Reversal of type 1 diabetes by a new MHC II‐peptide chimera: “Single‐epitope‐mediated suppression” to stabilize a polyclonal autoimmune T‐cell process

Polyclonality of self‐reactive CD4⁺ T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II‐peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T‐cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3 : 383–391]. Since most of the glutamic acid decarboxylase 65 (GAD65)‐specific CD4⁺ T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T‐regulatory cell population (GAD65_217–230‐specific CD4⁺ T cells) using a soluble I‐A/GAD65_217–230/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T‐cell process. Activated GAD65_217–230‐specific T cells and a fraction of the diabetogenic (B_9–23‐specific) T cells were polarized toward the IL‐10‐secreting T‐regulatory type 1‐like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post‐therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self‐reactive T‐cell process by a single epitope‐mediated bystander suppression. Dimeric MHC class II‐peptide chimeras‐like approach may provide rational grounds for the development of more efficient antigen‐specific therapies in type 1 diabetes.     

Schistosoma mansoni egg antigens induce Treg that participate in diabetes prevention in NOD mice

Schistosoma mansoni soluble egg antigens (SEA) profoundly regulate the infected host's immune system. We previously showed that SEA prevents type 1 diabetes in NOD mice and that splenocytes from SEA‐treated mice have reduced ability to transfer diabetes to NOD.scid recipients. To further characterize the mechanism of diabetes prevention we examined the cell types involved and showed that CD25⁺ T‐cell depletion of splenocytes from SEA‐treated donors restored their ability to transfer diabetes. Furthermore, SEA treatment increased the number and proportional representation of Foxp3⁺ T cells in the pancreas of NOD mice. We have used in vitro systems to analyze the effect of SEA on the development of NOD Foxp3⁺ T cells. We find that SEA can induce Foxp3 expression in naïve T cells in a TGF‐β‐dependent manner. Foxp3 induction requires the presence of DC, which we also show are modified by SEA to upregulate C‐type lectins, IL‐10 and IL‐2. Our studies show that SEA can have a direct effect on CD4⁺ T cells increasing expression of TGF‐β, integrin β8 and galectins. These effects of SEA on DC and T cells may act in synergy to induce Foxp3⁺ Treg in the NOD mouse.     

Abrogation of ICOS/ICOS ligand costimulation in NOD mice results in autoimmune deviation toward the neuromuscular system

NOD mice spontaneously develop insulin‐dependent diabetes around 10–40 wk of age. Numerous immune gene variants contribute to the autoimmune process. However, genes that direct the autoimmune response toward β cells remain ill defined. In this study, we provide evidence that the Icos and Icosl genes contribute to the diabetes process. Protection from diabetes in ICOS^?/? and ICOSL^?/? NOD mice was unexpectedly associated with the development of an autoimmune disorder of the neuro‐muscular system, characterized by myositis, sensory ganglionitis and, to a reduced extent, inflammatory infiltrates in the CNS. This syndrome was reproduced upon adoptive transfer of CD4⁺ and CD8⁺ T cells from diseased donors to naïve NOD.scid recipients. Our data further show that protection from diabetes results from defective activation of autoimmune diabetogenic effector T cells in ICOS^?/? NOD mice, whereas acceleration of diabetes in BDC2.5 ICOS^?/? NOD mice is induced by a dominant defect in Treg. Taken together, our findings indicate that costimulation signals play a key role in regulating immune tolerance in peripheral tissues and that the ICOS/ICOSL costimulatory pathway influences the balance between Treg and diabetogenic effector T cells.     

Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non‐obese diabetic mice

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of β cells and perforin deficiency effectively reduces diabetes in non‐obese diabetic (NOD) mice, it can be deduced that β cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non‐obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB^–/– mice developed diabetes spontaneously with kinetics similar to those of wild‐type NOD (wt‐NOD) mice. Adoptive transfer study with regulatory T cell (T_reg)‐depleted splenocytes (SPCs) into NOD‐SCID mice or in‐vivo T_reg depletion by anti‐CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB^–/– mice and wt‐NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre‐diabetic NOD.GzmB^–/– mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide‐promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4⁺, CD8⁺ and CD4⁺CD25⁺ T cells in SPCs from NOD.GzmB^–/– mice than those from wt‐NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for β cell destruction in NOD mice.     

Detection of vasostatin‐1‐specific CD8+ T cells in non‐obese diabetic mice that contribute to diabetes pathogenesis

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4⁺ T cells in a non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin‐1 is a naturally processed fragment of ChgA. We have now identified a novel H2‐K^d‐restricted epitope of vasostatin‐1, ChgA 36‐44, which elicits CD8⁺ T cell responses in NOD mice. By using ChgA 36‐44/K^d tetramers we have determined the frequency of vasostatin‐1‐specific CD8⁺ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin‐1‐specific CD4⁺ and CD8⁺ T cells constitute a significant fraction of islet‐infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36‐44 peptide‐primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin‐1‐specific CD8⁺ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.     

β‐cell‐specific IL‐35 therapy suppresses ongoing autoimmune diabetes in NOD mice

IL‐35 is a recently identified cytokine exhibiting potent immunosuppressive properties. The therapeutic potential and effects of IL‐35 on pathogenic T effector cells (Teff) and Foxp3⁺ Treg, however, are ill defined. We tested the capacity of IL‐35 to suppress ongoing autoimmunity in NOD mice. For this purpose, an adeno‐associated virus vector in which IL‐35 transgene expression is selectively targeted to β cells via an insulin promoter (AAV8mIP‐IL35) was used. AAV8mIP‐IL35 vaccination of NOD mice at a late preclinical stage of type 1 diabetes (T1D) suppressed β‐cell autoimmunity and prevented diabetes onset. Numbers of islet‐resident conventional CD4⁺ and CD8⁺ T cells, and DCs were reduced within 4 weeks of AAV8mIP‐IL35 treatment. The diminished islet T‐cell pool correlated with suppressed proliferation, and a decreased frequency of IFN‐γ‐expressing Teff. Ectopic IL‐35 also reduced islet Foxp3⁺ Treg numbers and proliferation, and protection was independent of induction/expansion of adaptive islet immunoregulatory T cells. These findings demonstrate that IL‐35‐mediated suppression is sufficiently robust to block established β‐cell autoimmunity, and support the use of IL‐35 to treat T1D and other T‐cell‐mediated autoimmune diseases.     

Peripherally induced regulatory T cells contribute to the control of autoimmune diabetes in the NOD mouse model

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells and is partly caused by deficiencies in the Foxp3⁺ regulatory T‐cell (Treg) compartment. Conversely, therapies that increase Treg function can prevent autoimmune diabetes in animal models. The majority of Tregs develop in the thymus (tTregs), but a proportion of Foxp3⁺ Tregs is generated in the periphery (pTregs) from Foxp3^?CD4⁺ T‐cell precursors. Whether pTregs play a distinct role in T1D has not yet been explored. We report here that pTregs are a key modifier of disease in the nonobese diabetic (NOD) mouse model for T1D. We generated NOD mice deficient for the Foxp3 enhancer CNS1 involved in pTreg induction. We show that CNS1 knockout decreased the frequency of pTregs and increased the risk of diabetes. Our results show that pTregs fulfill an important non‐redundant function in the prevention of beta cell autoimmunity that causes T1D.     

Plasmacytoid dendritic cells license regulatory T cells,upon iNKT‐cell stimulation,to prevent autoimmune diabetes

Invariant NKT (iNKT)‐cell stimulation with exogenous specific ligands prevents the development of type 1 diabetes (T1D) in NOD mice. Studies based on anti‐islet T‐cell transfer showed that iNKT cells prevent the differentiation of these T cells into effector T cells in the pancreatic lymph nodes (PLNs). We hypothesize that this defective priming could be explained by the ability of iNKT cells to induce tolerogenic dendritic cells (DCs) in the PLNs. We evaluated the effect of iNKT‐cell stimulation on T1D development by transferring naïve diabetogenic BDC2.5 T cells into proinsulin 2^?/? NOD mice treated with a long‐lasting α‐galactosylceramide regimen. In this context, iNKT cells induce the conversion of BDC2.5 T cells into Foxp3⁺ Treg cells in the PLNs accumulating in the pancreatic islets. Furthermore, tolerogenic plasmacytoid DCs (pDCs) characterized by low MHC class II molecule expression and TGF‐β production are critical in the PLNs for the recruitment of Treg cells into the pancreatic islets by inducing CXCR3 expression. Accordingly, pDC depletion in α‐galactosylceramide‐treated proinsulin 2^?/? NOD mice abrogates the protection against T1D. These findings reveal that upon repetitive iNKT‐cell stimulation, pDCs are critical for the recruitment of Treg cells in the pancreatic islets and the prevention of T1D development.     

The good turned ugly: immunopathogenic basis for diabetogenic CD8+ T cells in NOD mice

Summary: Type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is a T‐cell‐mediated autoimmune disease in which the insulin‐producing pancreatic islet β‐cells are selectively eliminated. As a result, glucose metabolism cannot be regulated unless exogenous insulin is administered. Both the CD4⁺ and the CD8⁺ T‐cell subsets are required for T1D development. Approximately 20 years ago, an association between certain class II major histocompatibility complex (MHC) alleles and susceptibility to T1D was reported. This finding led to enormous interest in the CD4⁺ T cells participating in the development of T1D, while the CD8⁺ subset was relatively ignored. However, the isolation of β‐cell‐autoreactive CD8⁺ T‐cell clones from the islets of NOD mice helped to generate interest in the pathogenic role of this subset, as has accumulating evidence that certain class I MHC alleles are additional risk factors for T1D development in humans. Three distinct diabetogenic CD8⁺ T‐cell populations have now been characterized in NOD mice. Here, we review recent investigations exploring their selection, activation, trafficking, and antigenic specificities. As CD8⁺ T cells are suspected contributors to β‐cell demise in humans, continued exploration of these critical areas could very possibly lead to tangible benefits for T1D patients and at‐risk individuals.     

FTY720-induced conversion of conventional Foxp3- CD4+ T cells to Foxp3+ regulatory T cells in NOD mice

Citation Sun Y, Wang W, Shan B, Di J, Chen L, Ren L, Li W, Li D‐J, Lin Y. FTY720‐induced conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in NOD mice. Am J Reprod Immunol 2011; 66: 349–362 Problem FTY720 is known as an agonist of sphingosine‐1‐phosphate (S1P) receptor, but little is known about the possibility that FTY720 induces the conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in non‐obese diabetic (NOD) mice. Method of study FTY720 treatment was performed using Foxp3^?CD4⁺ T cells purified from NOD mice. Results FTY720 caused an increase in Foxp3⁺ Treg cells in lymphoid organs in NOD mice. FTY720 effectively induced Foxp3 expression in Foxp3^?CD4⁺ T cells both in vitro and in vivo, an effect that was inhibited by a TGF‐β‐neutralizing antibody or the proinflammatory cytokine IL‐6. T‐cell‐mediated embryo rejection in NOD mice was prevented upon FTY720 treatment. Conclusions The use of FTY720 along with Ag administration may represent a useful therapeutic strategy to selectively expand Ag‐specific Foxp3⁺ Tregs to intervene autoimmune and infectious diseases.     

Gut immune deficits in LEW.1AR1‐iddm rats partially overcome by feeding a diabetes‐protective diet

The gut immune system and its modification by diet have been implicated in the pathogenesis of type 1 diabetes (T1D). Therefore, we investigated gut immune status in non‐diabetes‐prone LEW.1AR1 and diabetes‐prone LEW.1AR1‐iddm rats and evaluated the effect of a low antigen, hydrolysed casein (HC)‐based diet on gut immunity and T1D. Rats were weaned onto a cereal‐based or HC‐based diet and monitored for T1D. Strain and dietary effects on immune homeostasis were assessed in non‐diabetic rats (50–60 days old) and rats with recent‐onset diabetes using flow cytometry and immunohistochemistry. Immune gene expression was analysed in mesenteric lymph nodes (MLN) and jejunum using quantitative RT‐PCR and PCR arrays. T1D was prevented in LEW.1AR1‐iddm rats by feeding an HC diet. Diabetic LEW.1AR1‐iddm rats had fewer lymphoid tissue T cells compared with LEW.1AR1 rats. The percentage of CD4⁺ Foxp3⁺ regulatory T (Treg) cells was decreased in pancreatic lymph nodes (PLN) of diabetic rats. The jejunum of 50‐day LEW.1AR1‐iddm rats contained fewer CD3⁺ T cells, CD163⁺ M2 macrophages and Foxp3⁺ Treg cells. Ifng expression was increased in MLN and Foxp3 expression was decreased in the jejunum of LEW.1AR1‐iddm rats; Ifng/Il4 was decreased in jejunum of LEW.1AR1‐iddm rats fed HC. PCR arrays revealed decreased expression of M2‐associated macrophage factors in 50‐day LEW.1AR1‐iddm rats. Wheat peptides stimulated T‐cell proliferation and activation in MLN and PLN cells from diabetic LEW.1AR1‐iddm rats. LEW.1AR1‐iddm rats displayed gut immune cell deficits and decreased immunoregulatory capacity, which were partially corrected in animals fed a low antigen, protective HC diet consistent with other models of T1D.     

BAFF regulates activation of self‐reactive T cells through B‐cell dependent mechanisms and mediates protection in NOD mice

Targeting the BAFF/APRIL system has shown to be effective in preventing T‐cell dependent autoimmune disease in the NOD mouse, a spontaneous model of type 1 diabetes. In this study we generated BAFF‐deficient NOD mice to examine how BAFF availability would influence T‐cell responses in vivo and the development of spontaneous diabetes. BAFF‐deficient NOD mice which lack mature B cells, were protected from diabetes and showed delayed rejection of an allogeneic islet graft. Diabetes protection correlated with a failure to expand pathogenic IGRP‐reactive CD8⁺ T cells, which were maintained in the periphery at correspondingly low levels. Adoptive transfer of IGRP‐reactive CD8⁺ T cells with B cells into BAFF‐deficient NOD mice enhanced IGRP‐reactive CD8⁺ T‐cell expansion. Furthermore, when provoked with cyclophosphamide, or transferred to a secondary lymphopenic host, the latent pool of self‐reactive T cells resident in BAFF‐deficient NOD mice could elicit beta cell destruction. We conclude that lack of BAFF prevents the procurement of B‐cell‐dependent help necessary for the emergence of destructive diabetes. Indeed, treatment of NOD mice with the BAFF‐blocking compound, BR3‐Fc, resulted in a delayed onset and reduced incidence of diabetes.     

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B‐cell lymphopoiesis and implicates microRNA‐34a in diabetes protection

NOD.B10 Idd9.3 mice are congenic for the insulin‐dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)‐34a, from T1D‐resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen‐specific CD4⁺ T cells in T1D pathogenesis in non‐obese diabetic (NOD) mice. We show that early B‐cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR‐34a expression was significantly higher in B‐cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR‐34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B‐cell lymphopoiesis, which is directly repressed by miR‐34a. In addition, we show that islet‐specific CD4⁺ T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3‐encoded miR‐34a is a likely candidate in negatively regulating B‐cell lymphopoiesis, which may contribute to inefficient expansion of islet‐specific CD4⁺ T cells and to T1D protection in NOD.B10 Idd9.3 mice.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

Reversible lacrimal gland‐protective regulatory T‐cell dysfunction underlies male‐specific autoimmune dacryoadenitis in the non‐obese diabetic mouse model of Sjögren syndrome

CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells are required to maintain immunological tolerance; however, defects in specific organ‐protective Treg cell functions have not been demonstrated in organ‐specific autoimmunity. Non‐obese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity and are a well‐characterized model of Sjögren syndrome. Lacrimal gland disease in NOD mice is male‐specific, but the role of Treg cells in this sex‐specificity is not known. This study aimed to determine if male‐specific autoimmune dacryoadenitis in the NOD mouse model of Sjögren syndrome is the result of lacrimal gland‐protective Treg cell dysfunction. An adoptive transfer model of Sjögren syndrome was developed by transferring cells from the lacrimal gland‐draining cervical lymph nodes of NOD mice to lymphocyte‐deficient NOD‐SCID mice. Transfer of bulk cervical lymph node cells modelled the male‐specific dacryoadenitis that spontaneously develops in NOD mice. Female to female transfers resulted in dacryoadenitis if the CD4⁺ CD25⁺ Treg‐enriched population was depleted before transfer; however, male to male transfers resulted in comparable dacryoadenitis regardless of the presence or absence of Treg cells within the donor cell population. Hormone manipulation studies suggested that this Treg cell dysfunction was mediated at least in part by androgens. Surprisingly, male Treg cells were capable of preventing the transfer of dacryoadenitis to female recipients. These data suggest that male‐specific factors promote reversible dysfunction of lacrimal gland‐protective Treg cells and, to our knowledge, form the first evidence for reversible organ‐protective Treg cell dysfunction in organ‐specific autoimmunity.     

Inhibition of type 1 diabetes in filaria‐infected non‐obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3+ regulatory T cells

We sought to determine whether Litomosoides sigmodontis, a filarial infection of rodents, protects against type 1 diabetes in non‐obese diabetic (NOD) mice. Six‐week‐old NOD mice were sham‐infected or infected with either L3 larvae, adult male worms, or adult female worms. Whereas 82% of uninfected NOD mice developed diabetes by 25 weeks of age, no L. sigmodontis‐infected mice developed disease. Although all mice had evidence of ongoing islet cell inflammation by histology, L. sigmodontis‐infected mice had greater numbers of total islets and non‐infiltrated islets than control mice. Protection against diabetes was associated with a T helper type 2 (Th2) shift, as interleukin‐4 (IL‐4) and IL‐5 release from α‐CD3/α‐CD28‐stimulated splenocytes was greater in L. sigmodontis‐infected mice than in uninfected mice. Increased circulating levels of insulin‐specific immunoglobulin G1, showed that this Th2 shift occurs in response to one of the main autoantigens in diabetes. Multicolour flow cytometry studies demonstrated that protection against diabetes in L. sigmodontis‐infected NOD mice was associated with significantly increased numbers of splenic CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells. Interestingly, injection of crude worm antigen into NOD mice also resulted in protection against type 1 diabetes, though to a lesser degree than infection with live L. sigmodontis worms. In conclusion, these studies demonstrate that filarial worms can protect against the onset of type 1 diabetes in NOD mice. This protection is associated with a Th2 shift, as demonstrated by cytokine and antibody production, and with an increase in CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells.     

Alloantigen‐specific de novo‐induced Foxp3+ Treg revert in vivo and do not protect from experimental GVHD

Induced antigen‐specific Foxp3⁺ T cells (iTreg) are being discussed as a promising alternative to polyclonal natural Foxp3⁺ T cells (nTreg) for cell‐based therapies, particularly to achieve transplantation tolerance. Using Foxp3eGFP‐reporter mice, we here establish an efficient protocol to induce and expand alloantigen‐specific iTreg from Foxp3^?CD4⁺ T cells with cluster‐disrupted DC. These iTreg were mainly CD62L⁺ and showed efficient suppressive activity in vitro. However, in contrast to nTreg, adoptively transferred iTreg entirely failed to prevent lethal graft versus host disease (GVHD). Within irradiated recipients, the majority of adoptively transferred Foxp3⁺ iTreg, but not Foxp3⁺ nTreg quickly reverted to Foxp3^?CD4⁺ T cells. We therefore suggest that therapeutic approaches to treat GVHD should rely on nTreg, whereas the use of de novo alloantigen‐induced iTreg should be handled with caution since the stability of the regulatory phenotype of the iTreg could be of major concern.