CD4+CD25+ regulatory T cells control the progression from periinsulitis to destructive insulitis in murine autoimmune diabetes

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Abstract

Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic β-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-γ-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.

Introduction

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic islet cells. In the non-obese diabetic (NOD) mouse it is well established that this autoimmune process is mediated by T cells [1]. The NOD mouse spontaneously develops autoimmune diabetes and is a complex and well-studied model for human T1D. The spontaneous development of the autoimmune process makes the NOD mouse a very useful model to gain insight into human T1D and other spontaneously developing organ-specific autoimmune diseases. The development of diabetes in the NOD mouse is the result of a dysregulated immune system; defects have been shown on multiple levels of central and peripheral tolerance, including immune cells participating in the (auto)-immune response such as T cells and antigen presenting cells (APC) [2], [3]. Moreover, many experimental approaches have been successful in delaying and preventing diabetes onset or diabetes progression, mainly by skewing the Th1 polarized T-cell response towards Th2, suppressing T effector cell function or migration of T effector cells, and using non-specific substances with immunmodulatory effects [4]. Despite considerable progress in recent years, the initial trigger that leads to the priming of the islet cell antigen-specific T cells in the NOD mouse is still unknown, as is the identity of the primary target antigen(s) [5], [6], [7]. Moreover, the progression from non-destructive periinsulitis to invasive intrainsulitis (<50% of islet cells destroyed) and destructive insulitis (>50% of islet cells destroyed) that ultimately results in diabetes manifestation, is incompletely understood [3], [4], [8], [9]. Several mechanisms such as dysregulation of costimulation and upregulation of chemokines have been implicated in the control of this sequence of events, which ultimately leads to overt diabetes in the NOD mouse and presumably in human T1D [10].

The unususal MHC haplotype of the NOD mouse (KD, I-AG7, I-Enull, DB) is strongly associated with diabetes susceptibility. However, it is thought that diabetes in NOD mice is under polygenic control, with insulin-dependent diabetes (Idd) susceptibility loci playing an important role in the pathogenesis in addition to the MHC complex. The non-obese resistant (NOR) mouse is a NOD-related, MHC-congenic strain that does not develop diabetes (neither spontaneously nor cyclophosphamide-induced), despite the expression of the strongly diabetes-associated MHC I-AG7 molecule [11]. In addition to identical MHC molecules, NOR mice express the majority of the NOD gene pool (only a small fraction of the gene pool has been replaced by the BLKS/J strain) [11]. However, NOR mice can develop mild insulitis, suggesting that their T cells are being primed to autoantigens and invade the pancreatic islets, but are either incapable to exert their effector function (and sustain the autoimmune destruction) or are controlled by regulatory cells.

CD4+CD25+ regulatory cells have been described as important mediators of peripheral tolerance in a variety of experimental models including inflammatory bowel disease, EAE, and type 1 diabetes [12], [13]. CD4+CD25+ cells recently have been described as very efficient in the protection and cure of NOD mice from diabetes development [14], [15]. This class of T regulatory cells has been identified as being of thymic origin, anergic [16], dependent on IL-2 stimulation for their survival [17] as well as constitutively expressing CTLA-4, OX-40 [18], L-selectin [19], and the glucocorticoid induced tumor necrosis factor receptor (GITR) [20]. In vitro studies have shown that CD4+CD25+ regulatory T cells act cell contact-dependent while cytokines such as IL-10 and TGF-β appear not to be necessary for the suppression of CD4+CD25− cells [21], [22], [23]. However IL-10, IL-4, and TGF-β were required in vivo for the suppressive effect of CD4+CD25+ cells [24], [25], [26]. It has also been postulated that a different class of CD4+CD25+ cells, derived from CD4+CD25− cells in the periphery, is cell contact-independent and exerts its function through suppressive cytokines such as IL-10 and TNF-α [27].

Priming of naïve autoreactive T cells to a memory/effector state does not inevitably result in autoimmune disease. Several gate-keeping functions prevent autoreactive effector T cells from mediating immune pathology; control by CD4+CD25+ regulatory cells is one of these mechanisms. Therefore, it is conceivable that NOR mice do not develop T1D because, in contrast to NOD mice, their primed ICA (islet cell antigen)-reactive T cells are more effectively controlled by CD4+CD25+ regulatory cells. We here provide evidence that ICA-reactive T cells become spontaneously primed in NOR mice, but control by CD4+CD25+ regulatory cells prevents them from considerably damaging pancreatic islets.

Section snippets

Mice

NOD, NOD SCID, C57.BL/6, C57.BL/6 RAG KO, BALB/c, SJL/J, and B10.PL mice were purchased from the Jackson Laboratories (Bar Harbor, ME). NOR mice were purchased from Taconic (Germantown, NY). Mice were held under pathogen-free conditions in the Animal Resource Center at Case Western Reserve University.

Antigens

20-mer GAD65 peptides included GAD78–97 (GAD p6; KPCSCSKVDVNYAFLHATDL), GAD217–236 (GADp15; EYVTLKKMREIIGWPGGSGD), GAD479–498 (GAD p32; EYLYNIIKNREGYEMVFDGK), GAD524–543 (GADp35;

Spontaneous T-cell responses to GAD65 peptides arise in NOR mice

GAD65 is one of the major candidate autoantigens in NOD diabetes. In NOD mice, T-cell responses to several GAD65 peptides develop spontaneously and GAD65-specific T cells have been shown to induce diabetes in NOD SCID mice [32]. We tested whether T-cell responses to GAD65 peptides would also spontaneously develop in NOR mice. The production of IFN-γ and IL-5 by spleen cells from non-immunized NOD and NOR mice in response to a partial GAD65 9-mer peptide library and a set of previously defined

Discussion

NOR mice are resistant to the development of autoimmune diabetes despite their nearly identical genetic background to NOD mice (including the MHC haplotype).

T-cell mediated islet cell destruction in the NOD mouse is the result of a defect in control-mechanisms that normally prevent the emergence and effector functions of autoreactive T cells. This defect can occur on multiple levels during the generation of a T-cell response. We reasoned that by examining the T-cell compartments of both NOD and

Acknowledgments

This work was supported by grants to P.V.L. from the National Institutes of Health (DK-48799, AI-42635, and AI/DK 44484) and from the Juvenile Diabetes Foundation (1-2000-248). M.T.L. was supported by NIH Grant A147756, P.A.O. was supported by a fellowship of the Deutsche Forschungsgemeinschaft.

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