Updates on Immune Therapies in Type 1 Diabetes

European Endocrinology, 2016;12(2):89–95 DOI: http://doi.org/10.17925/EE.2016.12.02.89


Multiple clinical trials investigating the efficacy and safety of immunotherapeutic interventions in new onset type 1 diabetes (T1D) have failed to yield long term clinical benefit. Lack of efficacy has frequently been attributed to an incomplete understanding of the pathways involved in T1D and the use of single immunotherapeutic agents. Recent mechanistic studies have improved our knowledge of the complex etiopathogenesis of T1D. This in turn has provided the framework for new and ongoing clinical trials in new onset T1D patients and at-risk subjects. Focus has also shifted towards the potential benefits of synergistic combinatorial approaches, both in terms of efficacy and the potential for reduced side effects. These efforts seek to develop intervention strategies that will preserve β-cell function, and ultimately prevent and reverse clinical disease.
Keywords: T1D, immunotherapy, antibody, combination therapy, cytokine, Treg, tolerance
Disclosure: Bimota Nambam and Michael J Haller have nothing to disclose in relation to this article. No funding was received in the publication of this article.
Received: May 23, 2016 Accepted June 28, 2016
Correspondence: Michael J Haller, Professor and Chief, Pediatric Endocrinology University of Florida, Gainesville, US. E: hallemj@peds.ufl.edu
Open Access: This article is published under the Creative Commons Attribution Noncommercial License, which permits any non-commercial use, distribution, adaptation and reproduction provided the original author(s) and source are given appropriate credit.

Despite the introduction of newer and faster acting insulin analogues along with advances in glucose monitoring and insulin delivery technology, the majority of patients with type 1 diabetes (T1D) fail to achieve target glycemic control. There still remains a high burden of long term endorgan complications of T1D. Consequently, researchers continue to search for treatment modalities that not only preserve residual β-cell function, but also halt disease progression or even reverse the disease. An improved understanding of the complex immunological pathogenesis of T1D over the past decade has aided the identification of immunotherapeutics aimed at preserving residual β-cell function in high risk, and new onset T1D patients. However, previous intervention studies have not yielded adequate long term clinical benefit, a limitation many have suggested, results from our reliance on monotherapeutic approaches. Additionally, the task of employing safe and effective combination approaches has been challenging due to issues surrounding equipoise and an incomplete understanding of T1D etiopathogenesis. Herein, we provide a review of recently targeted pathways, drugs selected to augment those pathways, their respective clinical trials, relevant outcomes, and future directions.

Anti-CD3 antibodies
T-cells have been shown to have play an important role in the pathogenesis of T1D with autoreactive T effector cells (Teffs) bringing about islet cell destruction and suppressive T regulatory cells (Tregs) ameliorating autoimmunity. Hence, T-cells have been targeted in various immune interventions studies with the aim of preventing or delaying immune mediated destruction of β-cells. CD3, a transmembrane protein, acts as a co-receptor for the T-cell receptor (TCR), and is involved in activation and differentiation of naïve T-cells into pathogenic Teffs. Though not clearly understood, monoclonal antibodies against CD3 prevent activation and promote depletion of T-cells, with Teffs being more sensitive to the effects of anti-CD3 antibodies compared to Tregs. This leads to depletion of Teffs, restores the Teff/Treg ratio, and thus, promotes self- tolerance.1 Experimental studies in non-obese diabetic (NOD) mice have also shown that short term anti-CD3 antibody treatment can induce remission from disease.2,3

Otelixizumab is a humanised monoclonal antibody against CD3 with a mutation in the γFc portion, rendering it incapable of binding to the Fc receptor. The Fc mutation inhibits T-cell crosslinking, mitogenicity, and cytokine release. The Belgian Diabetes Registry conducted a randomised, placebo controlled, phase II study, where otelixizumab (48–64 mg) was administered over 6 days to new onset, T1D patients (12–39 yrs, T1D duration <4 weeks and positive for Epstein Barr virus [EBV] IgG). At 6, 12, and 18 months of follow up, subjects in the treatment group had a significantly higher stimulated C-peptide compared to placebo.4 At 36 months, those <27 years old in the treatment group continued to have higher C-peptide levels (80% higher) than in the placebo group of the same age range.5 Despite no significant differences in glycated haemoglobin (HbA1c) levels throughout the study, daily dose of insulin in the treatment group at all time points were significantly lower compared to placebo. However, one notable concern was the reactivation of EBV in more than 75% of the treatment group, though polymerase chain reaction (PCR) copy numbers returned to normal levels 5–10 weeks post treatment.4

The DEFEND-1 and -2 (Durable Response Therapy Evaluation for Early or New-Onset Type 1 Diabetes) trials, were multicentre studies in new onset T1D adults and adolescents respectively (12–45 years old, T1D duration ≤90 days), designed to explore the efficacy of low dose otelixizumab in preserving residual C-peptide (Table 1).6,7 With a goal of reducing rates of EBV reactivation, the DEFEND-1 and -2 investigators utilised a lower dose of otelixizumab (3.1 mg). EBV reactivation and cytokine release syndrome rates were insignificant in the treatment group, but these were achieved at a cost of lower clinical efficacy. There was no difference in the 2-hour C-peptide area under the curve (AUC), mean HbA1c, and mean daily dose of insulin between the placebo group and the treatment group at 12 months. A dose finding phase II, single blind, randomised, placebo controlled study is currently underway where the efficacy and tolerability of escalating doses of otelixizumab (9 to 36 mg in four different arms) is being investigated in new onset T1D patients (16–27 years old, disease duration less than 32 days).8 The primary outcome of this trial includes the incidence of adverse events such as cytokine release syndrome and reactivation of EBV during the study and the follow up period, while the secondary outcomes include change in C-peptide AUC from baseline till month 24.

Teplizumab is another anti-CD3 monoclonal antibody similar to otelixizumab but with two mutations in its Fc portion. The Protégé study, investigated the efficacy and safety of low and high doses of teplizumab, in new onset T1D patients between 8-35 years old (Table 1). Patients were enrolled from 14 countries and randomised to 4 groups: 14-day high dose (9034 mcg/m2), 14-day low dose (2985 mcg/m2), 6-day high dose (2426 mcg/m2), or 14-day placebo at baseline and at 26 weeks. After 1 year, there was no difference in the primary outcome- the percentage of patients with HbA1c <6.5% and insulin dose <0.5 U/kg/day, across the four groups.9 Notably, this was one of the first interventional studies in which the primary outcome was not based on C-peptide and as such, the lack of achieving significance largely overshadowed the study’s effects on C-peptide preservation. At year 2, despite having no difference in HbA1c levels and mean insulin use per day, the 14-day high dose subgroup had a higher mean AUC C-peptide (p=0.027) compared to placebo. This benefit was more pronounced in patients with the following characteristics: young age (8–17 years), disease duration <6 weeks, HbA1c <7.5%, insulin dose of <0.4 U/kg/day, baseline mean AUC C-peptide >0.2 nmol/L, and US residents.10

The AbATE (Autoimmunity-Blocking Antibody for Tolerance in Recently Diagnosed Type 1 Diabetes) team also undertook a similar study with teplizumab; this was a randomised, open label, study in new onset T1D subjects (8-30 years old, T1D duration ≤8 weeks) (Table 1). The treatment group received teplizumab at a cumulative median dose of 11.6 mg (IQ range 5.7 mg) over 14 days. After a year, those in the treatment group who had detectable C-peptide after mixed meal tolerance test (MMTT), and meeting additional criteria, received another dose of teplizumab (median cumulative dose 12.4 mg, IQ range 5.08 mg). The adjusted mean C-peptide AUC level at year 2 was 75% higher in the treatment group compared to controls, even though there was no significant difference in HbA1c between the groups during the entire study.11 A post hoc analysis revealed that clinical responders, defined as those in the treatment group with <40% of C-peptide loss from enrollment, had lower HbA1c and daily insulin use at baseline after adjusting for C-peptide AUC. They also had increased circulating CD8 + central memory (CM) T-cells. The same study group recently demonstrated that the increased CD8+CM T-cells were derived from naïve T-cells; naïve T-cells have been demonstrated to be stimulated by anti-CD3 antibodies. However, clinical responders from the drug treated group had a higher expression of genes involved in T-cell regulatory pathways, and lower expression of genes involved in pathogenic T-cell activation which may explain the observed beneficial outcome.12 These observations also highlight the importance of patient population heterogeneity possibly influencing study outcomes.

In summary, large clinical trials studying anti-CD3 monoclonal antibodies in new onset diabetes have been carried out with each one reporting slightly different outcomes. These variable outcomes likely relate to differences in drug dosing, disease duration at enrolment, and baseline metabolic, immunological, and genetic differences in the study groups. Though these agents delay progression to complete insulin deficiency in new onset diabetes, the effects are unfortunately not sustained. This highlights the need for studies that will address dose ranging as well as redosing, and identification of new biomarkers capable of predicting responders. In addition, efforts to utilise anti-CD3 based approaches in pre-T1D subjects may be more efficacious. A randomised, double-blind, placebo-controlled clinical trial under the collaboration of TrialNet and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) is currently investigating the efficacy of teplizumab in delaying or preventing clinical onset of T1D in non-diabetic, autoantibody positive, high risk individuals.13

1. Penaranda C, Tang Q, Bluestone JA, Anti-CD3 therapy promotes tolerance by selectively depleting pathogenic cells while preserving regulatory T cells, J Immunol, 2011;187:2015–22.
2. Bevier WC, Trujillo AL, Primbs GB, et al., Oral anti-CD3 monoclonal antibody delays diabetes in non-obese diabetic (NOD) mice: effects on pregnancy and offspring-a preliminary report, Diabetes Metab Res Rev, 2011;27:480–7.
3. Mehta DS, Rudy A. Christmas RA, et al., Partial and transient modulation of the CD3–T-cell receptor complex, elicited by lowdose regimens of monoclonal anti-CD3, is sufficient to induce disease remission in non-obese diabetic mice, Immunology, 2010;130:103–13.
4. Keymeulen B, Vandemeulebroucke E, Ziegler AG, et al., Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes, N Engl J Med, 2005;352:2598–608.
5. Keymeulen B, Walter M, Mathieu C, et al., Four-year metabolic outcome of a randomized controlled CD3-antibody trial in recent-onset type 1 diabetic patients depends on their age and baseline residual beta cell mass, Diabetologia, 2010;53:614–23.
6. Aronson R, Gottlieb PA, Christiansen JS, et al., Low-dose otelixizumab anti-CD3 monoclonal antibody DEFEND-1 study: results of the randomized phase III study in recent-onset human type 1 diabetes, Diabetes Care, 2014;37:2746–54.
7. Ambery P, Donner TW, Biswas N, et al., Efficacy and safety of low-dose otelixizumab anti-CD3 monoclonal antibody in preserving C-peptide secretion in adolescent type 1 diabetes: DEFEND-2, a randomized, placebo-controlled, double-blind, multi-centre study, Diabet Med, 2014;31:399–402
8. GlaxoSmithKline. A Single Blind, Randomised, Placebo Controlled, Repeat Dose, Dose Escalating Study Investigating Safety, Tolerability Pharmacokinetics, Pharmacodynamics and the Beta-Cell Preserving Effect of Otelixizumab in New-Onset, Autoimmune Type 1 Diabetes Mellitus Patients. In: ClinicalTrials. gov [Internet]. Bethesda, MD, National Library of Medicine, 2013. Available at: https://clinicaltrials.gov/ct2/show/NCT02000817 NLM Identifier: NCT02000817 (accessed 8 May 2016).
9. Sherry N, Hagopian W, Ludvigsson J, et al., Teplizumab for treatment of type 1 diabetes (Protégé study): 1-year results from a randomised, placebo-controlled trial, Lancet, 2011;378:487–97.
10. Hagopian W , Ferry RJ Jr, Sherry N, et al., Protégé Trial Investigators. Teplizumab preserves C-peptide in recentonset type 1 diabetes: two-year results from the randomized, placebo-controlled Protégé trial, Diabetes, 2013;62:3901–8.
11. Herold KC, Gitelman SE, Ehlers MR, et al., Teplizumab (anti-CD3 mAb) treatment preserves C-peptide responses in patients with new-onset type 1 diabetes in a randomized controlled trial: Metabolic and immunologic features at baseline identify a subgroup of responders, Diabetes, 2013;62:3766–74.
12. Tooley JE, Vudattu N, Choi J. et al., Changes in T-cell subsets identify responders to FcR-nonbinding anti-CD3 mAb (teplizumab) in patients with type 1 diabetes, Eur J Immunol, 2016; 46:230–41.
13. National Institute of Diabetes and Digestive and Kidney Diseases, AntiCD3 Mab (Teplizumab) For Prevention of Diabetes In Relatives At-Risk for Type 1 Diabetes Mellitus. In: ClinicalTrials. gov [Internet]. Bethesda, MD, National Library of Medicine, 2009. Available at: https://clinicaltrials.gov/ct2/show/NCT01030861 NLM Identifier: NCT01030861 (accessed 8 May 2016).
14. Orban T, Bundy B, Becker DJ, et al., Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled trial, Lancet, 2011;378:412–9.
15. Orban T, Beam CA, Xu P, et al., Reduction in CD4 central memory T-cell subset in costimulation modulator abatacepttreated patients with recent-onset type 1 diabetes is associated with slower C-peptide decline, Diabetes, 2014;63:3449–57.
16. Orban T, Bundy B, Becker DJ, et al., Costimulation modulation with abatacept in patients with recent-onset type 1 diabetes: follow-up 1 year after cessation of treatment, Diabetes Care, 2014;37:1069–75.
17. Ehlers MR, Rigby MR, Targeting memory T cells in type 1 diabetes, Curr Diab Rep, 2015;15: 84.
18. Rigby MR, Harris KM, Pinckney A, et al., Alefacept provides sustained clinical and immunological effects in new-onset type 1 diabetes patients, J Clin Invest, 2015;125:3285–96.
19. National Institute of Diabetes and Digestive and Kidney Diseases, CTLA4-Ig (abatacept) for prevention of abnormal glucose tolerance and diabetes in relatives at-risk for type 1 diabetes mellitus. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2010. Available at: https:// clinicaltrials.gov/ ct2/show/NCT01773707 NLM Identifier: NCT01773707 (accessed 8 May 2016).
20. Hinman RM1, Cambier JC, Role of B lymphocytes in the pathogenesis of type 1 diabetes, Curr Diab Rep, 2014;14:543.
21. Pescovitz MD , Greenbaum CJ, Krause-Steinrauf H, et al., Rituximab, B-lymphocyte depletion, and preservation of betacell function, N Engl J Med, 2009;361:2143–52.
22. Pescovitz MD, Greenbaum CJ, Bundy B, et al., B-lymphocyte depletion with rituximab and β-cell function: two-year results, Diabetes Care, 2014, 37:453-9.
23. Roncarolo MG, Zoppo M, Bacchetta R, et al., Interleukin-2 production and interleukin-2 receptor expression in children with newly diagnosed diabetes, Clin Immunol Immunopathol, 1988;49:53–62.
24. Tang Q, Adams JY, Penaranda C, central role of defective interleukin-2 production in the triggering of islet autoimmune destruction, Immunity, 2008;28:687–97.
25. Grinberg-Bleyer Y, Baeyens A, You S, et al., IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells, J Exp Med, 2010;207:1871-8.
26. Hartemann A, Bensimon G, Payan CA, et al., Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial, Lancet Diabetes Endocrinol, 2013;1:295-305.
27. National Institute of Diabetes and Digestive and Kidney Diseases, European Phase-IIb Clinical Trial Evaluating Efficacy of Low Dose rhIL-2 in Patients With Recently-diagnosed Type 1 Diabetes DIABIL-2. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2015. Available at: https:// clinicaltrials.gov/ ct2/show/NCT02411253 NLM Identifier: NCT02411253 (accessed 8 May 2016).
28. Waldron-Lynch F, Kareclas P, Irons K, Rationale and study design of the Adaptive study of IL-2 dose on regulatory T cells in type 1 diabetes (DILT1D): a non-randomised, open label, adaptive dose finding trial, BMJ Open, 2014;4:e005559.
29. University of British Columbia, Phase I/II Study of Ustekinumab in Patients With New-onset Type 1 Diabetes. In: ClinicalTrials. gov [Internet]. Bethesda, MD, National Library of Medicine, 2014. Available at: https://clinicaltrials.gov/ct2/show/ NCT02117765 NLM Identifier: NCT02117765 (accessed 8 May 2015).
30. Jewish General Hospital, A Single-center, Open-Label, Exploratory Study to Assess the Tolerability and Safety of the Addition of Ustekinumab to INGAP Peptide for 12 Weeks in Adult Patients With Type 1 Diabetes Mellitus. In: ClinicalTrials. gov [Internet]. Bethesda, MD, National Library of Medicine, 2014. Available at: https://clinicaltrials.gov/ct2 /show/NCT02204397 NLM Identifier NCT02204397 (accessed 8 May 2015).
31. National Institute of Allergy and Infectious Diseases, Preserving Beta-Cell Function With Tocilizumab in New-onset Type 1 Diabetes (ITN058AI). In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2014. Available from https:// clinicaltrials.gov/ ct2/show/NCT02293837 NLM Identifier NCT02293837 (accessed 8 May 2015).
32. Louvet C, Szot GL, Lang J, Tyrosine kinase inhibitors reverse type 1 diabetes in nonobese diabetic mice, Proc Natl Acad Sci USA, 2008;105:18895–900.
33. Huda MSB, Amiel SA, Ross P, Aylwin SJB, Tyrosine kinase inhibitor sunitinib allows insulin independence in long-standing type 1 diabetes, Diabetes Care, 2014;37:e87–e88.
34. University of California, San Francisco, Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2013. Available at: https:// clinicaltrials.gov/ct2/show/NCT01781975 NLM Identifier NCT01781975 (accessed 8 May 2015).
35. Ryba-Stanisławowska M, Rybarczyk-Kapturska K, My liwiec M, My liwska 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;37:1513–20.
36. Haseda F, Imagawa A, Murase-Mishiba Y, et al., CD4+ CD45RA− FoxP3high activated regulatory T cells are functionally impaired and related to residual insulin-secreting capacity in patients with type 1 diabetes, Clin Exp Immunol, 2013;173: 207–16.
37. 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;59:407–15.
38. Okubo Y, Torrey H, Butterworth J, Treg activation defect in type 1 diabetes: correction with TNFR2 agonism, Clin Transl Immunology, 2016;5:e56.
39. Marek-Trzonkowska N, Mysliwiec M, Dobyszuk A, Administration of CD4+CD25highCD127- regulatory T cells preserves β-cell function in type 1 diabetes in children, Diabetes Care, 2012;35:1817–20.
40. Bluestone JA, Buckner JH, Fitch M, Type 1 diabetes immunotherapy using polyclonal regulatory T cells, Sci Transl Med, 2015;7:315ra189.
41. Bluestone JA, T1DM Immunotherapy Using Polyclonal Tregs + IL-2 (TILT). In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2016. Available at: https://clinicaltrials.gov/ ct2/show/NCT02772679 NLM Identifier NCT02772679 (accessed 22 May 2016).
42. Pabst O, Mowat AM, Oral tolerance to food protein, Mucosal Immunol, 2012;5:232–9.
43. Skyler JS, Krischer JP, Wolfsdorf J, et al., Effects of oral insulin in relatives of patients with type 1 diabetes: the Diabetes Prevention Trial–Type 1, Diabetes Care, 2005;28:1068–76.
44. Vehik K, Cuthbertson D, Ruhlig H, DPT-1 and TrialNet Study Groups, Long-term outcome of individuals treated with oral insulin: diabetes prevention trial-type 1 (DPT-1) oral insulin trial, Diabetes Care, 2011;34:1585–90.
45. National Institute of Diabetes and Digestive and Kidney Diseases, Oral insulin for prevention of diabetes in relatives at risk for type 1 diabetes mellitus. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2007. Available at: https://clinicaltrials .gov/ct2/show/NCT00419562 NLM Identifier: NCT00419562 (accessed 8 May 2016).
46. Bonifacio E, Ziegler AG, Klingensmith G, et al., Effect of highdose oral insulin on immune responses in children at high risk for type 1 diabetes: the Pre-POINT randomized clinical trial, JAMA, 2015;313:1541–9.
47. National Institute of Diabetes and Digestive and Kidney Diseases, Exploring Immunologic Effects of Oral Insulin in Relatives at Risk for Type 1. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2007. Available at: https://clinicaltrials.gov /ct2/show/ NCT02580877 NLM Identifier: NCT02580877 (accessed 22 June 2016).
48. Cardiff University, Phase 1b Study of Proinsulin (PI) Peptide Immunotherapy in New-Onset Type 1 Diabetes. In: ClinicalTrials. gov [Internet]. Bethesda, MD, National Library of Medicine, 2012. Available at: https://clinicaltrials.gov/ct2/show/NCT01536431 NLM Identifier: NCT01536431 (accessed 8 May 2016).
49. Lund University, A Double-blind, Randomized Investigatorinitiated Study to Determine the Safety and the Effect of Diamyd® on the Progression to Type 1 Diabetes in Children With Multiple Islet Cell Autoantibodies. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2015. Available at: https://clinicaltrials.gov/ct2/show/study/ NCT01122446 NLM Identifier: NCT01122446 (accessed 21 May 2016).
50. Lund University, Double-blind, Investigator-initiated Study to Determine the Effect of Alum-GAD (Diamyd) in Combination With Vitamin D3 on the Progression to Type 1 Diabetes in Children With Multiple Islet Autoantibodies. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2015. Available at: https://clinicaltrials.gov/ct2/show/NCT02387164 NLM Identifier: NCT01536431 (accessed 8 May 2016).
51. Voltarelli JC, Couri CE, Stracieri AB, et al., Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus, JAMA, 2007;297:1568–76.
52. Voltarelli JC, Martinez ED, Burt RK, Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus, JAMA, 2009;302:624–5.
53. D’Addio F, Vasquez AV, Nasr MB, et al., Autologous nonmyeloablative , hematopoietic stem cell transplantation in new-onset type 1 diabetes: a multicenter analysis, Diabetes, 2014;63:3041–6.
54. 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, J Clin Invest, 2015;125 448–55.
55. National Institute of Diabetes and Digestive and Kidney Diseases, Antithymocyte Globulin (ATG) and Pegylated Granulocyte Colony Stimulating Factor (GCSF) in New Onset Type 1 Diabetes. In: ClinicalTrials.gov [Internet]. Bethesda, MD, National Library of Medicine, 2014. Available at: https:// clinicaltrials.gov/ ct2/show/NCT02215200 NLM Identifier: NCT02215200 (accessed 8 May 2016).
Keywords: T1D, immunotherapy, antibody, combination therapy, cytokine, Treg, tolerance