unpublished data, Treg-of-B cells did not express LAP. These indicate that Treg-of-B cells are different from Th3 cells.
Tr1 cells are characterized by IL-10-mediated suppression and the higher production of IL-10 [27]. In recent years, CD49b, LAG3, and CD226 were identified as the surface markers for human and mouse Tr1 cells [35]. In our results, Treg-of-B cells produced a higher amount of IL-10 compared with naïve CD4 + CD25 -T cells [50,64]. Repeated stimulation of B cells induced long-term Tregof-B cells with higher expression of ICOS, CTLA4, CD49b, and c-Maf, but not CD226. In addition to the difference in surface marker, IL-10 seems to be dispensable in the inhibitory mechanism of Treg-of-B cells and these would be described in the later section. These observations suggest that this Treg-of-B cell is a new type of regulatory T cells and different from Tr1 cells.
In addition to regulatory T cells, Treg-of-B cells did not share characteristics with follicular T helper (T FH ) cells. T FH cells expressed BCL-6, CXCR5, ICOS, PD1, and c-Maf and CXCR5 conferred T FH cells migration to B follicles [67,70]. Although Treg-of-B cells expressed ICOS, PD1 and c-Maf, they did not express the critical molecule BCL-6 and CXCR5 (data not shown). These indicate that Treg-of-B cells could not migrate into follicle to facilitate B cell as T FH cells did.

Application of Treg-of-B cells
The therapeutic effects of CD4 + Foxp3 -Treg-of-B cells has been described in several murine disease models (Fig. 2). Adoptive transfer of Treg-of-B cells prevented mice from graft-versus-host disease in a murine model of heart transplantation [65]. Peyer's patch B-cellinduced ovalbumin (OVA)-specific Treg-of-B cells protected mice from Th2-cell-mediated airway hyperresponsiveness (AHR), airway inflammation, and IgE hyperproduction in allergic asthma in an antigen-specific fashion [50]. In addition, splenic B-cell-induced OVA-specific Tregof-B cells shared several characteristics with oral antigen administration activated CD4 + CD25 + T cells, including elevated expression levels of ICOS, PD1, and CTLA4 and enhanced non-antigen-specific suppressive functions [69]. Monoclonal antibody-induced Treg-of-B cells prevented mice from osteolysis and joint inflammation in collageninduced arthritis [71]. Prophylactic transfer of Treg-of-B cells also protected mice from T-cell-induced Th1-and Th17-dominant inflammatory bowel disease [68]. Taken together, naïve B cell without cytokines or chemical supplements is able to induce functional CD4 + Foxp3regulatory T cells and that B-cell-induced regulatory T cells is an economical strategy for cellular therapy for different T-helpercell-dominant inflammatory diseases.
Treg-of-B cells possess both IL-10-dependent and IL-10independent suppressive functions IL-10 as an anti-inflammatory cytokine is an issue in Treg-of-B cells suppressive function. As described above, IL-10 does not play a crucial role in Treg-of-B cells differentiation. Chen and Chu et al. reported that LAG3 + Treg-of-B cells produced higher amount of IL-10 and both IL-10 and LAG3 play the roles in their inhibitory mechanisms [71,72]. Long-term Treg-of-B cells increased expression levels of CTLA4 and IL-10, both of which were involved in their suppressive functions [67]. IL-10-deficient mice were used to confirm the role of IL-10 in the regulation; however, IL-10-deficient Tregof-B cells remained suppressive activities [64,68]. IL-10 seems to be dispensable in the inhibitory mechanism of Treg-of-B cells. Although IL-10 plays a more important role in long-term Treg-of-B cells than in short-term Treg-of-B cells, three-day short-term culture is sufficient  Studies have demonstrated that ICOS controls IL-10 production and functional CTLA4 expression in Treg cells [73][74][75]. PD1 recruits SHP-1 and SHP-2 to intrinsically downregulate T cell receptor signaling, which maintains an anergic phenotype in Treg cells [76,77]. Mouse Treg cells constitutively expressed GITR and OX40 and involved the tTreg cells development as well as their functions [78][79][80]. All regulatory-T-related molecules on Treg-of-B cells, including IL-10, TGF-β, LAG3, CTLA4, ICOS, PD1, GITR, and OX40, might confer partial suppressive activities to compensate for single blockage or neutralization. The critical molecules controlling Treg-of-B cell phenotype and regulatory mechanisms remain priorities for investigation. The inhibitory functions of Treg-of-B cell depend on the suppressive molecules on the surface or soluble mediators that require short distance.

B-cell-induced CD4 + Foxp3 + regulatory T cells
Reports have revealed the role of B cells in the development of Treg cells. Naïve primary B cells preferentially induced the expansion of allogenic CD4 + Foxp3 + T cells rather than CD4 + Foxp3 -T cells [81,82]. Splenic B cells converted allogenic naïve T cells into Foxp3 + regulatory T cells in the presence of TGF-β and IL-2, and peritoneal B cells induce Th17 cells [83]. Human CD40activated B cells induced the differentiation of CD25 + Foxp3 + CD62L + regulatory T cells more efficiently than immature DCs [84,85]. In contrast, reports demonstrated that murine CD40-activated B cells promoted CD4 + T cell proliferation and effector functions [86,87]. Furthermore, the frequency of intrathymic B cells correlated with that of tTreg cells, and B cells colocalized with tTreg cells in the thymus [88,89]. Intrathymic B cells expressed autoimmune regulator (Aire), increased the levels of MHC class II and CD80, and contributed to T cell negative selection for central T cell tolerance [90,91]. Taken together, there are unknown criteria, such as MHC class II-TCR signaling, the B cell activation status, and different types of tissue resident B cells, that may fine-tune the expression of Foxp3 in B-cellinduced regulatory T cells.

Conclusions
To date, we know that naïve antigen-presenting B cell is sufficient to induce CD4 + Foxp3regulatory T cells without additional cytokines or chemicals in an IL-10-and IL-27-dispensable and cell-cell contact-dependent manner. The expression levels of characteristic molecules differentiate Treg-of-B cells from well-known T helper and regulatory T cells as a brand-new type of CD4 + Foxp3regulatory T cells (Fig. 1). Treg-of-B cells possess IL-10-depedent, IL-10-independent, and cell-cell contact-dependent suppressive abilities in antigenspecific and non-antigen specific fashions. Compared to long-term Treg-of-B cells, short-term Treg-of-B cells act through multiple suppressive pathways, and thus a blockade strategy would be more easily overcome through compensation by other pathways. Treg-of-B cells exhibit immunomodulatory effects in Th2-, Th1-, and Th17-medated diseases and even allogeneic transplantation. Nevertheless, the physiological conditions or cues necessary for Treg-of-B cell generation remain unknown. What is the fine-tuning mechanism for B cells to induce CD4 + Foxp3or expand CD4 + Foxp3 + T cells? What factors determine the kinetics, memory, and maintenance? And, most importantly, how could we use Treg-of-B cells in immunotherapy?