Autoimmune disease results from the dysregulation of basic tolerogenic processes designed to control self/non-self-discrimination. Approaches to treat autoimmunity have focused historically on potent immunosuppressives that block the activation and expansion of antigen-specific T cells before they differentiate into pathogenic T cell responses. These therapies are very efficient in reducing clonal expansion and altering early signaling pathways. However, once the pathogenic responses are established (i.e., autoimmunity), the interventions are less effective on activated and differentiated T cell subsets (including memory T cells) or acting in the presence of an inflammatory milieu to abort immune responses at the target tissue and systemically. Moreover, the current immunotherapies require continuous use because they do not redirect the immune system to a state of tolerance. The continuous treatment leads to long-term toxicities and can profoundly suppress protective immune responses targeted at viruses, bacteria, and other pathogens. Over the past decade, there have been tremendous advances in our understanding of the basic processes that control immune tolerance. Among the most exciting has been the identification of a professional regulatory T cell subset that has shown enormous potential in suppressing pathologic immune responses in autoimmune diseases, transplantation, and graft vs. host disease. In this review, we summarize current efforts to induce and maintain tolerance in the autoimmune diabetes setting by using therapeutic vaccination with CD4+CD25+ regulatory T cells. Emphasis will be placed on approaches to exploit regulatory T cells either directly or through the use of anti-CD3 immunotherapy.

-------------------------------------------------------------------------------- Regulation of the immune response to self-antigens is a complex process that depends on maintaining self-tolerance while retaining the capacity to mount a robust immune response. T cells specific for these autoantigens are present in most normal individuals but are kept under control by multiple diverse peripheral tolerance mechanisms. For at least 30 years, there has been the notion that in addition to T cells that mediate effector immune responses to combat infections and mediate graft rejection, there are classes of regulatory/suppressor T cells that exist to control immunity (reviewed in ref. 1). Early on, CD8+ T cells were identified that suppress immune responses through their direct cytotoxicity on antigen-bearing cells or through cryptic suppressor factors that were poorly characterized (2). However, during this early period, there were already hints that the quintessential helper T cells subset, CD4+ T cells, also may have regulatory activity. North and Awwad (3) showed that depletion of CD4+ T cells by using anti-CD4 mAbs resulted in tumor rejection. This modern view of CD4+ regulatory T cells (Tregs) was enhanced by the observations by Sakaguchi et al. (4, 5) that the adoptive transfer of T cells depleted of CD4+CD25+ cells induced multiorgan autoimmunity in the recipient animals. These studies complemented ongoing efforts by a number of groups in England and France who demonstrated antigen-specific Treg populations in mice and rats (6–9). In fact, multiple investigators provided compelling data to support the existence of Tregs in rodents, especially in those animals that had undergone certain immunotherapeutic interventions in the allogeneic transplant or autoimmune setting. For example, populations of CD4+ peripheral T cells and thymocytes were shown to prevent induction of autoimmune thyroiditis in an antigen-specific manner (10). The Tregs were shown to be driven by peripheral autoantigen and could be extracted from mice maintaining long-term allografts. Most importantly, Gershon and Kondo (11), and subsequently Cobbold and Waldmann (8), developed the concept of infectious tolerance, where cells from tolerant animals could be transferred to naïve recipients suppressing not only the original antigen specificities but other antigens linked through the same antigen-presenting cells. However, progress in this area was slow and tedious, often fraught with skepticism in the community because of difficulty in defining the precise phenotype of these cells, their antigen specificity, and the mechanistic basis for the suppressive activity.

Autoimmunity is caused by a loss of tolerance and results in an imbalance in immune homeostasis, particularly regulatory versus effector activities against self-antigens. Therapeutic approaches for the treatment of autoimmune diseases need to attack key components of the immune response to restore this balance. In this body of work I have worked toward this end using three distinct approaches to in the context of type 1 diabetes using the non-obese diabetic mouse model. First, I used a global approach to shift the balance of regulatory to effector T cells. I show that non-Fc receptor binding anti-CD3 monoclonal antibody treatment, which has been previously shown to reserve diabetes, alters the ratio of effector to regulatory T cells (Tregs) due to preferential depletion of activated effector T cells. In addition, treatment with anti-CD3 mAbs leads to increased expression of Helios in Tregs, suggesting stabilization of Tregs may account to the extended efficacy of this antibody. Second, I used FTY720 to "lock" lymphocytes in the lymph node and pancreas thereby isolating these locations. I show that continuous treatment with FTY720 prevents diabetes development but treatment withdrawal leads to rapid onset of disease. Furthermore, morphological changes that occur during the development of the disease in control and treated mice show that tertiary lymphoid organ development and their subsequent destruction correlate with disease progression suggesting that islet destruction maybe be due to loss of TLO integrity. Finally, I used a targeted approach to directly alter memory T cells. I show that treatment of NOD mice with anti-IL-7Rα monoclonal antibodies prevents and cures diabetes and induces expression of the inhibitory receptor PD-1 on memory T cells. Moreover, inhibiting the interaction of PD-1 with its ligand PD-L1 restores disease in cured mice. The data suggest that IL-7 contributes to the pathogenesis of autoimmune diabetes by keeping memory T cells in a functionally competent, tolerance-resistant state, and uncover a novel link between IL-7 and the PD-1/PD-L1 tolerance pathway. Together the data reveal biological the mechanisms at the basis of therapeutic interventions aimed at preventing or reversing diabetes through manipulating the immunological pathways underling the pathogenesis of the disease. The insights will improve our ability to translate these interventions to the treatment of patients with type 1 diabetes.

Regulatory T cell (Treg) therapy for type 1 diabetes is a novel approach to restore immune tolerance and halt the autoimmune destruction of the pancreatic beta cells. However, a majority of infused Tregs do not persist. Low-dose IL2 can enhance Treg survival, but off-Treg effects on NK and CD8+ T cells raises concerns of exacerbating autoimmune pathology. In this study, we investigated if orthogonal IL2 could induce selective IL2 signaling in Tregs engineered to express a corresponding orthogonal IL2 receptor (ortho-IL2R), thus enhance Treg persistence and function a NOD mouse model of type 1 diabetes (NOD CD28KO). To investigate this, we utilized in vitro and in vivo assays, flow cytometry, and type 1 diabetes mouse models. We compared two orthogonal IL2 variants, 1G12 or 3A10, to determine which provided greater selectivity and potency. 3A10 showed no reactivity to wildtype (WT) IL2R, thus was a true orthogonal ligand. However, a high concentration of 3A10, 100,000IU/ml, in vitro was required to induce STAT5 phosphorylation and proliferation, likely due to its relatively low affinity for the ortho-IL2R (KD= 0.2nM) (28). Consequently, a high dose of MSA-3A10 in vivo, 30ug once or twice a week, was needed to promote proliferation and lineage stability of ortho-IL2R expressing Tregs in NSG mice. Furthermore, in NOD CD28KO mice, high doses of MSA-3A10, 30ug once or twice a week, did not induce proliferation of ortho-IL2R+ Tregs and subsequently failed to aid 2,000 islet antigen-specific-ortho-IL2R+ Tregs in preventing of diabetes in NOD CD28KO mice. In lieu of these findings, we explored another ortho-IL2, 1G12, which has a high affinity for ortho-IL2R (KD=0.04nM). However, at high concentrations has the potential to induce IL2 signaling in Tregs expressing WT-IL2R (28). In vitro at low concentrations, below 1,000IU/ml, 1G12 selectively induced IL2 signaling in ortho-IL2R+ Tregs and in vivo a low dose of MSA-1G12 (7.6ug) induced proliferation and upregulation of Treg markers in ortho-IL2R+ Tregs. Moreover, in vivo a low dose of MSA-1G12, 7.6ug once a week, enhanced the potency of 2,000 islet antigen-specific-ortho-IL2R+ Tregs resulting in 100% diabetes prevention in NOD CD28KO mice. To investigate further the mechanism of prevention of T1D, we exogenously provided 7.6ug of MSA-1G12 to NOD CD28KO mice twice a week for two weeks and found that endogenous Tregs upregulated Treg markers in response to MSA-1G12. However, we are still exploring the dynamic nature of the main immune cell responder to ortho-IL2 during the progression of T1D. Collectively, these results show both ortho-IL2 ligands could preferentially enhance ortho-IL2R+ Tregs, however, their efficacy in enhancing infused Tregs was limited by their relative affinities for the orthogonal IL2R. Finally, we explored strategies to improve the orthogonal IL2R/IL2 system by creating an autocrine system that eliminates the need for low or high dosing approaches. In turn, we also investigated the consequences of constitutive IL2 signaling. We tethered the orthogonal IL2R with orthogonal IL2 (3A10 or 1G12) with a peptide linker (a truncated 26 amino acid portion of the extracellular domain of IL2RA). We report that selective IL2 signaling can be induced through the 3A10/oIL2R and 1G12/oIL2R tethers. However, IL2 signaling is induced at different degrees which results in differential proliferation rates. The 3A10/oIL2R tether induces greater proliferation and upregulation of Treg markers and pro-survival molecules such as Bcl-2 in vitro and in vivo. While in vivo, 1G12/oIL2R tether potentially induced survival. Efforts are underway to determine if islet-antigen specific Tregs with either tethers can prevent T1D and the mechanisms occurring.

The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

Regulatory T cells (Tregs) are part of the suppressive arm of the immune system, playing an important role in maintaining immune homeostasis and preventing excessive inflammatory responses. As autoimmune diseases become increasingly prevalent, it is important to clarify the role of Tregs in etiology and their potential for use in cell therapy. Characterization of Tregs has revealed specialized subsets that have unique functions in controlling tissue-specific inflammation. Of particular interest is the distinction between Tregs that differentiate within the thymus (tTregs) versus the periphery (pTregs). While tTregs recognize self-antigen, pTregs are generated to induce tolerance to non-self-antigens such as those from commensal microbiota and self-antigens not presented in the thymus. pTregs could play a unique non-redundant role in type 1 diabetes (T1D), a chronic autoimmune disease, based on recent work showing connections between microbiota and T1D incidence as well as the generation of neoantigens within the pancreas. Therefore, we created a new model to determine the importance of pTregs in T1D by deleting a key genetic enhancer of Foxp3 for pTreg generation (conserved noncoding sequence CNS1) within the autoimmune prone non-obese diabetic (NOD) mouse. CNS1 deletion does not alter Treg frequencies or characteristics in young female NOD mice in most organs except the large intestine lamina propria, where increased Helios expression and reduced RORt expression suggest a shift towards tTregs. Despite increased insulitis, these mice do not exhibit increased T1D incidence. Surprisingly, we also observed that Tregs in the islet do not recognize peripherally-derived neoantigens made from insulin fusion products, and the reactivity of Tregs and Tconvs is not changed in prediabetic NOD CNS1-/- females. Lastly, preliminary data suggest NOD CNS1-/- females do not show defects in maternal-fetal tolerance but could exhibit significantly reduced Treg frequencies in the spleen and increased frequencies in the pancreatic and mesenteric lymph nodes. We concluded that CNS1-dependent pTregs do not play a significant role in the NOD mouse model, with tTregs being the key regulators of T1D. Additionally, Tregs within pancreatic islets largely recognize thymically-presented antigens, and pTregs are not frequently generated against pancreatic neoantigens.