Chapter III. Re-establishing Tolerance

Clinical Potential of Antigen-Specific Therapies in Type 1 Diabetes

Ken T. Coppieters¹, Birgit Sehested Hansen², Matthias G. von Herrath^1,3

Manuscript submitted December 18, 2012; resubmitted January 21, 2013; accepted February 8, 2013.

Keywords: type 1 diabetes, immune tolerance, HSP60, DiaPep277, oral antigen, nasal antigen, regulatory T cell

Abstract

In type 1 diabetes (T1D), pancreatic beta-cells are attacked and destroyed by the immune system, which leads to a loss of endogenous insulin secretion. The desirable outcome of therapeutic intervention in autoimmune diseases is the restoration of immune tolerance to prevent organ damage. Past trials with immune suppressive drugs highlight the fact that T1D is in principle a curable condition. However, the barrier in T1D therapy in terms of drug safety is set particularly high because of the predominantly young population and the good prognosis associated with modern exogenous insulin therapy. Thus, there is a general consensus that chronic immune suppression is associated with unacceptable long-term safety risks. On the other hand, immune-modulatory biologicals have recently failed to confer significant protection in phase 3 clinical trials. However, the concept of antigen-specific tolerization may offer a unique strategy to safely induce long-term protection against T1D. In this review, we analyze the potential reasons for the failure of the different tolerization therapies, and describe how the concept of antigen-specific toleraization may overcome the obstacles associated with clinical therapy in T1D.

Abbreviations: ADA – American Diabetes Association; APC - antigen-presenting cell; APL – altered peptide ligand; CTL – cytotoxic T lymphocyte; CTLA4 – cytotoxic T lymphocyte antigen 4; DC – dendritic cell; DIAPREVENT – Diabetes Prevention Trial; DNA – deoxyribonucleic acid; DPT-1 – Diabetes Prevention Trial 1; GABA – gamma-aminobutyric acid; GAD – glutamate decarboxylase; GI – gastrointestinal; GST - glucagon-stimulated; HSP60 – heat shock protein 60 kDa; ICA – islet cell antibody; IFA – incomplete Freund's adjuvant; Ig – immunoglobulin; IL – interleukin; INIT-II – Intranasal Insulin Trial II; LADA – latent autoimmune diabetes of adulthood; MMTT – mixed meal tolerance test; MS – multiple sclerosis; NCT – national clinical trial; NOD – non-obese diabetic; nTreg – natural Treg; PDLN – pancreatic draining lymph node; POINT – Primary Oral/Intranasal Insulin Trial; T1D – type 1 diabetes; TGF-β – transforming growth factor beta; Th – T helper; TLR2 – toll-like receptor 2; TNF – tumor necrose factor; Treg – regulatory T cell

1. Immune suppression in T1D: the sledgehammer approach

At diagnosis, it is estimated that T1D patients possess only a limited pool of surviving beta-cells. Before diagnosis, the only consistent and reliable signs of ongoing autoimmunity are islet autoantibodies, but the pathophysiological processes at the level of the target organ remain incompletely characterized [1]. It is well known that T cells dominate the islet infiltrates [2], and that these cells are capable of killing beta-cells directly [3]. The beta-cells express high levels of MHC class I, and are therefore actively involved in their own demise. Autoreactive memory T cells also pose problems related to islet transplantation procedures, since these cells rapidly detect and destroy any transplanted beta-cell mass [4]. Therefore, a genuine cure for T1D needs to tackle the immune component of the disease, and should preferably be aimed at the induction of long term tolerance.

Indeed, the combined outcomes of several trials using immune-suppressive agents such as cyclosporin [5] and azathioprine [6] have proven that blocking T cell function in T1D leads to beta-cell preservation. Cyclosporin, for instance, secures prolonged periods of insulin independence in many patients, which is a rigorous efficacy outcome measure [5, 7]. Unfortunately, maintenance of remission requires continuous treatment, and chronic cyclosporin therapy results in accelerated renal dysfunction [8]. Similarly, aggressive therapies such as autologous hematopoietic stem cell transplantation involve similar risk-benefit considerations within the context of T1D [9]. It is therefore evident that this type of approach cannot be ethically justified for the prevention of T1D in young individuals at risk.

2. Immune modulation in T1D: improved safety, reduced efficacy

To reduce the risks associated with chronic immune suppression, biologicals directed against defined immune receptors could be envisioned to tackle the immune system in a more specific fashion. Agents directed at the co-inhibitory receptor CTLA-4 [10] (abatacept), the B cell-specific protein CD20 [11] (rituximab), and the T cell signaling receptor CD3 [12, 13] (teplizumab/otelixizumab) have all been tested in T1D trials. For instance, anti-CD3 therapy, contrary to pharmacological immunosuppression, does not affect subsequent infectious or iatrogenic immunization to antigens that are not related to beta-cells. On the other hand, anti-CD3 agents do trigger transient reactivation of Epstein-Barr virus [14]. The hope is that a short treatment course will trigger lasting tolerance, overcoming the need for chronic treatment which is afflicted with serious ethical considerations. Unfortunately, a recent series of immune modulation trials in new-onset T1D have been unable to permanently arrest the rate of C-peptide decline [10, 11, 15].

The rationale for the implementation of certain immune-modulatory biologicals in a recent-onset setting is not always evident. Non-mitogenic anti-CD3 agents are in fact the only class of biologicals that has consistently been able to reverse established hypoglycemia in several T1D models [16, 17]. Other agents such as CTLA-4Ig [18] and anti-TNF [19] only work in a prevention setting in animals, i.e. when given early and before the signs of overt hyperglycemia. It is therefore not entirely surprising that these approaches are unable to potently prevent the C-peptide decline in a recent-onset setting in humans.

We conclude that the suboptimal effects achieved with immune modulation may reflect a need for higher doses or longer treatment durations. The projected risk-benefit ratio may then approach the profile seen with general immunosuppression.

3. The concept of antigen-specific tolerance

The overall goal of antigen-specific tolerance is to present known autoantigens to the immune system in such a way that they are seen as non-harmful, and consequently provoke a regulatory response. This is in fact exactly the opposite approach to that used by ordinary vaccinations. The regulatory response can consist of an attenuation or deletion of autoreactive T cells and/or the expansion and functional activation of regulatory T cells (Tregs). Various types of these Tregs exist, one of which was termed 'Th3' which specifically referred to mucosal-derived regulatory CD4 T cells producing mainly TGF-β [20]. We know that many of these Treg subtypes contribute to mucosal antigen-specific tolerance, and that their key phenotypic and functional features often overlap. The generation of antigen-specific Tregs is intended to regulate local inflammatory responses, i.e. within the pancreatic draining lymph nodes (PDLNs) and islets in T1D. In oral antigen therapies, the Peyer's patches are crucial immunological niches, since mice lacking these structures do not develop tolerance [21].

How do these antigen-specific Tregs regulate the entire autoreactive T cell repertoire? Here, a process termed linked suppression, originally stemming from transplantation studies, comes into play (Figure 1). Essentially, this concept entails that an antigen-specific therapy based on a single islet antigen can control autoimmune responses against other islet antigens, provided only these antigens are co-presented on the same dendritic cell (DC) as the tolerizing antigen. Thus, the actual 'link' in this concept is the local DC, which ensures that suppression only occurs at the inflammatory target site, and that unwanted suppression of normal immune responses does not occur. This type of 'bystander suppression' holds another therapeutic advantage, namely "operational capacity" even when the antigenic specificities of the other autoareactive T cells are unknown [22]. Treg cells may orchestrate this bystander suppression by acting directly, through cytokine- or contact-dependent mechanisms or, more probably, indirectly on antigen-presenting cells (APCs) that in turn become tolerogenic. Recent evidence even shows that Tregs may be able to kill APCs directly, and as such prevent further T cell activation [23]. Since the concept of linked suppression enables to target the inter-individual variation of T1D patients with respect to their autoreactive T cell repertoire, this concept could aid in covering a broad spectrum of the population [2, 24]. Importantly, bystander tolerance (the term "infectious tolerance" is also used frequently) does not necessarily involve presentation of antigens by the same APC as in the linked tolerance model, since cytokine gradients can also limit tolerizing effects to a local environment.

Figure 1. Antigen-specific tolerance and "linked suppression". Antigen-specific regulatory T cells (Tregs) are raised under specific immunological conditions, for instance those that exist across the mucosal barrier. These Tregs may then regulate immune responses in the lymph nodes, for instance the pancreatic draining lymph nodes in T1D. Here, antigen-presenting cells (APCs) constantly drain the inflamed islets and shuttle islet antigens to the lymph nodes. The Tregs recognize the tolerizing antigens (here antigen A) and mediate immune suppression. The concept of "linked suppression" entails that any autoreactive T cell specific for another islet antigen (here antigen B) that is presented on the same APC will also be suppressed. Tregs mediate suppression via contact or cytokine expression on APC (1) or other T cells (2).

Finally, do we have evidence that stimulating the more focused, localized action of antigen-specific Tregs may provide clinical benefit over increasing the numbers of peripheral natural Treg (nTreg) numbers? It appears that there is no significant overall difference in peripheral nTreg numbers between T1D patients and healthy controls [25]. This argues against the idea of simply increasing their numbers by adding more polyclonal nTreg to the immune system. Recent clinical trial data appear to confirm this notion. For instance, by combining rapamycin and interleukin (IL)-2, nTreg numbers can be boosted in T1D patients [26]. However, this correlated with a fourfold exacerbation in C-peptide decline compared with historical control subjects. Therefore, it seems that polyclonal nTreg expansion does not necessarily correlate with beta-cell preservation, and that antigen-specific Tregs may in fact be preferable.

4. Antigen-specific tolerance in T1D: excellent safety, but efficacy to be improved

Antigen-specific oral tolerance induction has reliably suppressed disease in a range of animal models for autoimmunity, including arthritis [27], multiple sclerosis [28], and T1D [29]. Other modes of mucosal (nasal [30, 31]) and non-mucosal (subcutaneous [32]) antigen delivery, alone or in combination with specific adjuvants, were explored, and promising data were obtained showing that antigen-specific tolerance induction can prevent the development of T1D. However, as actual reversal of established disease is typically not achieved via these antigen-based approaches this treatment would be applicable in subjects at risk of T1D and possibly early-onset patients only. The overwhelming majority of studies revealed excellent safety profiles, with some notable exceptions in the diabetic BB rat model [33]. Due to significant cross-reactivity and endogenous mimicry effects [34] in adaptive responses, complete desired specificity of antigen-based intervention is not always guaranteed, and therefore should not be taken for granted.

Despite these robust data in animals, clinical studies in autoimmune disease patients have so far been less promising. In rheumatoid arthritis, collagen-specific tolerization therapy was tested in numerous trials, but with mixed outcomes [35]. Oral administration of myelin showed initial promise in multiple sclerosis [36], yet a subsequent phase 3 trial proved unsuccessful. Taken together, such trials probably highlight the notion that the outcome of antigen-specific therapy critically depends on a variety of factors, which also consistently influence treatment outcomes in animal models.

Looking at past experiences, we believe that the full potential of antigen-specific therapy for T1D has yet to be uncovered. Optimization of clinical translation could center on biomarker discovery and validation. We recently demonstrated that the presence of insulin autoantibodies prior to treatment with oral insulin/anti-CD3 combination therapy predicts efficacy in the NOD mouse [37]. To adequately apply potential immune biomarkers such as regulatory cytokine responses to islet antigens [38-40] or autoreactive T cell frequencies [41], in future trials, we need to better understand how these immune markers vary per individual during the natural course of T1D development and progression.

5. Examples of past and current antigen-specific therapy trials in T1D

5.1 Oral antigen administration

For obvious reasons, insulin is considered an important candidate for antigen-specific therapy in T1D. It is the only known beta-cell-restricted autoantigen as all other known protein targets are produced outside of the pancreas. Insulin is a major autoantigen in the NOD model [42], and evidence is mounting that insulin-reactive T cells play important roles in T1D [3]. Oral administration of whole insulin prevents autoimmune diabetes in spontaneous [29] and induced [43] animal models. One conclusion from these animal studies is that the dose needs to be carefully chosen, as efficacy decreases in both the low and high end of the dosing spectrum [44]. Additionally, the origin of the insulin influences the efficacy, as optimal doses in mouse models differ for example between porcine and human insulin [45]. It also shows that a structurally intact whole antigen holds the most promise [27].

Most studies on oral insulin therapy in mice found an optimal dose within the single digit milligram range, given intra-gastrically twice weekly in a relatively large (~0.5 ml) volume of buffer. The Diabetes Prevention Trial (DPT-1) first screened over 100.000 relatives of T1D patients for islet cell antibodies (ICAs), and subsequently tested the effect of oral insulin in a secondary prevention setting [46]. Specifically, 7.5 mg of human insulin crystals was administered daily in non-enterocoated capsules or sprinkled over food. It was believed by the authors that the breakdown of insulin in the gastrointestinal (GI) tract was desirable to avoid any hypoglycemic effects. However, no beneficial outcome was reported, although post-hoc analysis showed a projected delay of 4.5 to 5 years in those patients with insulin autoantibody titers of over 80 nU/ml.

Many researchers in the field believe that some important factors could be altered to improve the efficacy of oral insulin in a prevention setting. First, the dose that can be extrapolated from mouse studies is several hundred folds higher than the one used in the DPT-1 trial. Given the importance of accurate dosing, as outlined above, this most likely is a pivotal variable. Second, insulin is extremely sensitive to denaturation and degradation, and it is unsure whether any naturally presented autoantigens will reach the relevant sites in the lower GI tract. Finally, this type of therapy may only work in patients with ongoing anti-insulin autoimmunity, and thus prior insulin autoantibody screening is required, as mouse studies appear to confirm [37]. A TrialNet study is underway to test this latter hypothesis (NCT00419562). All trials with oral insulin in a recent-onset setting, including the IMDIAB [47], Diabète Insuline Orale group [48], and the trial by MacLaren et al. [49] were unsuccessful. Importantly, all used low daily insulin doses and lacked any entero-protective formulation. Our studies using the in silico predictive platform developed by Entelos Inc. showed, at least for nasal insulin peptide tolerization, that low frequency administration, as done in mice, was more effective than high frequency dosing [50]. We conclude that oral insulin therapy in T1D may still hold clinical potential, provided that parameters such as dosing, formulation methods, and inclusion criteria are optimized.

5.2 Nasal antigen administration

An alternative mode of mucosal administration, nasal insulin inhalation, was characterized in mouse models [30]. In contrast to the unpredictable bioavailability in the small intestine after oral administration, nasal administration could aid in better preserving the antigenic structure for optimal presentation to the mucosal immune system. A pilot study showed that nasal insulin treatment is safe and tends to induce immune changes consistent with the establishment of tolerance to insulin [51].

However, a large trial of daily nasal insulin in islet autoantibody-positive children at very high risk for type 1 diabetes found no effect on progression to diabetes [52]. A recent trial in new-onset patients also demonstrated no effect on C-peptide preservation, yet revealed promising immunological changes suggestive of tolerance induction [53]. A large nasal insulin trial (INIT-II, NCT00336674) in relatives of individuals with T1D is underway in Australia and New Zealand. The Pre-POINT trial which is currently being enrolled aims to identify optimal timing, disease stage, dose, and route of administration by intervening with oral or nasal insulin in genetically at-risk children before the appearance of islet autoantibodies [54].

5.3 Subcutaneous antigen administration

(Prepro)Insulin. Subcutaneous administration of insulin prior to diagnosis has been explored as a secondary prevention strategy, based on encouraging data in animal models [32] and pilot trials [55]. In addition to the effects on the immune system, it was hypothesized that insulin may act through metabolic effects, offering much needed ‘rest’ to the stressed beta-cells in at-risk individuals [56]. Recent data mechanistically support this assumption as beta-cells producing higher levels of endogenous insulin were shown to be more vulnerable to recognition and killing by preproinsulin-specific CTLs [3].

The DPT-1 study group assessed the outcome of parenteral insulin administration in a large cohort of high-risk individuals, and failed to demonstrate any effect on functional beta-cell preservation [57]. Alternative approaches include the use of insulin B chain administration in incomplete Freund's adjuvant (IFA) and proinsulin DNA vaccination [38, 39]. While some immunological evidence exists that supports their tolerogenic capacity, these therapies remain to be validated in large controlled trials.

An altered peptide ligand (APL) of insulin B9-23, an important autoantigen, was evaluated in a four-arm trial in recent-onset T1D. Patients were injected with 3 different doses or placebo at randomization, 2 weeks, 4 weeks, and thereafter monthly until 24 months. Maintenance of beta-cell function was not observed at any of the doses tested [58]. Since disease exacerbation with APL was seen in multiple sclerosis (MS), extreme caution is obviously advised in any future trials involving APL [59].

Glutamic acid decarboxylase (GAD). GAD is an enzyme that catalyzes the rate-limiting step in the biosynthesis of the inhibitory neurotransmitter GABA (γ-aminobutyric acid) [60]. The protein is broadly expressed in the nervous system and in other tissues such as the beta-cells. Two mammalian isoforms of GAD with different molecular weights were identified, GAD65 and GAD67, with GAD65 being the main immunogenic isoform in T1D [61]. GAD autoreactivity predicts clinical onset, is closely associated with recent-onset T1D [62], and thus represents an important diagnostic tool for clinicians. Patients suffering from a rare neurological condition called stiff man syndrome also exhibit GAD autoreactivity. Stiff-man syndrome is characterized by severe muscle stiffness with accompanying muscle spasms. A high prevalence of diabetes in patients with stiff man syndrome has been documented [63]. However, stiff man syndrome patients frequently show different GAD-specific T cell repertoires and distinct humoral responses (isotype) to GAD, suggesting fundamentally different underlying immunopathology in both diseases [64].

Not unlike insulin, GAD autoreactivity plays an essential role in the disease process in the NOD model [65]. A variety of tolerization therapies against GAD proved highly successful in NOD mice [31, 66, 67]. The treatment regimen was taken forward into controlled trials consisting of subcutaneous injections of GAD protein in aluminium hydroxide (alum), a common vaccine adjuvant. This adjuvant is known to skew Th1-dominated cellular immune responses, as in T1D, towards a Th2 humoral response [68]. An optimal dose of 20 micrograms (given twice, four weeks apart, C-peptide measurement at 24 weeks) was found in latent autoimmune diabetes of adulthood (LADA) patients, whereas both lower and higher doses were inefficacious [69]. The beneficial effects on fasting C-peptide of the 20 microgram dosing were still measurable after five years, which indicates that long-term tolerance was established [70]. A phase II study, in a recent onset design, was then conducted, and showed improvement of C-peptide preservation, particularly in T1D patients with short disease duration, which was again maintained for at least 4 years [71, 72]. GAD65-specific Tregs [73], decreased antigen-specific Th1 responses [74], and T cell inhibitory pathways upon antigen stimulation [75] were all demonstrated in the GAD-treated patients.

Unfortunately, a recent-onset TrialNet study subsequently failed to confirm these positive outcomes [76]. Two phase 3 trials were recently conducted, one in Europe [77] and one in the U.S. (DIAPREVENT, NCT00751842), without significant evidence of beta-cell preservation. While much of the optimism for GAD-alum treatment for diagnosed T1D patients has vanished, an ongoing secondary prevention trial (DIAPREV-IT; NCT01122446) should provide more insight into the question of whether the drug can be used to protect high-risk individuals.

60 kDa heat-shock protein (HSP60, p277, DiaPep277). HSP60 has long been known as an autoantigen in the NOD mouse [78]. Specifically, a HSP60-derived peptide (p277) harbors the potential to protect mice from diabetes [79, 80], and has the ability to induce disease in non-susceptible strains after vaccination [81]. The protein appears to be a relatively unimportant autoantigen in patients, although some evidence exists that autoreactivity arises during the natural course of T1D [82].

The peptide used in clinical trials (DiaPep277, TEVA Pharmaceuticals) differs from the native p277 sequence in two amino acid positions that were introduced for stabilization purposes. A pilot study tested the efficacy of three subcutaneous 1 mg p277 injections in patients with recent-onset T1D [83]. Glucagon-stimulated (GST) C-peptide was determined at 10 months, and was higher in the DiaPep277 treated group, an effect that was sustained with follow-up to 18 months [84]. Mechanistically, DiaPep277 skews T cell reactivity to hsp60 and p277 towards a Th2 functional phenotype, but binds directly to toll-like receptor 2 (TLR2) on Tregs, which provokes better regulatory potential [85].
A number of other small trials with DiaPep277 were conducted, which all showed excellent safety, yet were not powered to offer conclusive evidence of efficacy [86-89]. The study by Huurman and coworkers revealed that the proliferative T cell response to p277 is able to distinguish between treatment group and placebo, and may serve as a future biomarker [40]. These immune biomarkers need to be further developed, but the latter data suggest that efficacy, at least in part, correlates with an altered immune response against the immunizing antigen.
A successful phase 3 trial has now been completed, and data presented at the 2012 ADA Annual Sessions reported that C-peptide at 24 months showed an improvement with the glucagon stimulation test (GST), but not with the mixed meal tolerance test (MMTT). A press release also announced a reduction of hypoglycemia frequencies and insulin usage in the treatment group (http://www.andromedabio.com/news.php). A second phase 3 trial is underway, and has recently completed recruitment. The phase data support the hypothesis that antigen-specific therapies represent a safe and powerful approach, even during the advanced phases of disease. Interestingly, the dose used in the phase 3 trial (1 mg DiaPep277) was chosen based on reduced hypoglycemia frequencies, whereas the study by Huurman et al. showed that C-peptide preservation was most significant at a dose of 2.5 mg [86]. These results suggest that further dose optimization may lead to even better efficacy.

6. The future of antigen-specific therapy

Trials with immune-suppressive agents have taught us that tackling the immune-related component of T1D is an effective strategy to preserve functional beta-cell mass. The safety profile of these therapies, however, is unsatisfactory given the high quality of life that can be achieved with modern insulin therapy. Furthermore, we have come to the conclusion that long-term tolerance is preferable to short-term remission achieved by immunosuppression. New biologicals such as anti-CD3 have yet to meet the high expectations that were raised by animal studies and phase 2 trials. The phase 3 study with DiaPep277 in recent-onset T1D provides a glimmer of hope that antigen-specific tolerization could be used to treat late-stage disease at around the time of clinical diagnosis.

It is still puzzling why the impressive efficacy record of antigen-specific therapy in animal models does not translate to the clinic. To find answers, first it should be kept in mind that most antigen-specific therapies only work in a prevention setting in animal studies. As outlined above, a combination of ethical considerations, shortened trial durations, and cost minimization requirements urge trial sponsors to prefer a recent-onset setting. Quite clearly, this setup implicates a high stringency for efficacy testing (end-stage disease), as compared to a secondary prevention setting, and offers a less meaningful endpoint (C-peptide) in comparison with the delay of diabetes. Additionally, current preclinical animal research focuses almost entirely on devising tolerogenic strategies for a naïve specific repertoire. However, the challenge for tolerogenic strategies in clinical autoimmune settings is to understand how regulation can be produced on the background of an ongoing chronic inflammatory response against the same antigens to which tolerance is to be produced. Ignoring this challenge may, for example, explain the surprising deleterious effects of the rapamycin/IL-2 trial mentioned above.

Second, important variables such as antigen dose, formulation, and frequency of administration have often been characterized in detail in animal models, yet were largely ignored in the inception of clinical trials. Despite all their limitations, we argue that animal models should continue to contribute in making better informed decisions on the path to clinical translation. Third, suitable immune biomarkers are needed to better identify the correct target population, and to correlate immune function with clinical benefit. Finally, it is believed that for the adequate treatment of established disease, combination therapies are warranted, although generally more practical obstacles need to be overcome than with monotherapies [90].

Disclosure: The authors report no conflict of interests.

References

1. Coppieters KT, von Herrath MG. Histopathology of type 1 diabetes: old paradigms and new insights. Rev Diabet Stud 2009. 6(2):85-96. [DOD] [CrossRef]
2. Coppieters KT, Dotta F, Amirian N, Campbell PD, Kay TW, Atkinson MA, Roep BO, von Herrath MG. Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients. J Exp Med 2012. 209(1):51-60. [DOD] [CrossRef]
3. Skowera A, Ellis RJ, Varela-Calvino R, Arif S, Huang GC, Van-Krinks C, Zaremba A, Rackham C, Allen JS, Tree TI, et al. CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope. J Clin Invest 2008. 118(10):3390-3402. [DOD] 
4. Tyden G, Reinholt FP, Sundkvist G, Bolinder J. Recurrence of autoimmune diabetes mellitus in recipients of cadaveric pancreatic grafts. N Engl J Med 1996. 335(12):860-863. [DOD] [CrossRef]
5. Feutren G, Papoz L, Assan R, Vialettes B, Karsenty G, Vexiau P, Du RH, Rodier M, Sirmai J, Lallemand A, et al. Cyclosporin increases the rate and length of remissions in insulin-dependent diabetes of recent onset. Results of a multicentre double-blind trial. Lancet 1986. 2(8499):119-124. [DOD] [CrossRef]
6. Harrison LC, Colman PG, Dean B, Baxter R, Martin FI. Increase in remission rate in newly diagnosed type I diabetic subjects treated with azathioprine. Diabetes 1985. 34(12):1306-1308. [DOD] [CrossRef]
7. Cyclosporin-induced remission of IDDM after early intervention. Association of 1 yr of cyclosporin treatment with enhanced insulin secretion. The Canadian-European Randomized Control Trial Group. Diabetes 1988. 37(11):1574-1582. [DOD] [CrossRef]
8. Parving HH, Tarnow L, Nielsen FS, Rossing P, Mandrup-Poulsen T, Osterby R, Nerup J. Cyclosporine nephrotoxicity in type 1 diabetic patients. A 7-year follow-up study. Diabetes Care 1999. 22(3):478-483. [DOD] [CrossRef]
9. Voltarelli JC, Couri CE, Stracieri AB, Oliveira MC, Moraes DA, Pieroni F, Coutinho M, Malmegrim KC, Foss-Freitas MC, Simoes BP, et al. Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. JAMA 2007. 297(14):1568-1576. [DOD] [CrossRef]
10. Orban T, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, Gottlieb PA, Greenbaum CJ, Marks JB, Monzavi R, 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(9789):412-419. [DOD] [CrossRef]
11. Pescovitz MD, Greenbaum CJ, Krause-Steinrauf H, Becker DJ, Gitelman SE, Goland R, Gottlieb PA, Marks JB, McGee PF, Moran AM, et al. Rituximab, B-lymphocyte depletion, and preservation of beta-cell function. N Engl J Med 2009. 361(22):2143-2152. [DOD] [CrossRef]
12. Herold KC, Hagopian W, Auger JA, Poumian-Ruiz E, Taylor L, Donaldson D, Gitelman SE, Harlan DM, Xu D, Zivin RA, et al. Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med 2002. 346(22):1692-1698. [DOD] [CrossRef]
13. Keymeulen B, Vandemeulebroucke E, Ziegler AG, Mathieu C, Kaufman L, Hale G, Gorus F, Goldman M, Walter M, Candon S, et al. Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. N Engl J Med 2005. 352(25):2598-2608. [DOD] [CrossRef]
14. Keymeulen B, Candon S, Fafi-Kremer S, Ziegler A, Leruez-Ville M, Mathieu C, Vandemeulebroucke E, Walter M, Crenier L, Thervet E, et al. Transient Epstein-Barr virus reactivation in CD3 monoclonal antibody-treated patients. Blood 2010. 115(6):1145-1155. [DOD] [CrossRef]
15. Sherry N, Hagopian W, Ludvigsson J, Jain SM, Wahlen J, Ferry RJ Jr , Bode B, Aronoff S, Holland C, Carlin D, et al. Teplizumab for treatment of type 1 diabetes (Protege study): 1-year results from a randomised, placebo-controlled trial. Lancet 2011. 378(9790):487-497. [DOD] [CrossRef]
16. Chatenoud L, Thervet E, Primo J, Bach JF. Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Proc Natl Acad Sci USA 1994. 91(1):123-127. [DOD] [CrossRef]
17. von Herrath MG, Coon B, Wolfe T, Chatenoud L. Nonmitogenic CD3 antibody reverses virally induced (rat insulin promoter-lymphocytic choriomeningitis virus) autoimmune diabetes without impeding viral clearance. J Immunol 2002. 168(2):933-941. [DOD] 
18. Lenschow DJ, Ho SC, Sattar H, Rhee L, Gray G, Nabavi N, Herold KC, Bluestone JA. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J Exp Med 1995. 181(3):1145-1155. [DOD] [CrossRef]
19. Yang XD, Tisch R, Singer SM, Cao ZA, Liblau RS, Schreiber RD, McDevitt HO. Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process. J Exp Med 1994. 180(3):995-1004. [DOD] [CrossRef]
20. Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner HL. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 1994. 265(5176):1237-1240. [DOD] [CrossRef]
21. Fujihashi K, Dohi T, Rennert PD, Yamamoto M, Koga T, Kiyono H, McGhee JR. Peyer's patches are required for oral tolerance to proteins. Proc Natl Acad Sci USA 2001. 98(6):3310-3315. [DOD] [CrossRef]
22. von Herrath MG, Harrison LC. Antigen-induced regulatory T cells in autoimmunity. Nat Rev Immunol 2003. 3(3):223-232. [DOD] [CrossRef]
23. Tree TI, Lawson J, Edwards H, Skowera A, Arif S, Roep BO, Peakman M. Naturally arising human CD4 T-cells that recognize islet autoantigens and secrete interleukin-10 regulate proinflammatory T-cell responses via linked suppression. Diabetes 2010. 59(6):1451-1460. [DOD] [CrossRef]
24. Coppieters KT, Amirian N, von Herrath MG. Incidental CD8 T cell reactivity against caspase-cleaved apoptotic self-antigens from ubiquitously expressed proteins in islets from prediabetic human leucocyte antigen-A2 transgenic non-obese diabetic mice. Clin Exp Immunol 2011. 165(2):155-162. [DOD] [CrossRef]
25. Brusko T, Wasserfall C, McGrail K, Schatz R, Viener HL, Schatz D, Haller M, Rockell J, Gottlieb P, Clare-Salzler M, et al. No alterations in the frequency of FOXP3+ regulatory T-cells in type 1 diabetes. Diabetes 2007. 56(3):604-612. [DOD] [CrossRef]
26. Long SA, Rieck M, Sanda S, Bollyky JB, Samuels PL, Goland R, Ahmann A, Rabinovitch A, Aggarwal S, Phippard D, et al. Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs beta-cell function. Diabetes 2012. 61(9):2340-2348. [DOD] [CrossRef]
27. Nagler-Anderson C, Bober LA, Robinson ME, Siskind GW, Thorbecke GJ. Suppression of type II collagen-induced arthritis by intragastric administration of soluble type II collagen. Proc Natl Acad Sci USA 1986. 83(19):7443-7446. [DOD] [CrossRef]
28. Higgins PJ, Weiner HL. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J Immunol 1988. 140(2):440-445. [DOD] 
29. Zhang ZJ, Davidson L, Eisenbarth G, Weiner HL. Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc Natl Acad Sci USA 1991. 88(22):10252-10256. [DOD] [CrossRef]
30. Harrison LC, Dempsey-Collier M, Kramer DR, Takahashi K. Aerosol insulin induces regulatory CD8 gamma delta T cells that prevent murine insulin-dependent diabetes. J Exp Med 1996. 184(6):2167-2174. [DOD] [CrossRef]
31. Tian J, Atkinson MA, Clare-Salzler M, Herschenfeld A, Forsthuber T, Lehmann PV, Kaufman DL. Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med 1996. 183(4):1561-1567. [DOD] [CrossRef]
32. Atkinson MA, Maclaren NK, Luchetta R. Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy. Diabetes 1990. 39(8):933-937. [DOD] [CrossRef]
33. Bellmann K, Kolb H, Rastegar S, Jee P, Scott FW. Potential risk of oral insulin with adjuvant for the prevention of Type I diabetes: a protocol effective in NOD mice may exacerbate disease in BB rats. Diabetologia 1998. 41(7):844-847. [DOD] [CrossRef]
34. Krishnamoorthy G, Saxena A, Mars LT, Domingues HS, Mentele R, Ben-Nun A, Lassmann H, Dornmair K, Kurschus FC, Liblau RS, et al. Myelin-specific T cells also recognize neuronal autoantigen in a transgenic mouse model of multiple sclerosis. Nat Med 2009. 15(6):626-632. [DOD] [CrossRef]
35. Macfarlane GJ, El-Metwally A, De SV, Ernst E, Dowds GL, Moots RJ. Evidence for the efficacy of complementary and alternative medicines in the management of rheumatoid arthritis: a systematic review. Rheumatology (Oxford) 2011. 50(9):1672-1683. [DOD] 
36. Weiner HL, Mackin GA, Matsui M, Orav EJ, Khoury SJ, Dawson DM, Hafler DA. Double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis. Science 1993. 259(5099):1321-1324. [DOD] [CrossRef]
37. Mamchak AA, Manenkova Y, Leconet W, Zheng Y, Chan JR, Stokes CL, Shoda LK, von HM, Bresson D. Preexisting autoantibodies predict efficacy of oral insulin to cure autoimmune diabetes in combination with anti-CD3. Diabetes 2012. 61(6):1490-1499. [DOD] [CrossRef]
38. Thrower SL, James L, Hall W, Green KM, Arif S, Allen JS, Van-Krinks C, Lozanoska-Ochser B, Marquesini L, Brown S, et al. Proinsulin peptide immunotherapy in type 1 diabetes: report of a first-in-man Phase I safety study. Clin Exp Immunol 2009. 155(2):156-165. [DOD] [CrossRef]
39. Orban T, Farkas K, Jalahej H, Kis J, Treszl A, Falk B, Reijonen H, Wolfsdorf J, Ricker A, Matthews JB, et al. Autoantigen-specific regulatory T cells induced in patients with type 1 diabetes mellitus by insulin B-chain immunotherapy. J Autoimmun 2010. 34(4):408-415. [DOD] [CrossRef]
40. Huurman VA, van der Meide PE, Duinkerken G, Willemen S, Cohen IR, Elias D, Roep BO. Immunological efficacy of heat shock protein 60 peptide DiaPep277 therapy in clinical type I diabetes. Clin Exp Immunol 2008. 152(3):488-497. [DOD] [CrossRef]
41. Velthuis JH, Unger WW, Abreu JR, Duinkerken G, Franken K, Peakman M, Bakker AH, Reker-Hadrup S, Keymeulen B, Drijfhout JW, et al. Simultaneous detection of circulating autoreactive CD8+ T-cells specific for different islet cell-associated epitopes using combinatorial MHC multimers. Diabetes 2010. 59(7):1721-1730. [DOD] [CrossRef]
42. Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, Yu L, Wegmann DR, Hutton JC, Elliott JF, et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature 2005. 435(7039):220-223. [DOD] [CrossRef]
43. von Herrath MG, Dyrberg T, Oldstone MB. Oral insulin treatment suppresses virus-induced antigen-specific destruction of beta cells and prevents autoimmune diabetes in transgenic mice. J Clin Invest 1996. 98(6):1324-1331. [DOD] [CrossRef]
44. Petersen JS, Bregenholt S, Apostolopolous V, Homann D, Wolfe T, Hughes A, De JK, Wang M, Dyrberg T, von Herrath MG. Coupling of oral human or porcine insulin to the B subunit of cholera toxin (CTB) overcomes critical antigenic differences for prevention of type I diabetes. Clin Exp Immunol 2003. 134(1):38-45. [DOD] [CrossRef]
45. Homann D, Dyrberg T, Petersen J, Oldstone MB, von Herrath MG. Insulin in oral immune "tolerance": a one-amino acid change in the B chain makes the difference. J Immunol 1999. 163(4):1833-1838. [DOD] 
46. Skyler JS, Krischer JP, Wolfsdorf J, Cowie C, Palmer JP, Greenbaum C, Cuthbertson D, Rafkin-Mervis LE, Chase HP, Leschek E. Effects of oral insulin in relatives of patients with type 1 diabetes: The Diabetes Prevention Trial - Type 1. Diabetes Care 2005. 28(5):1068-1076. [DOD] [CrossRef]
47. Pozzilli P, Pitocco D, Visalli N, Cavallo MG, Buzzetti R, Crino A, Spera S, Suraci C, Multari G, Cervoni M, et al. No effect of oral insulin on residual beta-cell function in recent-onset type I diabetes (the IMDIAB VII). IMDIAB Group. Diabetologia 2000. 43(8):1000-1004. [DOD] [CrossRef]
48. Chaillous L, Lefevre H, Thivolet C, Boitard C, Lahlou N, Atlan-Gepner C, Bouhanick B, Mogenet A, Nicolino M, Carel JC, et al. Oral insulin administration and residual beta-cell function in recent-onset type 1 diabetes: a multicentre randomised controlled trial. Diabete Insuline Orale group. Lancet 2000. 356(9229):545-549. [DOD] [CrossRef]
49. Ergun-Longmire B, Marker J, Zeidler A, Rapaport R, Raskin P, Bode B, Schatz D, Vargas A, Rogers D, Schwartz S, et al. Oral insulin therapy to prevent progression of immune-mediated (type 1) diabetes. Ann N Y Acad Sci 2004. 1029:260-277. [DOD] [CrossRef]
50. Fousteri G, Chan JR, Zheng Y, Whiting C, Dave A, Bresson D, Croft M, von Herrath M. Virtual optimization of nasal insulin therapy predicts immunization frequency to be crucial for diabetes protection. Diabetes 2010. 59(12):3148-3158. [DOD] [CrossRef]
51. Harrison LC, Honeyman MC, Steele CE, Stone NL, Sarugeri E, Bonifacio E, Couper JJ, Colman PG. Pancreatic beta-cell function and immune responses to insulin after administration of intranasal insulin to humans at risk for type 1 diabetes. Diabetes Care 2004. 27(10):2348-2355. [DOD] [CrossRef]
52. Nanto-Salonen K, Kupila A, Simell S, Siljander H, Salonsaari T, Hekkala A, Korhonen S, Erkkola R, Sipila JI, Haavisto L, et al. Nasal insulin to prevent type 1 diabetes in children with HLA genotypes and autoantibodies conferring increased risk of disease: a double-blind, randomised controlled trial. Lancet 2008. 372(9651):1746-1755. [DOD] [CrossRef]
53. Fourlanos S, Perry C, Gellert SA, Martinuzzi E, Mallone R, Butler J, Colman PG, Harrison LC. Evidence that nasal insulin induces immune tolerance to insulin in adults with autoimmune diabetes. Diabetes 2011. 60(4):1237-1245. [DOD] [CrossRef]
54. Achenbach P, Barker J, Bonifacio E. Modulating the natural history of type 1 diabetes in children at high genetic risk by mucosal insulin immunization. Curr Diab Rep 2008. 8(2):87-93. [DOD] [CrossRef]
55. Keller RJ, Eisenbarth GS, Jackson RA. Insulin prophylaxis in individuals at high risk of type I diabetes. Lancet 1993. 341(8850):927-928. [DOD] [CrossRef]
56. Bowman MA, Campbell L, Darrow BL, Ellis TM, Suresh A, Atkinson MA. Immunological and metabolic effects of prophylactic insulin therapy in the NOD-scid/scid adoptive transfer model of IDDM. Diabetes 1996. 45(2):205-208. [DOD] [CrossRef]
57. Effects of insulin in relatives of patients with type 1 diabetes mellitus. N Engl J Med 2002. 346(22):1685-1691. [DOD] [CrossRef]
58. Walter M, Philotheou A, Bonnici F, Ziegler AG, Jimenez R. No effect of the altered peptide ligand NBI-6024 on beta-cell residual function and insulin needs in new-onset type 1 diabetes. Diabetes Care 2009. 32(11):2036-2040. [DOD] [CrossRef]
59. Bielekova B, Goodwin B, Richert N, Cortese I, Kondo T, Afshar G, Gran B, Eaton J, Antel J, Frank JA, et al. Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand. Nat Med 2000. 6(10):1167-1175. [DOD] [CrossRef]
60. Baekkeskov S, Aanstoot HJ, Christgau S, Reetz A, Solimena M, Cascalho M, Folli F, Richter-Olesen H, De CP. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990. 347(6289):151-156. [DOD] [CrossRef]
61. Velloso LA, Kampe O, Hallberg A, Christmanson L, Betsholtz C, Karlsson FA. Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression. J Clin Invest 1993. 91(5):2084-2090. [DOD] [CrossRef]
62. De Aizpurua HJ, Wilson YM, Harrison LC. Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes. Proc Natl Acad Sci USA 1992. 89(20):9841-9845. [DOD] [CrossRef]
63. Solimena M, Folli F, Aparisi R, Pozza G, De CP. Autoantibodies to GABA-ergic neurons and pancreatic beta cells in stiff-man syndrome. N Engl J Med 1990. 322(22):1555-1560. [DOD] [CrossRef]
64. Lohmann T, Hawa M, Leslie RD, Lane R, Picard J, Londei M. Immune reactivity to glutamic acid decarboxylase 65 in stiffman syndrome and type 1 diabetes mellitus. Lancet 2000. 356(9223):31-35. [DOD] [CrossRef]
65. Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L, McDevitt HO. Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 1993. 366(6450):72-75. [DOD] [CrossRef]
66. Petersen JS, Karlsen AE, Markholst H, Worsaae A, Dyrberg T, Michelsen B. Neonatal tolerization with glutamic acid decarboxylase but not with bovine serum albumin delays the onset of diabetes in NOD mice. Diabetes 1994. 43(12):1478-1484. [DOD] [CrossRef]
67. Tisch R, Wang B, Weaver DJ, Liu B, Bui T, Arthos J, Serreze DV. Antigen-specific mediated suppression of beta cell autoimmunity by plasmid DNA vaccination. J Immunol 2001. 166(3):2122-2132. [DOD] 
68. Ludvigsson J. Therapy with GAD in diabetes. Diabetes Metab Res Rev 2009. 25(4):307-315. [DOD] [CrossRef]
69. Agardh CD, Cilio CM, Lethagen A, Lynch K, Leslie RD, Palmer M, Harris RA, Robertson JA, Lernmark A. Clinical evidence for the safety of GAD65 immunomodulation in adult-onset autoimmune diabetes. J Diabetes Complications 2005. 19(4):238-246. [DOD] [CrossRef]
70. Agardh CD, Lynch K, Palmer M, Link K, Lernmark A. GAD65 vaccination significantly reduces insulin dependence at five years follow-up in a dose escalating study in adult-onset autoimmune diabetes patients. Diabetologia 2008. 51:S230-S230. [DOD] 
71. Ludvigsson J, Faresjo M, Hjorth M, Axelsson S, Cheramy M, Pihl M, Vaarala O, Forsander G, Ivarsson S, Johansson C, et al. GAD treatment and insulin secretion in recent-onset type 1 diabetes. N Engl J Med 2008. 359(18):1909-1920. [DOD] [CrossRef]
72. Ludvigsson J, Hjorth M, Cheramy M, Axelsson S, Pihl M, Forsander G, Nilsson NO, Samuelsson BO, Wood T, Aman J, et al. Extended evaluation of the safety and efficacy of GAD treatment of children and adolescents with recent-onset type 1 diabetes: a randomised controlled trial. Diabetologia 2011. 54(3):634-640. [DOD] [CrossRef]
73. Hjorth M, Axelsson S, Ryden A, Faresjo M, Ludvigsson J, Casas R. GAD-alum treatment induces GAD65-specific CD4+CD25highFOXP3+ cells in type 1 diabetic patients. Clin Immunol 2011. 138(1):117-126. [DOD] [CrossRef]
74. Axelsson S, Hjorth M, Ludvigsson J, Casas R. Decreased GAD(65)-specific Th1/Tc1 phenotype in children with Type 1 diabetes treated with GAD-alum. Diabet Med 2012. 29(10):1272-1278. [DOD] [CrossRef]
75. Axelsson S, Cheramy M, Hjorth M, Pihl M, Akerman L, Martinuzzi E, Mallone R, Ludvigsson J, Casas R. Long-lasting immune responses 4 years after GAD-alum treatment in children with type 1 diabetes. Plos One 2011. 6(12):e29008. [DOD] [CrossRef]
76. Wherrett DK, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R, Gottlieb PA, Greenbaum CJ, Herold KC, Marks JB, et al. Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-blind trial. Lancet 2011. 378(9788):319-327. [DOD] [CrossRef]
77. Ludvigsson J, Krisky D, Casas R, Battelino T, Castano L, Greening J, Kordonouri O, Otonkoski T, Pozzilli P, Robert JJ, et al. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. N Engl J Med 2012. 366(5):433-442. [DOD] [CrossRef]
78. Elias D, Markovits D, Reshef T, van der Zee R, Cohen IR. Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc Natl Acad Sci USA 1990. 87(4):1576-1580. [DOD] [CrossRef]
79. Elias D, Reshef T, Birk OS, van der Zee R, Walker MD, Cohen IR. Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc Natl Acad Sci USA 1991. 88(8):3088-3091. [DOD] [CrossRef]
80. Elias D, Cohen IR. Peptide therapy for diabetes in NOD mice. Lancet 1994. 343(8899):704-706. [DOD] [CrossRef]
81. Elias D, Marcus H, Reshef T, Ablamunits V, Cohen IR. Induction of diabetes in standard mice by immunization with the p277 peptide of a 60-kDa heat shock protein. Eur J Immunol 1995. 25(10):2851-2857. [DOD] [CrossRef]
82. Abulafia-Lapid R, Elias D, Raz I, Keren-Zur Y, Atlan H, Cohen IR. T cell proliferative responses of type 1 diabetes patients and healthy individuals to human hsp60 and its peptides. J Autoimmun 1999. 12(2):121-129. [DOD] [CrossRef]
83. Raz I, Elias D, Avron A, Tamir M, Metzger M, Cohen IR. Beta-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial. Lancet 2001. 358(9295):1749-1753. [DOD] [CrossRef]
84. Raz I, Avron A, Tamir M, Metzger M, Symer L, Eldor R, Cohen IR, Elias D. Treatment of new-onset type 1 diabetes with peptide DiaPep277 is safe and associated with preserved beta-cell function: extension of a randomized, double-blind, phase II trial. Diabetes Metab Res Rev 2007. 23(4):292-298. [DOD] [CrossRef]
85. Zanin-Zhorov A, Cahalon L, Tal G, Margalit R, Lider O, Cohen IR. Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling. J Clin Invest 2006. 116(7):2022-2032. [DOD] [CrossRef]
86. Huurman VA, Decochez K, Mathieu C, Cohen IR, Roep BO. Therapy with the hsp60 peptide DiaPep277 in C-peptide positive type 1 diabetes patients. Diabetes Metab Res Rev 2007. 23(4):269-275. [DOD] [CrossRef]
87. Lazar L, Ofan R, Weintrob N, Avron A, Tamir M, Elias D, Phillip M, Josefsberg Z. Heat-shock protein peptide DiaPep277 treatment in children with newly diagnosed type 1 diabetes: a randomised, double-blind phase II study. Diabetes Metab Res Rev 2007. 23(4):286-291. [DOD] [CrossRef]
88. Schloot NC, Meierhoff G, Lengyel C, Vandorfi G, Takacs J, Panczel P, Barkai L, Madacsy L, Oroszlan T, Kovacs P, et al. Effect of heat shock protein peptide DiaPep277 on beta-cell function in paediatric and adult patients with recent-onset diabetes mellitus type 1: two prospective, randomized, double-blind phase II trials. Diabetes Metab Res Rev 2007. 23(4):276-285. [DOD] [CrossRef]
89. Buzzetti R, Cernea S, Petrone A, Capizzi M, Spoletini M, Zampetti S, Guglielmi C, Venditti C, Pozzilli P. C-peptide response and HLA genotypes in subjects with recent-onset type 1 diabetes after immunotherapy with DiaPep277: an exploratory study. Diabetes 2011. 60(11):3067-3072. [DOD] [CrossRef]
90. von Herrath M. Combination therapies for type 1 diabetes: why not now? Immunotherapy 2010. 2(3):289-291. [DOD] 

This article has been cited by other articles:

	Metabolic syndrome - Removing roadblocks to therapy: Antigenic immunotherapies Coppieters KT, von Herrath MG Mol Metab 2014. 3(3):275-283