In this study we report the generation and characterization of an insulin-reactive T regulatory TCR transgenic NOD mouse in which autoimmune diabetes mellitus development is completely prevented in both spontaneous and adoptive transfer model systems. The protection is mediated by the signaling of TGF-β in both paracrine and autocrine fashion.
T1D onset is the result of ultimate failure in immune tolerance to islet autoantigens. Proinsulin/insulin, glutamic acid decarboxylase (GAD), and a tyrosine phosphatase–like protein known as IA-2 are major islet autoantigens targeted by both T cell– and B cell–mediated autoimmune responses (33
). Recently, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) has also been identified as a target for diabetogenic CD8+
T cells (34
). Insulin, a key hormone for survival, is produced exclusively by pancreatic β cells in large quantities. Insulin is a major autoantigen in the disease (35
) and not only appears, however, to promote diabetes development but also has the capacity to generate regulatory cells as well. To better understand how such regulatory cells might develop, we generated an insulin-reactive, regulatory TCR transgenic NOD mouse — the 2H6 NOD mouse. It is noteworthy that most, if not all, CD4+
T cells from TCR transgenic mouse strains have a pathogenic phenotype in autoimmune diabetes and other autoimmune disease models (30
). Tarbell et al. have recently reported a TCR transgenic NOD mouse in which the T cells are specific for a GAD peptide. However, the mouse does not develop diabetes in spite of the fact that its lymphocytes proliferate and make IFN-γ, IL-2, TNF-α, and IL-10 when stimulated in vitro with GAD65 peptide (40
). Apart from the antigen specificity, the biological properties of the original T cell clone that was used to generate this transgenic mouse are unknown, as the TCR was cloned from a T cell hybridoma. Here, we describe the generation and characterization of a bona fide T regulatory TCR transgenic mouse derived from a known insulin-reactive T regulatory clone. The importance of this regulatory TCR transgenic mouse is that it is a unique tool that will permit the elucidation of the role for autoantigen-reactive and TGF-β–producing Tregs in autoimmune diabetes and for similar Tregs in other autoimmune conditions.
There are 3 isoforms of TGF-β — β1, β2, and β3 — that have overlapping and distinct biological functions. TGF-β1 is the most studied isoform in immunology and is also the isoform produced by 2H6 T cells. TGF-β1–deficient mice express a severe inflammatory and autoimmune phenotype in multiple organs (41
). TGF-β receptor II is the high-affinity receptor for TGF-β1, and mice deficient in TGF-β receptor II specifically in T cells showed a phenotype very similar to that of systemic TGF-β1–deficient mice (32
). This suggests that TGF-β1 plays an important role in immune tolerance, especially mediated by T cells. Thus, TGF-β1 is generally considered to be an immune regulatory cytokine with strong therapeutic potential for treatment of human autoimmune or chronic inflammatory diseases. In several animal models of human autoimmune disease, including T1D, administration of TGF-β1 has demonstrated promising therapeutic applications (42
). Furthermore, induction and/or enhancement of endogenous TGF-β1 production, such as through oral tolerance, also produced a striking inhibitory effect on chronic inflammatory and autoimmune diseases, including T1D (14
). It has been reported that TGF-β1 exerts immune regulatory function through cell contact–dependent or –independent mechanisms (47
). It is not clear which of these pathways 2H6 T cells use to mediate their islet-specific immune tolerance in vivo, and our in vitro data suggest that cell contact–dependent mechanisms may play a major role. However, it is clear that regulation within the immune system is possible, and if manipulation can increase regulatory cell activity, then this has important implications for both prevention and treatment of autoimmune diseases; a recent study has provided evidence for proof of this principle (49
Many insulin-reactive CD4+
T cells in the NOD mouse recognize the insulin B chain peptide 9–23 and are diabetogenic, i.e., capable of adoptively transferring diabetes (9
). It is interesting that a very potent diabetogenic CD8+
T cell clone, derived from a young NOD mouse, also recognizes insulin B chain 15–23 (7
). Those studies suggested that T cells reactive to this region of the insulin B chain are mostly pathogenic. It is intriguing, therefore, that 2H6 T cells recognize insulin B chain peptides 12–25 and 9–23 (24
), a region similar to that recognized by the insulin-reactive pathogenic T cells. In addition, 2H6 T cells share the same Jα sequence, KLTFGKGT, with diabetogenic CD4+
T cell clones (9
), and the same Jβ sequence, YFGSGTRLTVL, with a diabetogenic CD8+
T cell clone (7
). However, unlike those pathogenic insulin-reactive T cells, 2H6 T cells are diabetes protective and express a Treg phenotype, and the phenotype of 2H6 TCR transgenic T cells mirrors the parental cloned 2H6 cells. Our data provide evidence that, firstly, Tregs can be antigen specific; and secondly, both pathogenic and regulatory T cells can recognize a similar, if not identical, antigenic region. This is important as the information may have an impact on the design of antigen-based immunotherapy for the disease.
Insulin- or proinsulin-reactive T cells have also been found in patients with T1D (11
). However, the in vivo biological function of those T cells is not known. Interestingly, some of the insulin- or proinsulin-reactive T cells found in patients with T1D showed an IL-4– and/or IL-10–producing profile (50
), implying protective or regulatory properties. This suggests that the autoimmune response may be downregulated. The PLN origin of 2H6 cells and their isolation from a diabetic mouse indicate that Tregs are present even when pathogenic T cells have destroyed most of the β cells (24
). 2H6 TCR transgenic NOD mice are completely protected from development of diabetes. 2H6 transgenic cells also blocked the disease induced by diabetic spleen cells in the adoptive transfer model. Moreover, 2H6 TCR transgenic cells were able to inhibit diabetes development in an accelerated disease model. It is interesting that the protection is more likely mediated by peripheral 2H6 cells, as neither 2H6 thymocytes nor 2H6 BM provided disease protection.
There is no single marker that identifies a population of Tregs. Tregs are a heterogeneous group of cells. However, naturally occurring Tregs found in both humans and mice are commonly CD4+
and express FoxP3. 2H6 transgenic T cells do not express CD25 ex vivo. However, almost all the 2H6 transgenic T cells become CD25+
after stimulation in vitro. Thus, CD25 expression on 2H6 cells is an activation marker, not the signature of conventional Tregs. FoxP3, highly expressed in CD4+
cells, has recently been considered as a master transcription factor for Tregs (17
). FoxP3 has also been reported to be induced by TGF-β (26
). It is interesting that 2H6 cells express very little FoxP3 even after anti-CD3 stimulation, which induces a large amount of TGF-β production by 2H6 cells. This suggests that the immune regulation, in particular diabetes protection mediated by 2H6 cells, is not associated with FoxP3. It is possible that the 2H6 cell represents a nonclassical naturally occurring Treg. It is also conceivable that 2H6 cells represent a novel type of Treg that might have developed in the periphery and has an important immune regulatory role at the site of the immune response, given that the 2H6 T cell clone was derived from diabetic PLN cells after adoptive transfer (24
Like the parental clone, 2H6 transgenic cells produce a significant amount of TGF-β in response to TCR ligation. It is noteworthy that a high level of TGF-β was found in 2H6 transgenic mice even without stimulation, likely because 2H6 cells are also autoreactive. Increasing evidence suggests that TGF-β is an important mediator of T regulatory function; however, it is not clear how TGF-β mediates the immune regulation, such as in the case of diabetes protection by 2H6 cells. Using the TGF-βDNRII model system, we were able to determine that TGF-β signaling in the target pathogenic cells is critical for the disease suppression. It is interesting that a similar finding was reported very recently for a colitis model (53
). The authors showed that TGF-β receptor–deficient CD4+
T cells escape control by disease-suppressive CD4+
T cells in vivo. Lack of TGF-β signaling in diabetogenic T cells in our system somewhat enhanced their pathogenicity in both NOD and BDC2.5 NOD mice in spontaneous diabetes development and also allowed them to escape from the immune regulation by Tregs. On the other hand, the lack of TGF-β signaling in the 2H6 Tregs did not change their diabetes-protective properties during the natural history of autoimmune diabetes but disarmed their ability to protect from disease development in the adoptive transfer model system.
In conclusion, our TCR transgenic model has demonstrated the mechanisms by which insulin-specific, nonclassical Tregs inhibit both spontaneous and induced diabetes development. These PLN-derived cells arise naturally and do not share the common features of classical Tregs, such as the expression of CD25 and FoxP3. Instead, they represent more a Th3 phenotype and mediate immune regulation through both the paracrine and the autocrine action of TGF-β. This model system has the potential to elucidate how insulin-specific T cells downregulate islet-specific autoimmunity.