Regulatory T-Cells in Treatment of Type-1 Diabetes: Types and Approaches

Naresh Sachdeva, PhD

Assistant Professor Department of Endocrinology Post Graduate Institute of Medical Education and Research (PGIMER) Chandigarh – 160012, India; Tel. +91-172-2755282; Fax: +91-172-2744401; E-mail: naresh_pgi@hotmail.com

INTRODUCTION

Type 1 Diabetes (T1D) is mainly a T-cell mediated autoimmune disease characterized by the destruction of pancreatic beta (β) cells leading to insulin deficiency. Regardless of the predisposing factors and environmental triggers, the main pathogenic mechanism leading to T1D is the priming of CD8+ T-cells by the autoreactive CD4+ T-cells. These CD8+ T-cells further recognize and destroy pancreatic β cells by releasing cytotoxic granules mainly containing granzymes and perforin molecule.¹ Such autoreactive CD8+ T-cells can be easily detected from the peripheral blood of T1D subjects, as they are more differentiated and express central memory markers.^2,3,4 In healthy individuals these autoreactive T-cells are either eliminated in thymus or suppressed by regulatory T-cells (Tregs) in the peripheral circulation.

Regulatory T-cells

These cells also called as suppressor T-cells, are a subpopulation of T-cells that play an important role in regulation of exaggerated immune response to self/foreign antigens.⁵ Tregs are important in induction and maintenance of self-tolerance.⁶ They comprise 1-10% of the T Helper (TH) cell population in healthy adult humans and mice.^5,7,8 These cells express high levels of surface marker CD25, Fork head box P3 (FoxP3)9 along with low CD127^10,11,12 which together have been suggested as reliable markers for Tregs. Tregs have the capacity to actively block immune responses, inflammation and tissue destruction by suppressing the functions of various cell types and processes, including classical TH cells, B-cell antibody production, affinity maturation, CD8+ Cytotoxic T Lymphocyte (CTL) granule release and Antigen Presenting Cell (APC) function and maturation state.13,14,15 Tregs mediate these functions mainly by 4 mechanisms including; 1) production of suppressive cytokines, 2) direct cytolytic activity, 3) cytokine (IL-2) deprivation, and 4) cell contact-induced cell modulation.^16,17

Based on acquisition of CD25, Tregs can be divided into two subsets: natural Treg (nTreg) cells and adaptive or induced Treg (iTreg) cells. nTregs acquire expression of CD25 in thymus whereas iTregs acquire CD25 expression in the periphery. However, utility of CD25 as a marker of Tregs is limited because of its expression on activated T-cells as well. iTregs are generated extra-thymically and IL-2 is essential for their generation both in vivo and in vitro. Tr1 and Th3 cells represent other subsets of suppressor T-cells. Tr1 cells do not express FoxP3, but produce high level of immunosuppressive cytokine, IL-10,^18,19 whereas Th3 cells produce TGF-β, which also has immunosuppressive role.²⁰ Phenotypically, it is difficult to differentiate nTregs from iTregs as both subsets have similar characteristics and suppressive function. Both Treg subsets express CD25, FoxP3, Glucocorticoid-induced TNF- receptor (GITR) and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) whereas, nTregs exhibit a higher expression of Programmed cell death-1 (PD1), Neuropilin 1 (Nrp-1) and Helios compared to iTregs.²¹ It has been reported that nTregs are generated when there is a need to control inflammatory responses to autoantigens, whereas iTregs are generated in response to stimulation with foreign antigens such as intestinal flora and food allergens.²² The features distinguishing nTregs from iTregs have been summarized in Table 1.

Table 1: Characteristic features of natural and induced Tregs.

Alteration in the Frequency and Function of Tregs in T1D

Several groups have reported alterations in the frequency, function and phenotype of Tregs in patients with T1D. Subjects with T1D may harbor lower frequency of Tregs in the peripheral blood.^11,42,43,44 Ryba-Stanisławowska, et al. showed that patients with T1D had a decreased percentage of circulating CD4+CD25hi Tregs and elevated levels of serum IL12 and IL-18 in comparison to their healthy controls.⁴⁵ However, a few studies have also reported no alteration in the frequency of the Tregs in peripheral blood of T1D subjects.^46,47 A recent study by Xufré, et al. reported that the frequency of peripheral CD4+CD25hi Treg cells are similar between T1D subjects and healthy controls.⁴⁷ However, the yield of sorted Treg cells was found to be significantly lower in T1D subjects than in controls. Again, upon comparison of Treg cell phenotype between the two groups, the only difference observed was the low expression of GITR in T1D subjects.⁴⁷ Zoka, et al. studied the expression of CD25 on CD4+FoxP3+ cells and reported that T1D subjects have higher proportion of CD25-/low cells among CD4+FoxP3+ Treg cells.⁴⁸ Willcox, et al. analyzed postmortem pancreatic samples from T1D subjects.³⁰ FoxP3+ Tregs were only found in islets from a single subject, suggesting that the lack of local Treg cells might be important in the pathogenesis of T1D.⁴⁹

Besides numbers, many studies have reported that Tregs isolated from peripheral blood of the T1D patients are defective in suppressive function.^50,51,52,53 Ferraro, et al. showed that Tregs from peripheral blood of T1D subjects have normal suppressive activity but Tregs isolated from Pancreatic Lymph Nodes (PLN) of same subjects are functionally defective.¹² It has also been reported that Tregs are unstable in T1D subjects since they lose the expression of FoxP3 due to defect in IL-2R signalling.⁵³

Another study showed that T1D subjects harbor substantial percentage of cells with transient or unstable expression of FoxP3. These exFoxP3 cells produce inflammatory cytokines, indicative of a high degree of plasticity in Treg phenotype.^41,54 It has also been reported that the Teff cell population in T1D subjects are resistant to suppression by Tregs.^55,56 Thus, it is still unclear whether Treg cells from T1D patients have intrinsic defective function or whether the responder T-cells are resistant to suppression, warranting the need for additional studies. Moreover, studies on the role of Tregs in T1D were performed on peripheral blood rather than pancreas or PLN, therefore the defects in local Tregs are not well known.

Potential of Tregs in Treatment of T1D

There are many evidences which show that Tregs have the potential to prevent destruction of pancreatic islets, thereby protecting from T1D. Hence, strategies to increase Treg cell numbers and/or function are being explored as potential therapeutic approaches in treating T1D. Most of the treatment regimens to reverse diabetes in NOD mice worked via induction of Tregs or proliferation of Tregs.^{57,58,59,60,61,62} Therapy of T1D subjects with Tregs has been shown to prolong survival of pancreatic islets.⁶³ At the same time, the knowledge on use of different type of Tregs for their clinical applications has increased tremendously. Today, several groups are engaged in exploring different Treg cell types, sources, induction procedures and experimental systems in pursuit of generation of highly efficacious and stable Tregs for immunotherapy of T1D.

Approaches used for in vitro Induction of Tregs

Clinical use of Tregs is hindered by their low frequency in peripheral blood.64 Therefore several methods have been developed for induction and expansion of Tregs (Table 2), few of which have led to trials in T1D subjects with varying success rates (Table 3). Generation of iTregs from CD25- T-cells in vivo is still not fully understood. However it has been established that it requires TCR stimulation, IL-2 and TGF-β both in vitro and in vivo. Supplementation of other compounds such as rapamycin and All Trans Retinoic Acid (ATRA)^25,65 increase the yield and purity of Tregs.66 Addition of TGF-β induces transcription of FoxP3 by a mechanism that involves transcription factors STAT3 and NFAT at the FoxP3 gene enhancer element.^67,68 Tregs induced in the presence of rapamycin/TGF-β are more stable than ATRA/TGF-β iTregs.⁶⁶ However, upon re-stimulation the expression of FoxP3 decreases in both the iTregs; which in turn may lead to loss of suppressive activity.⁶⁶ nTregs expanded in presence of rapamycin maintain FoxP3 expression and are highly suppressive than IL-2 expanded nTregs. Tregs expanded with anti CD3 and anti CD28 infused into T1D subjects have been shown to prolong the survival of pancreatic islets.⁶³ Recently Lu, et al. reported that iTregs induced from CD39+ naive T-cells demonstrated enhanced proliferative and suppressive ability.⁶⁹ However, expanded nTregs are shown to be superior to fresh nTregs since in vitro expansion improves their in vivo regulation.⁷ 

Table 2: Approaches used for in vitro induction of Tregs.

Table 3: Immunotherapeutic approaches involving induction/use of Tregs in T1D subjects.

Antigen Specific Tregs are More Potent than Polyclonal Tregs

Harnessing Tregs is a promising approach for treating autoimmune disease. Administration of polyclonal Tregs may be associated with significant off-target effects, including a global immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Therefore, the objective of research in recent times has shifted to antigen specific therapeutic approaches that can reverse the disease by selectively halting the harmful immune response without requiring lifelong immune suppression. Adoptive transfer studies suggest that antigen specificity is required by Tregs for trafficking and maintenance in inflammatory tissues such as the pancreas in T1D.^98,99 Antigen specific Tregs are required at less number and are more efficient than polyclonal Tregs in suppressing autoimmune diabetes.^85,100,101 Previous studies have shown that small number of in vitro expanded antigen specific Tregs are sufficient to reverse T1D in comparison to large numbers of polyclonal Tregs.¹⁰⁰ Antigen specific Tregs have been reported to exhibit a much lower threshold for activation and may be activated by a broad range of loosely-defined analogs of their cognate antigen; normally it is conceivable that the polyclonal Tregs may have received sufficient signaling within the pancreas to become suppressive.¹⁰² Besides, the site specific mode of action, antigen specific Tregs have the ability to act as bystander suppressor locally in the organ under attack. It has also been shown in mice that antigen-specific Tregs treat autoimmunity without compromising antibacterial immune response.¹⁰³ However, isolation of sufficient number of antigen specific Tregs is a major challenge, particularly when sampling is limited to peripheral blood. Moreover, success in inducing antigen-specific tolerance has been hampered by the inability to identify peptides triggering the diabetogenic versus the regulatory response.

Generation of Antigen Specific Tregs

Several protocols have been established to induce antigen specific Tregs. Groux, et al. described induction of antigen specific Tregs by stimulating CD4+ T-cells with antigen and IL-10 in vitro. This resulted in generation of antigen specific IL-10 producing Tr1 cells.⁷¹ Immature Dendritic Cells (DCs) as well as plasmacytoid DCs exhibit regulatory functions.^{78,104,105,106} Therefore, these DCs, have been used to induce antigen specific CD4+ Tregs from CD4+CD25-T-cells.78,79 Walker, et al. used the mature DCs loaded with hemagglutinin (306-319, PKYVKQNTLKLAT) to generate Influenza hemagglutinin epitopes specific Tregs from CD4+CD25- T-cells.75 CD40 activated B cells are more potent than immature DCs for the induction of antigen specific Tregs.^86,107 Wenwei, et al. developed a method for expansion of alloantigen specific Tregs using CD40 activated B cells as APCs.¹⁰⁷ Alice, et al. generated the islet antigen specific Tregs from CD4+CD25- T-cells by growing them in presence of GAD65 and IL-2 and observed that GAD65 derived epitope specific Tregs exhibit bystander suppression in the presence of antigen. In the suppression assay these epitope specific Tregs suppressed not only proliferation of GAD specific Teff cells but also of Tetanus Toxoid (TT) specific Teff cells in the presence of GAD. However, this bystander suppression was not observed in absence of GAD65 peptides or when TT was present alone.⁸⁴ Therefore these observations indicate that it might be possible to reverse autoimmune diabetes by small number of epitope specific Tregs rather than having Tregs specific for all the diabetes associated antigens. Brusko, et al. used lentiviral T-cell Receptor (TCR) gene transfer system to generate antigen specific Tregs from murine nTregs.⁸⁵ Tregs generated using this approach effectively blocked antigen-specific Teff cell activity. Also, DCs treated with glucocorticoids, upregulate Glucocorticoid-induced leucine zipper (GILZ). GILZ expressing DCs in the presence of IL-10 induce antigen specific CD25hi CTLA4+ Tregs.⁷⁹ Recently Akbarpour, et al. transferred an immunodominant insulin epitope (B9-23) expressing lentivirus vector in hepatocytes of NOD mice. The therapy induced insulin specific Tregs that inhibited immune cell infiltration in the pancreatic islets and halted diabetes development.⁸⁹ While induction of antigen specific Tregs is difficult, analysis of their characteristics is also technically challenging. Following induction, either ex or in vivo, antigen specific Tregs can be sorted using MHC class II tetramers loaded with peptide of interest.⁸⁴ Latency-associated peptide (LAP) and Glycoprotein A Repetitions Predominant (GARP) protein have also been reported as markers to identify human antigen-specific Tregs.¹⁰⁸

Stability of Tregs

Clinical usage of Treg cells is hindered due to their instability. Tregs have been shown to lose FoxP3 expression under inflammatory environment.^{54,109,110,111,112,113} Proinflammatory environment may abrogate the suppressive activity of Tregs^114,115,116 or cause Teff cells resistant to suppression.¹¹⁷ There are certain reports that show that plasticity of Tregs might play important role in pathogenesis of autoimmune diseases. Indeed, increased frequency of IFN-γ+FoxP3+ cells has been reported in subjects with T1D.⁴¹ Th17 cells originating from FoxP3+ T-cells have shown to play a key role in the pathogenesis of autoimmune arthritis.¹¹⁸ Stable Tregs can be distinguished from the unstable ones on the basis of epigenetic modifications in the CpG-rich Treg Specific Demethylated Region (TSDR) of the FoxP3 locus.66 Demethylation of the TSDR region correlates with the stability of FoxP3 gene. Strong methylation in the TSDR of FoxP3 promoter may be associated with unstable phenotype of Tregs. Analyzing the demethylation status of the TSDR in the FoxP3 may aid in distinguishing the stable Tregs from unstable Tregs.³⁶

Role of Colonic Tregs

The gut immune system plays an important role in autoimmune diabetes. One of the most influential environmental factors that influences gut immune system is the gut microbiota. The development of clinical diabetes is preceded by intestinal alterations such as an aberrant intestinal microbiota, a leaky intestinal mucosal barrier and an altered mucosal immune system.¹¹⁹ Therefore, a hygiene hypothesis has been postulated which suggested a reduction in childhood exposure to infections leading to the accelerated development of T1D.¹²⁰ The gut microbiota shape the mucosal immune system by controlling many types of T-cells including the colonic regulatory T-cells (cTregs) which are a type of induced Tregs. It has been proposed that pathogenic microbes promote T1D development by enhancing self-reactive T-cells,¹¹⁹ while many microbial species such as Clostridia species has been shown to be potent inducers of cTregs. The gut microbiota modulates local immune system by acting on various immune cells including DCs. These lamina propria CD103+ CD11c+ DCs direct the antigens that cross the epithelial barrier to the Gut Associated Lymphoid Tissue (GALT), and enable the differentiation of naïve CD4+ T-cells to cTregs via TGF-β and retinoic acid.¹⁰⁴,¹²¹ These Tregs control inflammation via anti-inflammatory agents such as IL10 and TGF-β.¹²² Studies in T1D subjects have reported low frequency of FoxP3+ Tregs and an impaired differentiation of FoxP3+ Treg cells by intestinal CD103+CD11c+DCs.¹¹ Due to the immunological connection between the GALT and the PLN, the immunological changes taking place in the gut are reflected in the pancreas. Thus the impaired generation of Tregs in the gut alters the Teff/Treg cell balance in PLN and islets thereby promoting Teff cell responses against pancreatic self-antigens.^123,124 This leads to failure of self-tolerance and development of autoimmunity.

Strategies to Induce/Increase Abundance of cTregs

cTregs play a critical role in limiting the intestinal inflammation. They are constitutively present in the intestinal mucosa as well as the GALT and thus maintain immune homeostasis. However the breakdown of gut immune system leads to many autoimmune diseases including T1D. Hence various strategies have been developed to generate cTregs.

Animal studies have shown that intestinal colonization with commensal bacteria activate and expand Treg cells, as well as de novo generate cTregs. The colonization of germ free mice with Altered Schaedler Flora (ASF) species resulted in the generation of Tregs in colonic lamina propria. These Tregs limited the proliferation of Th1 and Th17 cells.¹²⁵ Furthermore a defined cocktail of 17 strains of clostridium species within the cluster IV, XIVa and XVIII of Clostridia strains has been shown to trigger the expression of TGF-β in the intestinal epithelial cells, thereby promoting the accumulation of FoxP3+ Tregs.¹²⁶ Also Polysaccharide A (PSA) secreted by Bacteroides fragilis has been shown to act via TLR2 expressed on CD4+ T-cells which enable their conversion to FoxP3+ T-cells that produce IL-10.⁹⁴ The specific Bifidobacterium strains present in healthy microbiota provides protection against pathogens; accordingly the early administration of Bifidobacterium infantis to mice attenuated the severity of colitis by the induction of Tregs in the Mesenteric Lymph Nodes (MLN).¹²⁷ With encouraging reports, several groups have come up with probiotics, live beneficial microorganisms that when administered continuously can induce gut immunity. In an important study, oral administration of probiotic VSL#3 to NOD mice during the early stages of life showed a delay in the progression of diabetes. This prevention was associated with the generation of IL-10 producing Tregs in the GALT.¹²⁸ Autoantigen specific therapies also hold great promise in the reversal of T1D by induction of oral tolerance. One such approach involved the administration of Lactococcus lactis for controlled secretion of GAD65 and IL10 in the gut, which favored the induction of Tregs.¹²⁹

Besides bacteria, their metabolic products such as, Short Chain Fatty Acids (SCFA) have been shown to affect the colonic health as they can penetrate the intestinal epithelium and restore intestinal immune responses. The administration of SCFA such as acetate, propionate and butyrate enabled the restoration of cTregs in germ free mice and significantly increased the expression of IL-10 and TGF-β in cTregs.¹³⁰ Among the SCFA, butyrate has received a lot of attention due to its effect on colonic function. The dietary administration of butyrylated high amylose maize starches to mice showed an increase in the frequency of cTregs.¹³¹ Butyrate is also well known to epigenetically modify the FoxP3 gene by inhibiting the class I and IIa of histone deacetylases, thereby increasing the FoxP3 expression and differentiation of Treg cells.^132,133,134 The colonic DCs and macrophages express the cell surface receptor Gpr109a.^135,136 Butyrate acts via these receptors and induces the expression of anti-inflammatory molecules such as IL-10 and aldehyde dehydrogenase (Aldh1a), thereby supporting the differentiation of cTregs.^137,138 Additionally, intervention strategies such as dietary supplementation with 1,2 dihydroxyvitamin D (1,25(OH)2D3), an active form of vitamin D promotes the development of FoxP3+ Treg cells and inhibits the differentiation of Th1 and Th17 cells.¹³⁹ High doses of vitamin D3 safely reduced diabetes development by preventing insulitis and preserving β cell mass in NOD mice.¹⁴⁰ Also the deficiency of Vitamin B9 or folic acid derived from diet and commensal bacteria showed marked reduction in gut FoxP3+ Treg cells.¹⁴¹ In addition to induction approaches, homing of T lymphocytes to the gut is also important in induction of cTregs and impaired homing of T-cells is implicated in many inflammatory diseases. For example, GPR15, an orphan heterotrimeric guanine nucleotide-binding protein (G protein) coupled receptor, controls the specific homing of FoxP3 Tregs, to the large intestine lamina propria, and its expression can be modulated by gut microbiota and TGF-β.¹⁴²

Despite the difficulties in characterization of induced Tregs, there is an increasing awareness about the importance of induction of immune tolerance via gut and generation of cTregs have come to the forefront as an actively pursued area of research, in prevention of autoimmune diseases like T1D.

CONCLUSIONS

Many immunotherapeutic approaches including selfantigens and immune modulating agents have been tried to tackle autoimmunity observed in T1D. Most of these treatment strategies have failed to prevent or improve the clinical outcome of the disease. There are multiple etiologies that are known to cause β cell destruction in T1D. Hence targeting a single factor may not provide a lifelong preservation of the β cell mass in T1D. While the defects in number and function of Tregs in T1D were known long ago, research on application of Tregs in T1D has picked up more in recent years. Today there are several choices available in immunotherapeutic approaches with Tregs, ranging from their type (natural versus induced), source (peripheral versus colonic) or specificity (polyclonal versus antigen-specific) or methods of induction (direct versus indirect) and expansion (invitro versus in-vivo), each of which has its specific advantages and limitations. Regardless of the variety, Tregs have opened up new vistas in treatment of T1D. With growing understanding about the generation of different types of Tregs and their clinical applications, the use of Tregs in future treatment of T1D looks quite promising. We believe, Tregs might provide benefit in the form of a combination therapy that attenuates autoimmunity towards the pancreas, ultimately preserving β cell mass.

CONFLICTS OF INTEREST: None.

1. Knight RR, Kronenberg D, Zhao M, et al. Human β-cell killing by autoreactive preproinsulin-specific CD8 T-cells is predominantly granule-mediated with the potency dependent upon T-cell receptor avidity. Diabetes. 2013; 62(1): 205-213. doi: 10.2337/db12-0315

2. Luce S, Lemonnier F, Briand J-P, et al. Single insulin-specific CD8+ T-cells show characteristic gene expression profiles in human type 1 diabetes. Diabetes. 2011; 60(12): 3289-3299. doi: 10.2337/db11-0270

3. Skowera A, Ladell K, McLaren JE, et al. β-cell-specific CD8 T-cell phenotype in type 1 diabetes reflects chronic autoantigen exposure. Diabetes. 2014; DB-140332. doi: 10.2337/db14-0332

4. Sachdeva N, Paul M, Badal D, et al. Preproinsulin specific CD8+ T-cells in subjects with latent autoimmune diabetes show lower frequency and different pathophysiological characteristics than those with type 1 diabetes. Clinical Immunology. 2015. doi: 10.1016/j.clim.2015.01.005

5. Sakaguchi S. Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T-cells in immunological tolerance to self and non-self. Nature immunology. 2005; 6(4): 345-352. doi: 10.1038/ni1178

6. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Pillars article: immunologic self-tolerance maintained by activated T-cells expressing IL-2 receptor α-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 1995.

7. Maloy KJ, Powrie F. Regulatory T-cells in the control of immune pathology. Nature immunology. 2001; 2(9): 816-822. doi: 10.1038/ni0901-816

8. Shevach EM. CD4+ CD25+ suppressor T-cells: more questions than answers. Nature Reviews Immunology. 2002; 2(6): 389-400. doi: 10.1038/nri821

9. Ramsdell F. Foxp3 and natural regulatory T-cells: key to a cell lineage? Immunity. 2003; 19(2): 165-168. doi: 10.1016/s1074-7613(03)00207-3

10. Liu W, Putnam AL, Xu-Yu Z, et al. CD127 expression in- versely correlates with FoxP3 and suppressive function of hu- man CD4+ T reg cells. The Journal of experimental medicine. 2006; 203(7): 1701-1711. doi: 10.1084/jem.20060772

11. Badami E, Sorini C, Coccia M, et al. Defective differentiation of regulatory FoxP3+ T-cells by small-intestinal dendritic cells in patients with type 1 diabetes. Diabetes. 2011; 60(8): 2120- 2124. doi: 10.2337/db10-1201

12. Ferraro A, Socci C, Stabilini A, et al. Expansion of Th17 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes. Diabetes. 2011; 60(11): 2903-2913. doi: 10.2337/db11-0090

13. Taams LS, Boot EP, van Eden W, Wauben MH. Anergic T-cells modulate the T-cell activating capacity of antigen- presenting cells. Journal of autoimmunity. 2000; 14(4): 335-341. doi: 10.1006/jaut.2000.0372

14. Eddahri F, Oldenhove G, Denanglaire S, Urbain J, Leo O, Andris F. CD4+ CD25+ regulatory T-cells control the magnitude of T-dependent humoral immune responses to exogenous antigens. European journal of immunology. 2006; 36(4): 855- 863. doi: 10.1002/eji.200535500

15. Mempel TR, Pittet MJ, Khazaie K, et al. Regulatory T-cells reversibly suppress cytotoxic T-cell function independent of effector differentiation. Immunity. 2006; 25(1): 129-141. doi: 10.1016/j.immuni.2006.04.015

16. Bluestone JA, Tang Q. How do CD4+ CD25+ regulatory T-cells control autoimmunity? Current opinion in immunology. 2005; 17(6): 638-642. doi: 10.1016/j.coi.2005.09.002

17.TangQ,BluestoneJA.TheFoxp3+regulatoryT-cell:ajack of all trades, master of regulation. Nature immunology. 2008; 9(3): 239-244. doi: 10.1038/ni1572

18. Roncarolo MG, Bacchetta R, Bordignon C, Narula S, Levings MK. Type 1 T regulatory cells. Immunological reviews. 2001; 182(1): 68-79. doi: 10.1034/j.1600-065X.2001.1820105.x

19. Pot C, Apetoh L, Kuchroo VK, editors. Type 1 regulatory T-cells (Tr1) in autoimmunity. Seminars in immunology. Elsevier, 2011. doi: 10.1016/j.smim.2011.07.005

20. Okamura T, Fujio K, Sumitomo S, Yamamoto K. Roles of LAG3 and EGR2 in regulatory T-cells. Annals of the rheumatic diseases. 2012; 71(Suppl 2): i96-i100. doi: 10.1136/annrheumdis-2011-200588

21. Yadav M, Louvet C, Davini D, et al. Neuropilin-1 distinguishes natural and inducible regulatory T-cells among regulatory T-cell subsets in vivo. The Journal of experimental medicine. 2012; 209(10): 1713-1722. doi: 10.1084/jem.20120822

22. de Lafaille MAC, Lafaille JJ. Natural and adaptive foxp3+ regulatory T-cells: more of the same or a division of labor? Immunity. 2009; 30(5): 626-635. doi: 10.1016/j. immuni.2009.05.002

23. Lu L, Ma J, Li Z, et al. All-trans retinoic acid promotes TGF- β-induced Tregs via histone modification but not DNA demethy- lation on Foxp3 gene locus. PloS one. 2011; 6(9): e24590. doi: 10.1371/journal.pone.0024590

24. Tai X, Cowan M, Feigenbaum L, Singer A. CD28 costimulation of developing thymocytes induces Foxp3 expression and regulatory T-cell differentiation independently of interleukin 2. Nature immunology. 2005; 6(2): 152-162. doi: 10.1038/ni1160

25. Benson MJ, Pino-Lagos K, Rosemblatt M, Noelle RJ. All- trans retinoic acid mediates enhanced T reg cell growth, differ- entiation, and gut homing in the face of high levels of co-stim- ulation. The Journal of experimental medicine. 2007; 204(8): 1765-1774. doi: 10.1084/jem.20070719

26. Shevach EM, Tran DQ, Davidson TS, Andersson J. The critical contribution of TGF-β to the induction of Foxp3 expression and regulatory T-cell function. European journal of immunology. 2008; 38(4): 915-917. doi: 10.1002/eji.200738111

27. Bilate AM, Lafaille JJ. Induced CD4+ Foxp3+ regulatory T-cells in immune tolerance. Annual review of immunology. 2012; 30: 733-758. doi: 10.1146/annurev-immunol-020711-075043

28. Salomon B, Lenschow DJ, Rhee L, et al. B7/CD28 costimulation is essential for the homeostasis of the CD4+ CD25+ immunoregulatory T-cells that control autoimmune diabetes. Immunity. 2000; 12(4): 431-440. doi: 10.1016/s1074-7613(00)80195-8

29. Cheng G, Yu A, Dee MJ, Malek TR. IL-2R signaling is essential for functional maturation of regulatory T-cells during thymic development. The Journal of Immunology. 2013; 190(4): 1567-1575. doi: 10.4049/jimmunol.1201218

30. Stritesky GL, Jameson SC, Hogquist KA. Selection of self- reactive T-cells in the thymus. Annual review of immunology. 2012; 30: 95. doi: 10.1146/annurev-immunol-020711-075035

31. Hsieh C-S, Lee H-M, Lio C-WJ. Selection of regulatory T-cells in the thymus. Nature Reviews Immunology. 2012; 12(3): 157-167. doi: 10.1038/nri3155

32. Josefowicz SZ, Lu L-F, Rudensky AY. Regulatory T-cells: mechanisms of differentiation and function. Annual review of immunology. 2012; 30: 531-564. doi: 10.1146/annurev. immunol.25.022106.141623

33. Lee H-M, Bautista JL, Scott-Browne J, Mohan JF, Hsieh C-S. A broad range of self-reactivity drives thymic regulatory T-cell selection to limit responses to self. Immunity. 2012; 37(3): 475-486. doi: 10.1016/j.immuni.2012.07.009

34. Round JL, Mazmanian SK. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proceedings of the National Academy of Sciences. 2010; 107(27): 12204-12209. doi: 10.1073/pnas.0909122107

35. Lathrop SK, Bloom SM, Rao SM, et al. Peripheral education of the immune system by colonic commensal microbiota. Na- ture. 2011; 478(7368): 250-254. doi: 10.1038/nature10434

36. Lin X, Chen M, Liu Y, et al. Advances in distinguishing natural from induced Foxp3+ regulatory T-cells. International journal of clinical and experimental pathology. 2013; 6(2): 116.

37. Bruder D, Probst-Kepper M, Westendorf AM, et al. Frontline: Neuropilin-1: a surface marker of regulatory T-cells. European journal of immunology. 2004; 34(3): 623-630. doi: 10.1002/ eji.200324799

38. Thornton AM, Korty PE, Tran DQ, et al. Expression of He- lios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. The Journal of Immunology. 2010; 184(7): 3433-3441. doi: 10.4049/jimmunol.0904028

39. Kim YC, Bhairavabhotla R, Yoon J, et al. Oligodeoxynucle- otides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion. Blood. 2012; 119(12): 2810- 2818. doi: 10.1182/blood-2011-09-377895

40. Lal G, Bromberg JS. Epigenetic mechanisms of regulation of Foxp3 expression. Blood. 2009; 114(18): 3727-3735. doi: 10.1182/blood-2009-05-219584

41. McClymont SA, Putnam AL, Lee MR, et al. Plasticity of human regulatory T-cells in healthy subjects and patients with type 1 diabetes. The Journal of Immunology. 2011; 186(7): 3918-3926. doi: 10.4049/jimmunol.1003099

42. Kukreja A, Cost G, Marker J, et al. Multiple immuno-regula- tory defects in type-1 diabetes. The Journal of clinical investiga- tion. 2002; 109(109 (1)): 131-140. doi: 10.1172/JCI13605

43. Ryba M, Rybarczyk-Kapturska K, Zorena K, Myśliwiec M, Myśliwska J. Lower Frequency of CD62L high and Higher Fre- quency of TNFR2. Mediators of inflammation. 2011; 2011. doi: 10.1155/2011/645643

44. Ryba-Stanisławowska M, Skrzypkowska M, Myśliwska J, Myśliwiec M. The serum IL-6 profile and Treg/Th17 peripheral cell populations in patients with type 1 diabetes. Mediators of inflammation. 2013; 2013. doi: 10.1155/2013/205284

45. Ryba-Stanisławowska M, Rybarczyk-Kapturska K, Myśliwiec M, Myśliwska J. Elevated Levels of Serum IL-12 and IL-18 are Associated with Lower Frequencies of CD4+ CD25highFOXP3+ Regulatory T-cells in Young Patients with Type 1 Diabetes. Inflammation. 2014; 37(5): 1513-1520. doi: 10.1007/s10753-014-9878-1

46. Brusko T, Wasserfall C, McGrail K, et al. No alterations in the frequency of FOXP3+ regulatory T-cells in type 1 diabetes. Diabetes. 2007; 56(3): 604-612. doi: 10.2337/db06-1248

47. Xufré C, Costa M, Roura-Mir C, et al. Low frequency of GITR+ T-cells in ex vivo and in vitro expanded Treg cells from type 1 diabetic patients. International immunology. 2013: dxt020. doi: 10.1093/intimm/dxt020

48. Zóka A, Barna G, Somogyi A, et al. Extension of the CD4+ Foxp3+ CD25-/low regulatory T-cell subpopulation in type 1 diabetes mellitus. Autoimmunity. 2014; 1-9. doi: 10.3109/08916934.2014.992518

49. Willcox A, Richardson S, Bone A, Foulis A, Morgan N. Analysis of islet inflammation in human type 1 diabetes. Clini- cal & Experimental Immunology. 2009; 155(2): 173-181. doi: 10.1111/j.1365-2249.2008.03860.x

50. Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TI. Defective suppressor function in CD4+ CD25+ T-cells from patients with type 1 diabetes. Diabetes. 2005; 54(1): 92-99. doi: 10.2337/diabetes.54.1.92

51. Brusko TM, Wasserfall CH, Clare-Salzler MJ, Schatz DA, Atkinson MA. Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes. Diabetes. 2005; 54(5): 1407-1414. doi: 10.2337/diabetes.54.5.1407

52. Haseda F, Imagawa A, Murase-Mishiba Y, Terasaki J, Hanafusa T. CD4+ CD45RA− FoxP3high activated regulatory T-cells are functionally impaired and related to residual insulin- secreting capacity in patients with type 1 diabetes. Clinical & Experimental Immunology. 2013; 173(2): 207-216. doi: 10.1111/cei.12116

53. Long SA, Cerosaletti K, Bollyky PL, et al. Defects in IL- 2R signaling contribute to diminished maintenance of FOXP3 expression in CD4+ CD25+ regulatory T-cells of type 1 diabetic subjects. Diabetes. 2010; 59(2): 407-415. doi: 10.2337/db09- 0694

54. Zhou X, Bailey-Bucktrout SL, Jeker LT, et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T-cells in vivo. Nature immunology. 2009; 10(9): 1000-1007. doi: 10.1038/ni.1774

55. Schneider A, Rieck M, Sanda S, Pihoker C, Greenbaum C, Buckner JH. The effector T-cells of diabetic subjects are resistant to regulation via CD4+ FOXP3+ regulatory T-cells. The Journal of Immunology. 2008; 181(10): 7350-7355. doi: 10.4049/jimmunol.181.10.7350

56. Lawson J, Tremble J, Dayan C, et al. Increased resistance to CD4+ CD25hi regulatory T-cell-mediated suppression in patients with type 1 diabetes. Clinical & Experimental Immunology. 2008; 154(3): 353-359. doi: 10.1111/j.1365-2249.2008.03810.x

57. Zhang J, Gao W, Yang X, et al. Tolerogenic vaccination reduced effector memory CD4 T-cells and induced effector memory Treg cells for type I diabetes treatment. PloS one. 2013; 8(7): e70056. doi: 10.1371/journal.pone.0070056

58. Johnson MC, Garland AL, Nicolson SC, et al. β-Cell–Specific IL-2 Therapy Increases Islet Foxp3+ Treg and Suppresses Type 1 Diabetes in NOD Mice. Diabetes. 2013; 62(11): 3775-3784. doi: 10.2337/db13-0669

59. Bilbao D, Luciani L, Johannesson B, Piszczek A, Rosenthal N. Insulin-like growth factor-1 stimulates regulatory T-cells and suppresses autoimmune disease. EMBO molecular medicine. 2014; e201303376. doi: 10.15252/emmm.201303376

60. Tian J, Dang H, Nguyen AV, Chen Z, Kaufman DL. Combined therapy with GABA and proinsulin/alum acts synergistically to restore long-term normoglycemia by modulating T-Cell autoimmunity and promoting β-Cell replication in newly diabetic NOD mice. Diabetes. 2014; 63(9): 3128-3134. doi: 10.2337/db13-1385

61. Turner MS, Isse K, Fischer DK, Turnquist HR, Morel PA. Low TCR signal strength induces combined expansion of Th2 and regulatory T-cell populations that protect mice from the development of type 1 diabetes. Diabetologia. 2014; 57(7): 1428-1436. doi: 10.1007/s00125-014-3233-9

62. Lin G-J, Sytwu H-K, Yu J-C, et al. Dimethyl sulfoxide inhibits spontaneous diabetes and autoimmune recurrence in non-obese diabetic mice by inducing differentiation of regulatory T-cells. Toxicology and applied pharmacology. 2015; 282(2): 207-214. doi: 10.1016/j.taap.2014.11.012

63. Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, et al. Therapy of type 1 diabetes with CD4+ CD25 high CD127- regulatory T-cells prolongs survival of pancreatic islets-Results of one year follow-up. Clinical Immunology. 2014; 153(1): 23- 30. doi: 10.1016/j.clim.2014.03.016

64. Bluestone JA. Regulatory T-cell therapy: is it ready for the clinic? Nature Reviews Immunology. 2005; 5(4): 343-349. doi: 10.1038/nri1574

65. Hill JA, Hall JA, Sun C-M, et al. Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+ CD44 hi cells. Immunity. 2008; 29(5): 758-770. doi: 10.1016/j. immuni.2008.09.018

66. Rossetti M, Spreafico R, Saidin S, et al. Ex vivo-expanded but not in vitro-induced human regulatory T-cells are candidates for cell therapy in autoimmune diseases thanks to stable demethylation of the FOXP3 regulatory T-cell-specific demethylated region. The Journal of Immunology. 2015; 194(1): 113-124. doi: 10.4049/jimmunol.1401145

67. Fantini M, Becker C, Monteleone G, Pallone F, Galle P, Neurath M. TGF-beta induces a regulatory phenotype in CD4+ CD25-T-cells through FoxP3 induction and dowuregulation of Smad7. Gastroenterology. Wb Saunders Co Independence Square West Curtis Center, Ste 300, Philadelphia, PA 19106- 3399 USA, 2004 .

68. Josefowicz SZ, Rudensky A. Control of regulatory T-cell lineage commitment and maintenance. Immunity. 2009; 30(5): 616-625. doi: 10.1016/j.immuni.2009.04.009

69. Lu Y, Gu J, Lu H, et al. iTreg induced from CD39+ naive T-cells demonstrate enhanced proliferate and suppressive ability. International immunopharmacology. 2015. doi: 10.1016/j. intimp.2015.03.039

70. Chai J-G, Coe D, Chen D, Simpson E, Dyson J, Scott D. In vitro expansion improves in vivo regulation by CD4+ CD25+ regulatory T-cells. The Journal of Immunology. 2008; 180(2): 858-869. doi: 10.4049/jimmunol.180.2.858

71. Groux H, O’Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997; 389(6652): 737-742. doi: 10.1038/39614

72. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. Induction of interleukin 10–producing, nonproliferating CD4+ T-cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. The Journal of experimental medicine. 2000; 192(9): 1213-1222. doi: 10.1084/jem.192.9.1213

73. Gad M, Kristensen NN, Kury E, Claesson MH. Characteriza- tion of T-regulatory cells, induced by immature dendritic cells, which inhibit enteroantigen-reactive colitis-inducing T-cell re- sponses in vitro and in vivo. Immunology. 2004; 113(4): 499- 508. doi: 10.1111/j.1365-2567.2004.01977.x

74. Rao PE, Petrone AL, Ponath PD. Differentiation and expansion of T-cells with regulatory function from human peripheral lymphocytes by stimulation in the presence of TGF-β. The Journal of Immunology. 2005; 174(3): 1446-1455. doi: 10.4049/jimmunol.174.3.1446

75. Walker MR, Carson BD, Nepom GT, Ziegler SF, Buckner JH. De novo generation of antigen-specific CD4+ CD25+ regulatory T-cells from human CD4+ CD25–cells. Proceedings of the National Academy of Sciences of the United States of America. 2005; 102(11): 4103-4108. doi: 10.1073/pnas.0407691102

76. DiPaolo RJ, Brinster C, Davidson TS, Andersson J, Glass D, Shevach EM. Autoantigen-specific TGFβ-induced Foxp3+ regulatory T-cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T-cells. The Journal of Immunology. 2007; 179(7): 4685-4693. doi: 10.4049/jimmunol.179.7.4685

77. Zhao C, Shi G, Vistica BP, et al. Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC. Cellular immunology. 2014; 290(2): 179- 184. doi: 10.1016/j.cellimm.2014.06.004

78. Kang H-K, Liu M, Datta SK. Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T-cells and contraction of inflammatory Th17 cells. The Journal of Immunology. 2007; 178(12): 7849-7858. doi: 10.4049/jimmunol.178.12.7849

79. Hamdi H, Godot V, Maillot M-C, et al. Induction of antigen- specific regulatory T lymphocytes by human dendritic cells ex- pressing the glucocorticoid-induced leucine zipper. Blood. 2007; 110(1): 211-219. doi: 10.1182/blood-2006-10-052506

80. Tarbell KV, Petit L, Zuo X, et al. Dendritic cell–expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T-cells restore normoglycemia in diabetic NOD mice. The Journal of experimental medicine. 2007; 204(1): 191-201. doi: 10.1084/jem.20061631

81. Pillai V, Ortega SB, Wang C, Karandikar NJ. Transient regulatory T-cells: a state attained by all activated human T- cells. Clinical immunology. 2007; 123(1): 18-29. doi: 10.1016/j. clim.2006.10.014

82. Prevosto C, Zancolli M, Canevali P, Zocchi MR, Poggi A. Generation of CD4+ or CD8+ regulatory T-cells upon mesenchymal stem cell-lymphocyte interaction. Haematologica. 2007; 92(7): 881-888. doi: 10.3324/haematol.11240

83. Francisco LM, Salinas VH, Brown KE, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T-cells. The Journal of experimental medicine. 2009; 206(13): 3015-3029. doi: 10.1084/jem.20090847

84. Long SA, Walker MR, Rieck M, et al. Functional islet-specific Treg can be generated from CD4+ CD25- T-cells of healthy and type 1 diabetic subjects. European journal of immunology. 2009; 39(2): 612-620. doi: 10.1002/eji.200838819

85. Brusko TM, Koya RC, Zhu S, et al. Human antigen-specific regulatory T-cells generated by T-cell receptor gene transfer. PloS one. 2010; 5(7): e11726. doi: 10.1371/journal.pone.0011726

86. Zheng J, Liu Y, Lau Y-L, Tu W. CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4+ regulatory T-cells. Cellular & molecular immunology. 2010; 7(1): 44-50. doi: 10.1038/cmi.2009.103

87.MotaC,Nunes-SilvaV,PiresAR,etal.Delta-like1-mediated notch signaling enhances the in vitro conversion of human memory CD4 T-cells into FOXP3-expressing regulatory T-cells. The Journal of Immunology. 2014; 193(12): 5854-5862. doi: 10.4049/jimmunol.1400198

88. Romani R, Pirisinu I, Calvitti M, et al. Stem cells from hu- man amniotic fluid exert immunoregulatory function via se- creted indoleamine 2, 3-dioxygenase1. Journal of cellular and molecular medicine. 2015. doi: 10.1111/jcmm.12534

89. Akbarpour M, Goudy KS, Cantore A, et al. Insulin B chain 9-23 gene transfer to hepatocytes protects from type 1 diabetes by inducing Ag-specific FoxP3+ Tregs. Science Translational Medicine. 2015; 7(289): 289ra81-289ra81. doi: 10.1126/sci-translmed.aaa3032

90. Chatenoud L, Bluestone JA. CD3-specific antibodies: a por- tal to the treatment of autoimmunity. Nature Reviews Immunol- ogy. 2007; 7(8): 622-632. doi: 10.1038/nri2134

91. Orban T, Farkas K, Jalahej H, et al. Autoantigen-specific regulatory T-cells induced in patients with type 1 diabetes mellitus by insulin B-chain immunotherapy. Journal of autoimmunity. 2010; 34(4): 408-415. doi: 10.1016/j.jaut.2009.10.005

92. Bock G, Prietl B, Mader JK, et al. The effect of vitamin D supplementation on peripheral regulatory T-cells and β cell function in healthy humans: a randomized controlled trial. Diabetes/metabolism research and reviews. 2011; 27(8): 942- 945. doi: 10.1002/dmrr.1276

93. Hjorth M, Axelsson S, Rydén A, Faresjö M, Ludvigsson J, Casas R. GAD-alum treatment induces GAD 65-specific CD4+ CD25 high FOXP3+ cells in type 1 diabetic patients. Clinical immunology. 2011; 138(1): 117-126. doi: 10.1016/j.clim.2010.10.004

94. Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, et al. Administration of CD4+ CD25 high CD127-regulatory T-cells preserves β-Cell function in Type 1 Diabetes in children. Diabetes care. 2012; 35(9): 1817-1820. doi: 10.2337/dc12-0038

95. Long SA, Rieck M, Sanda S, et al. Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs β-cell function. Diabetes. 2012; 61(9): 2340-2348. doi: 10.2337/db12-0049

96. Haller MJ, Gitelman SE, Gottlieb PA, et al. Anti-thymocyte globulin/G-CSF treatment preserves β cell function in patients with established type 1 diabetes. The Journal of clinical investi- gation. 2014; 125(125(1)). doi: 10.1172%2FJCI78492

97. Bonifacio E, Ziegler A-G, Klingensmith G, et al. Effects of high-dose oral insulin on immune responses in children at high risk for type 1 diabetes: the Pre-POINT randomized clinical trial. JAMA. 2015; 313(15): 1541-1549. doi: 10.1001/jama.2015.2928

98. Penaranda C, Bluestone JA. Is antigen specificity of autoreactive T-cells the key to islet entry? Immunity. 2009; 31(4): 534-536. doi: 10.1016/j.immuni.2009.09.006

99. Lennon GP, Bettini M, Burton AR, et al. T-cell islet accumulation in type 1 diabetes is a tightly regulated, cell- autonomous event. Immunity. 2009; 31(4): 643-653. doi: 10.1016/j.immuni.2009.07.008

100. Tang Q, Henriksen KJ, Bi M, et al. In vitro-expanded antigen-specific regulatory T-cells suppress autoimmune diabetes. The Journal of experimental medicine. 2004; 199(11): 1455-1465. doi: 10.1084/jem.20040139

101. Bluestone JA, Tang Q. Therapeutic vaccination using CD4+ CD25+ antigen-specific regulatory T-cells. Proceedings of the National Academy of Sciences. 2004; 101(Suppl 2): 14622- 14626. doi: 10.1073/pnas.0405234101

102. Larkin J, Picca CC, Caton AJ. Activation of CD4+ CD25+ regulatory T-cell suppressor function by analogs of the selecting peptide. European journal of immunology. 2007; 37(1): 139- 146. doi: 10.1002/eji.200636577

103. Kasagi S, Zhang P, Che L, et al. In vivo–generated antigen-specific regulatory T-cells treat autoimmunity without compromising antibacterial immune response. Science translational medicine. 2014; 6(241): 241ra78-241ra78. doi: 10.1126/scitranslmed.3008895

104. Coombes JL, Siddiqui KR, Arancibia-Cárcamo CV, et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T-cells via a TGF-β–and retinoic acid–dependent mechanism. The Journal of experimental medicine. 2007; 204(8): 1757-1764. doi: 10.1084/jem.20070590

105. Ito T, Yang M, Wang Y-H, et al. Plasmacytoid dendritic cells prime IL-10–producing T regulatory cells by inducible co- stimulator ligand. The Journal of experimental medicine. 2007; 204(1): 105-115. doi: 10.1084/jem.20061660

106. Yamazaki S, Dudziak D, Heidkamp GF, et al. CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T-cells. The Journal of Immunology. 2008; 181(10): 6923-6933. doi: 10.4049/jimmunol.181.10.6923

107. Tu W, Lau Y-L, Zheng J, et al. Efficient generation of human alloantigen-specific CD4+ regulatory T-cells from naive precursors by CD40-activated B cells. Blood. 2008; 112(6): 2554-2562. doi: 10.1182/blood-2008-04-152041

108. Noyan F, Lee YS, Zimmermann K, et al. Isolation of human antigen-specific regulatory T-cells with high suppressive function. European journal of immunology. 2014; 44(9): 2592- 2602. doi: 10.1002/eji.201344381

109. Yang XO, Nurieva R, Martinez GJ, et al. Molecular antagonism and plasticity of regulatory and inflammatory T-cell programs. Immunity. 2008; 29(1): 44-56. doi: 10.1016/j.immuni.2008.05.007

110. Komatsu N, Mariotti-Ferrandiz ME, Wang Y, Malissen B, Waldmann H, Hori S. Heterogeneity of natural Foxp3+ T-cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proceedings of the National Academy of Sciences. 2009; 106(6): 1903-1908. doi: 10.1073/pnas.0811556106

111. Oldenhove G, Bouladoux N, Wohlfert EA, et al. Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity. 2009; 31(5): 772-786. doi: 10.1016/j.immuni.2009.10.001

112. Miyao T, Floess S, Setoguchi R, et al. Plasticity of Foxp3+ T-cells reflects promiscuous Foxp3 expression in conventional T-cells but not reprogramming of regulatory T-cells. Immunity. 2012; 36(2): 262-275. doi: 10.1016/j.immuni.2011.12.012

113. Yurchenko E, Shio MT, Huang TC, et al. Inflammation- driven reprogramming of CD4+ Foxp3+ regulatory T-cells into pathogenic Th1/Th17 T effectors is abrogated by mTOR inhibition in vivo. PloS one. 2012; 7(4): e35572. doi: 10.1371/journal.pone.0035572

114. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4+ CD25+ T-cell-mediated suppression by dendritic cells. Science. 2003; 299(5609): 1033-1036. doi: 10.1126/science.1078231

115. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+ CD25hi T-regulatory cells. Blood. 2006; 108(1): 253-261. doi: 10.1182/blood-2005-11-4567

116. Peluso I, Fantini MC, Fina D, et al. IL-21 counteracts the regulatory T-cell-mediated suppression of human CD4+ T lymphocytes. The Journal of Immunology. 2007; 178(2): 732- 739. doi: 10.4049/jimmunol.178.2.732

117. Ruprecht CR, Gattorno M, Ferlito F, et al. Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T-cells in inflamed synovia. The Journal of experimental medicine. 2005; 201(11): 1793-1803. doi: 10.1084/jem.20050085

118. Komatsu N, Okamoto K, Sawa S, et al. Pathogenic conversion of Foxp3+ T-cells into TH17 cells in autoimmune arthritis. Nature medicine. 2014; 20(1): 62-68. doi: 10.1038/nm.3432

119. Vaarala O, Atkinson MA, Neu J. The Perfect storm for type 1 diabetes the complex interplay between intestinal micro- biota, gut permeability, and mucosal immunity. Diabetes. 2008; 57(10): 2555-2562. doi: 10.2337/db08-0331

120. Kukreja A, Maclaren NK. NKT-cells and type-1 diabetes and the” hygiene hypothesis” to explain the rising incidence rates. Diabetes technology & therapeutics. 2002; 4(3): 323-333. doi: 10.1089/152091502760098465

121. Sun C-M, Hall JA, Blank RB, et al. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. The Journal of experimental medicine. 2007; 204(8): 1775-1785. doi: 10.1084/jem.20070602

122. Coombes JL, Powrie F. Dendritic cells in intestinal immune regulation. Nature Reviews Immunology. 2008; 8(6): 435-446. doi: 10.1038/nri2335

123. Jaakkola I, Jalkanen S, Hänninen A. Diabetogenic T-cells are primed both in pancreatic and gut-associated lymph nodes in NOD mice. European journal of immunology. 2003; 33(12): 3255-3264. doi: 10.1002/eji.200324405

124. Hänninen A, Nurmela R, Maksimow M, Heino J, Jalkanen S, Kurts C. Islet β-cell-specific T-cells can use different homing mechanisms to infiltrate and destroy pancreatic islets. The American journal of pathology. 2007; 170(1): 240-250. doi: 10.2353/ajpath.2007.060142

125. Geuking MB, Cahenzli J, Lawson MA, et al. Intestinal bacterial colonization induces mutualistic regulatory T-cell responses. Immunity. 2011; 34(5): 794-806. doi: 10.1016/j.immuni.2011.03.021

126. Atarashi K, Tanoue T, Oshima K, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 2013; 500(7461): 232-236. doi: 10.1038/ nature12331

127. Zuo L, Yuan K-T, Yu L, Meng Q-H, Chung PC-K, Yang D-H. Bifidobacterium infantis attenuates colitis by regulating T-cell subset responses. World journal of gastroenterology: WJG. 2014; 20(48): 18316. doi: 10.3748/wjg.v20.i48.18316

128. Calcinaro F, Dionisi S, Marinaro M, et al. Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse. Diabetologia. 2005; 48(8): 1565-1575. doi: 10.1007/s00125-005-1831-2

129. Robert S, Gysemans C, Takiishi T, et al. Oral delivery of Glutamic Acid Decarboxylase (GAD)-65 and IL10 by Lactococ- cus lactis reverses diabetes in recent-onset NOD mice. Diabetes. 2014; 63(8): 2876-287. doi: 10.2337/db13-1236

130. Smith PM, Howitt MR, Panikov N, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science. 2013; 341(6145): 569-573. doi: 10.1126/science.1241165

131. Furusawa Y, Obata Y, Fukuda S, et al. Commensal microbe- derived butyrate induces the differentiation of colonic regulatory T-cells. Nature. 2013; 504(7480): 446-450. doi: 10.1038/nature12721

132. Ferrante RJ, Kubilus JK, Lee J, et al. Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington’s disease mice. The Journal of neuroscience. 2003; 23(28): 9418-9427. doi: 10.1523/jneurosci.23-28-09418.2003

133. Tao R, de Zoeten EF, Özkaynak E, et al. Deacetylase inhibition promotes the generation and function of regulatory T-cells. Nature medicine. 2007; 13(11): 1299-1307. doi: 10.1038/nm1652

134. De Zoeten EF, Wang L, Sai H, Dillmann WH, Hancock WW. Inhibition of HDAC9 increases T regulatory cell function and prevents colitis in mice. Gastroenterology. 2010; 138(2): 583-594. doi: 10.1053/j.gastro.2009.10.037

135. Manicassamy S, Reizis B, Ravindran R, et al. Activation of β-catenin in dendritic cells regulates immunity versus toler- ance in the intestine. Science. 2010; 329(5993): 849-853. doi: 10.1126/science.1188510

136. Ganapathy V, Thangaraju M, Prasad PD, Martin PM, Singh N. Transporters and receptors for short-chain fatty acids as the molecular link between colonic bacteria and the host. Current opinion in pharmacology. 2013; 13(6): 869-874. doi: 10.1016/j.coph.2013.08.006

137. Blad CC, Tang C, Offermanns S. G protein-coupled recep- tors for energy metabolites as new therapeutic targets. Nature Reviews Drug Discovery. 2012; 11(8): 603-619. doi: 10.1038/nrd3777

138. Singh N, Gurav A, Sivaprakasam S, et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity. 2014; 40(1): 128-139. doi: 10.1016/j.immuni.2013.12.007

139. Kang SW, Kim SH, Lee N, et al. 1, 25-Dihyroxyvitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved noncoding sequence region. The Jour- nal of Immunology. 2012; 188(11): 5276-5282. doi: 10.4049/jimmunol.1101211

140. Takiishi T, Ding L, Baeke F, et al. Dietary supplementation with high doses of regular vitamin D3 safely reduces diabetes in- cidence in nod mice when given early and long-term. Diabetes. 2014; DB-131559. doi: 10.2337/db13-1559

141. Kunisawa J, Hashimoto E, Ishikawa I, Kiyono H. A pivotal role of vitamin B9 in the maintenance of regulatory T-cells in vitro and in vivo. PloS one. 2012; 7(2): e32094. doi: 10.1371/journal.pone.0032094

142. Kim SV, Xiang WV, Kwak C, et al. GPR15-mediated homing controls immune homeostasis in the large intestine mucosa. Science. 2013; 340(6139): 1456-1459. doi: 10.1126/sci- ence.1237013