Recruitment and activation of macrophages by pathogenic CD4 T cells in type 1 diabetes: evidence for involvement of CCR8 and CCL1

Adoptive transfer of diabetogenic CD4 Th1 T cell clones into young NOD or NOD.scid recipients rapidly induces onset of diabetes and also provides a system for analysis of the pancreatic infiltrate. Although many reports have suggested a role for macrophages in the inflammatory response, there has been little direct characterization of macrophage activity in the pancreas. We showed previously that after migration to the pancreas, diabetogenic CD4 T cell clones produce a variety of inflammatory cytokines and chemokines, resulting in the recruitment of macrophages. In this study, we investigated mechanisms by which macrophages are recruited and activated by T cells. Analysis of infiltrating cells after adoptive transfer by the diabetogenic T cell clone BDC-2.5 indicates that large numbers of cells staining for both F4/80 and CD11b are recruited into the pancreas where they are activated to make IL-1beta, TNF-alpha, and NO, and express the chemokine receptors CCR5, CXCR3, and CCR8. Diabetogenic CD4 T cell clones produce several inflammatory chemokines in vitro, but after adoptive transfer we found that the only chemokine that could be detected ex vivo was CCL1. These results provide the first evidence that CCR8/CCL1 interaction may play a role in type 1 diabetes through macrophage recruitment and activation.     

Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently "naive" in surface phenotype, are highly activated functionally and do not resemble CD4(+) T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4(+) T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.     

MHC-mismatched islet allografts are vulnerable to autoimmune recognition in vivo

When transplanted into type 1a diabetic recipients, islet allografts are subject both to conventional allograft immunity and, presumably, to recurrent autoimmune (islet-specific) pathogenesis. Importantly, CD4 T cells play a central role both in islet allograft rejection and in autoimmune disease recurrence leading to the destruction of syngeneic islet transplants in diabetic NOD mice. However, it is unclear how NOD host MHC class II (I-A(g7))-restricted, autoreactive CD4 T cells may also contribute to the recognition of allogeneic islet grafts that express disparate MHC class II molecules. We hypothesized that islet-specific CD4 T cells can target MHC-mismatched islet allografts for destruction via the "indirect" (host APC-dependent) pathway of Ag recognition. To test this hypothesis, we determined whether NOD-derived, islet-specific CD4 T cells (BDC-2.5 TCR transgenic cells) could damage MHC-mismatched islets in vivo independent of conventional allograft immunity. Results demonstrate that BDC-2.5 CD4 T cells can vigorously destroy MHC class II-disparate islet allografts established in NOD.scid recipients. Tissue injury is tissue-specific in that BDC-2.5 T cells destroy donor-type islet, but not thyroid allografts established in the same NOD.scid recipient. Furthermore, BDC-2.5 CD4 T cells acutely destroy MHC class II-deficient islet allografts in vivo, indicating that autoimmune pathogenesis can be completely independent of donor MHC class II expression. Taken together, these findings indicate that MHC-mismatched islet allografts can be vulnerable to autoimmune pathogenesis triggered by autoreactive CD4 T cells, presumably through indirect autoantigen recognition in vivo.     

Blockade of late stages of autoimmune diabetes by inhibition of the receptor for advanced glycation end products

Ligation of the receptor for advanced glycation end products (RAGE) occurs during inflammation. Engagement of RAGE results in enhanced expression of addressins and it is therefore, not surprising that previous studies have shown a role of RAGE/ligand interactions in immune responses including cell/cell contact but the role of RAGE in spontaneous autoimmunity has not been clearly defined. To study the role of RAGE/ligand interactions in autoimmune diabetes, we tested the ability of soluble RAGE, a scavenger of RAGE ligands, in late stages of diabetes development in the NOD mouse-disease transferred with diabetogenic T cells and recurrent disease in NOD/scid recipients of syngeneic islet transplants. RAGE expression was detected on CD4(+), CD8(+), and B cells from diabetic mice and transferred to NOD/scid recipients. RAGE and its ligand, S100B, were found in the islets of NOD/scid mice that developed diabetes. Treatment of recipient NOD/scid mice with soluble RAGE prevented transfer of diabetes and delayed recurrent disease in syngeneic islet transplants. RAGE blockade was associated with increased expression of IL-10 and TGF-beta in the islets from protected mice. RAGE blockade reduced the transfer of disease with enriched T cells, but had no effect when diabetes was transferred with the activated CD4(+) T cell clone, BDC2.5. We conclude that RAGE/ligand interactions are involved in the differentiation of T cells to a mature pathogenic phenotype during the late stages of the development of diabetes.     

Effector function of diabetogenic CD4 Th1 T cell clones: a central role for TNF-alpha

Effector function of T cells in autoimmune diabetes has been widely studied with mixed populations of lymphoid T cells stimulated ex vivo, but this approach does not permit evaluation of the contribution by a single T cell clone in the inflammatory site during pathogenesis. We have investigated cytokine production both in vitro and in vivo in a panel of diabetogenic CD4 Th1 T cell clones derived from the NOD mouse. SuperArray analysis showed a common pattern of mRNA expression for inflammatory cytokines and receptors in vitro after TCR stimulation. Ex vivo intracellular cytokine staining demonstrated that two important inflammatory cytokines, IFN-gamma and TNF-alpha, were being made by these T cells recovered from the pancreas 6 days following adoptive transfer. TNF-alpha produced in the pancreas by pathogenic T cell clones and recruited macrophages was not the membrane-bound form. Secreted TNF-alpha can lead to production of multiple inflammatory chemokines, as were observed in the pathogenic clones by intracellular cytokine staining. Our results not only define the nature of an inflammatory cytokine response critical to development of diabetes, but also suggest its role in the regulation of other events during pathogenesis induced by CD4 T cells. Similar analyses in other models demonstrated that disease induced by CD4 T cell clones closely resembles spontaneous autoimmune diabetes in which both CD4 and CD8 T cells are required. Thus, cloned T cells in effect amplify effector function of T cells which otherwise may be difficult to detect without ex vivo stimulation.     

Different diabetogenic potential of autoaggressive CD8+ clones associated with IFN-gamma-inducible protein 10 (CXC chemokine ligand 10) production but not cytokine expression, cytolytic activity, or homing characteristics

Type 1 diabetes mellitus is an autoimmune disease characterized by T cell-mediated destruction of the insulin-producing beta cells in the islets of Langerhans. From studies in animal models, CD8(+) T cells recognizing autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit-related protein, insulin, or glutamic acid decarboxylase (GAD) are believed to play important roles in both the early and late phases of beta cell destruction. In this study, we investigated the factors governing the diabetogenic potential of autoreactive CD8(+) clones isolated from spleens of NOD mice that had been immunized with GAD65(515-524) or insulin B-chain(15-23) peptides. Although these two clones were identical in most phenotypic and functional aspects, for example cytokine production and killing of autologous beta cells, they differed in the expression of IFN-gamma-inducible protein-10, which was only produced at high levels by the insulin-specific clone, but not by the GAD65-specific clone, and other autoantigen-specific nonpathogenic CD8 T cell clones. Interestingly, upon i.p. injection into neonatal mice, only the insulin B-chain(15-23)-reactive CD8(+) T clone accelerated diabetes in all recipients after 4 wk, although both insulin- and GAD-reactive clones homed to pancreas and pancreatic lymph nodes with similar kinetics. Diabetes was associated with increased pancreatic T cell infiltration and, in particular, recruitment of macrophages. Thus, secretion of IFN-gamma-inducible protein-10 by autoaggressive CD8(+) lymphocytes might determine their diabetogenic capacity by affecting recruitment of cells to the insulitic lesion.     

Paralytic autoimmune myositis develops in nonobese diabetic mice made Th1 cytokine-deficient by expression of an IFN-gamma receptor beta-chain transgene

Nonobese diabetic (NOD) mice and some human type 1 diabetes (T1D) patients manifest low to high levels of other autoimmune pathologies. Skewing their cytokine production from a Th1 (primarily IFN-gamma) to a Th2 (primarily IL-4 and IL-10) pattern is a widely proposed approach to dampen the pathogenicity of autoreactive diabetogenic T cells. However, it is important that altered cytokine balances not enhance any other autoimmune proclivities to dangerous levels. Murine CD4 T cells are characterized by a reciprocal relationship between the production of IFN-gamma and expression of the beta-chain component of its receptor (IFN-gamma RB). Thus, NOD mice constitutively expressing a CD2 promoter-driven IFN-gamma RB transgene in all T cells are Th1-deficient. Unexpectedly, NOD.IFN-gamma RB Tg mice were found to develop a lethal early paralytic syndrome induced by a CD8 T cell-dependent autoimmune-mediated myositis. Furthermore, pancreatic insulitis levels were not diminished in 9-wk-old NOD.IFN-gamma RB Tg females, and overt T1D developed in the few that survived to an older age. Autoimmune-mediated myositis is only occasionally detected in standard NOD mice. Hence, some manipulations diminishing Th1 responses can bring to the forefront what are normally secondary autoimmune pathologies in NOD mice, while also failing to dependably abrogate pancreatic beta cell destruction. This should raise a cautionary note when considering the use of protocols that induce alterations in cytokine balances as a means of blocking progression to overt T1D in at-risk humans.     

Targeting of a T cell agonist peptide to lysosomes by DNA vaccination induces tolerance in the nonobese diabetic mouse

CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides to the lysosomal compartment on a monospecific T cell population termed 2.5mi(+) T cells that shares reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse. MHC class II tetramer analysis showed that repeated administrations were necessary to expand 2.5mi(+) T cells in vivo. This expansion was independent of Ag presentation by B cells. A single peptide epitope was sufficient to induce protection against T1D, which was not due to Ag-specific T cell anergy. Typical Th2 cytokines such as IL-10 or IL-4 were undetectable in 2.5mi(+) T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi(+) T cell population produced IFN-γ similar to 2.5mi(+) T cells from naive mice. Protection against T1D by DNA treatment was completely lost in NOD.CD28(-/-) mice which are largely deficient of natural regulatory T cells (Treg). Although Ag-specific Foxp3(+) Treg did not expand in response to DNA treatment, diabetes onset was delayed in Treg-reconstituted and DNA-treated NOD.SCID mice. These observations provide evidence for a Treg-mediated protective mechanism that is independent of the expansion or de novo generation of Ag-specific Treg.     

Cutting edge: interactions through the IL-10 receptor regulate autoimmune diabetes.

BDC2.5/nonobese diabetic (NOD) transgenic mice express a TCR from a diabetogenic T cell clone yet do not spontaneously develop diabetes at high incidence. Evidence exists showing that in the absence of endogenous TCR alpha-chain rearrangements this transgenic mouse spontaneously develops diabetes and that CTLA-4 negatively regulates diabetes onset. This strongly suggests that onset of diabetes in BDC2.5/NOD mice is governed by T cell regulation. We addressed the mechanism of immune regulation in BDC2.5/NOD mice. We find that activated spleen cells from young, but not old, BDC2.5/NOD mice are able to transfer diabetes to NOD-scid recipients. We have used anti-IL-10R to show that the failure of splenocytes from older mice to transfer diabetes is due to dominant regulation. We furthermore found that diabetes developed following anti-IL-10R treatment of 6-wk old BDC2.5/NOD mice indicating that endogenous IL-10 plays a key role in the regulation of diabetes onset in this transgenic mouse.     

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.     

Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice.

B cells can serve dual roles in modulating T cell immunity through their potent capacity to present Ag and induce regulatory tolerance. Although B cells are necessary components for the initiation of spontaneous T cell autoimmunity to beta cell Ags in nonobese diabetic (NOD) mice, the role of activated B cells in the autoimmune process is poorly understood. In this study, we show that LPS-activated B cells, but not control B cells, express Fas ligand and secrete TGF-beta. Coincubation of diabetogenic T cells with activated B cells in vitro leads to the apoptosis of both T and B lymphocytes. Transfusion of activated B cells, but not control B cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta cell autoantigens. Furthermore, this treatment induced mononuclear cell apoptosis predominantly in the spleen and temporarily impaired the activity of APCs. Cotransfer of activated B cells with diabetogenic splenic T cells prevented the adoptive transfer of type I diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of NOD mice treated with control B cells developed T1DM within 27 wk, <20% of the NOD mice treated with activated B cells became hyperglycemic up to 1 year of age. Our data suggest that activated B cells can down-regulate pathogenic Th1 immunity through triggering the apoptosis of Th1 cells and/or inhibition of APC activity by the secretion of TGF-beta. These findings provide new insights into T-B cell interactions and may aid in the design of new therapies for human T1DM.     

Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice

Nonobese diabetic (NOD) mice carrying a transgenic TCR from an islet Ag-specific CD4 T cell clone, BDC2.5, do not develop diabetes. In contrast, the same transgenic NOD mice on the SCID background develop diabetes within 4 wk after birth. Using a newly developed mAb specific for the BDC2.5 TCR, we examined the interaction between diabetogenic T cells and regulatory T cells in NOD.BDC transgenic mice. CD4 T cells from NOD.BDC mice, expressing high levels of the clonotype, transfer diabetes to NOD.SCID recipients. In contrast, CD4 T cells expressing low levels due to the expression of both transgenic and endogenous TCR alpha-chains inhibit diabetes transfer. The clonotype-low CD4 T cells appear late in the ontogeny in the thymus and peripheral lymphoid organs, coinciding with resistance to cyclophosphamide-induced diabetes. These results demonstrate that diabetic processes in NOD.BDC mice are regulated by a balance between diabetogenic T cells and regulatory T cells. In the absence of specific manipulation, regulatory T cell function seems to be dominant and mice remain diabetes free. Understanding of mechanisms by which regulatory T cells inhibit diabetogenic processes would provide means to prevent diabetes development in high-risk human populations.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.     

A glutamic acid decarboxylase 65-specific Th2 cell clone immunoregulates autoimmune diabetes in nonobese diabetic mice

Several studies have provided indirect evidence in support of a role for beta cell-specific Th2 cells in regulating insulin-dependent diabetes (IDDM). Whether a homogeneous population of Th2 cells having a defined beta cell Ag specificity can prevent or suppress autoimmune diabetes is still unclear. In fact, recent studies have demonstrated that beta cell-specific Th2 cell clones can induce IDDM. In this study we have established Th cell clones specific for glutamic acid decarboxylase 65 (GAD65), a known beta cell autoantigen, from young unimmunized nonobese diabetic (NOD) mice. Adoptive transfer of a GAD65-specific Th2 cell clone (characterized by the secretion of IL-4, IL-5, and IL-10, but not IFN-gamma or TGF-beta) into 2- or 12-wk-old NOD female recipients prevented the progression of insulitis and subsequent development of overt IDDM. This prevention was marked by the establishment of a Th2-like cytokine profile in response to a panel of beta cell autoantigens in cultures established from the spleen and pancreatic lymph nodes of recipient mice. The immunoregulatory function of a given Th cell clone was dependent on the relative levels of IFN-gamma vs IL-4 and IL-10 secreted. These results provide direct evidence that beta cell-specific Th2 cells can indeed prevent and suppress autoimmune diabetes in NOD mice.     

Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.     

Induction of autoantigen-specific Th2 and Tr1 regulatory T cells and modulation of autoimmune diabetes

Autoantigen-based immunotherapy can modulate autoimmune diabetes, perhaps due to the activation of Ag-specific regulatory T cells. Studies of these regulatory T cells should help us understand their roles in diabetes and aid in designing a more effective immunotherapy. We have used class II MHC tetramers to isolate Ag-specific T cells from nonobese diabetic (NOD) mice and BALB/c mice treated with glutamic acid decarboxylase 65 peptides (p206 and p221). Based on their cytokine secretion profiles, immunization of NOD mice with the same peptide induced different T cell subsets than in BALB/c mice. Treatment of NOD mice induced not only Th2 cells but also IFN-gamma/IL-10-secreting T regulatory type 1 (Tr1) cells. Adoptive transfer experiments showed that isolated tetramer(+) T cells specific for p206 or p221 could inhibit diabetes development. These cells were able to suppress the in vitro proliferation of other NOD mouse T cells without cell-cell contact. They performed their regulatory functions probably by secreting cytokines, and Abs against these cytokines could block their suppressive effect. Interestingly, the presence of both anti-IL-10 and anti-IFN-gamma could enhance the target cell proliferation, suggesting that Tr1 cells play an important role. Further in vivo experiments showed that the tetramer(+) T cells could block diabetogenic T cell migration into lymph nodes. Therefore, treatment of NOD mice with autoantigen could induce Th2 and Tr1 regulatory cells that can suppress the function and/or block the migration of other T cells, including diabetogenic T cells, and inhibit diabetes development.     

B cells are crucial for determinant spreading of T cell autoimmunity among beta cell antigens in diabetes-prone nonobese diabetic mice

The determinant spreading of T cell autoimmunity plays an important role in the pathogenesis of type 1 diabetes and in the protective mechanism of Ag-based immunotherapy in NOD mice. However, little is known about the role of APCs, particularly B cells, in the spreading of T cell autoimmunity. We studied determinant spreading in NOD/scid or Igmu(-/-) NOD mice reconstituted with NOD T and/or B cells and found that mice with mature B cells (TB NOD/scid and BMB Igmu(-/-) NOD), but not mice that lacked mature B cells (T NOD/scid and BM Igmu(-/-) NOD), spontaneously developed Th1 autoimmunity, which spread sequentially among different beta cell Ags. Immunization of T NOD/scid and BM Igmu(-/-) NOD mice with a beta cell Ag could prime Ag-specific Th1 or Th2 responses, but those T cell responses did not spread to other beta cell Ags. In contrast, immunization of TB NOD/scid and BMB Igmu(-/-) NOD mice with a beta cell Ag in IFA induced Th2 responses, which spread to other beta cell Ags. Furthermore, we found that while macrophages and dendritic cells could evoke memory and effector T cell responses in vitro, B cells significantly enhanced the detection of spontaneously primed and induced Th1 responses to beta cell Ags. Our data suggest that B cells, but not other APCs, mediate the spreading of T cell responses during the type 1 diabetes process and following Ag-based immunotherapy. Conceivably, the modulation of the capacity of B cells to present Ag may provide new interventions for enhancing Ag-based immunotherapy and controlling autoimmune diseases.     

CD8 T cells mediate direct biliary ductule damage in nonobese diabetic autoimmune biliary disease

We previously described the NOD.c3c4 mouse, which is protected from type 1 diabetes (T1D) because of protective alleles at multiple insulin-dependent diabetes (Idd) genes, but develops autoimmune biliary disease (ABD) resembling primary biliary cirrhosis (PBC). In this paper, we characterize the NOD.ABD strain, which is genetically related to the NOD.c3c4 strain but develops both ABD and T1D. Histologically, NOD.ABD biliary disease is indistinguishable from that in NOD.c3c4 mice. The frequency of effector memory (CD44(+)CD62L(-)) and central memory (CD44(+)CD62L(+)) CD8 T cells is significantly increased in the intrahepatic lymphocyte fraction of NOD.ABD mice, and NOD.ABD CD8 T cells produce more IFN-γ and TNF-α, compared with controls. NOD.ABD splenocytes can transfer ABD and T1D to NOD.c3c4 scid mice, but only T1D to NOD scid mice, suggesting that the genetic origin of the target organ and/or its innate immune cells is critical to disease pathogenesis. The disease transfer model, importantly, shows that biliary duct damage (characteristic of PBC) and inflammation precede biliary epithelial cell proliferation. Unlike T1D where both CD4 and CD8 T cells are required for disease transfer, purified NOD.ABD CD8 T cells can transfer liver inflammation into NOD.c3c4 scid recipients, and disease transfer is ameliorated by cotransferring T regulatory cells. Unlike NOD.c3c4 mice, NOD.ABD mice do not develop anti-nuclear or anti-Smith autoantibodies; however, NOD.ABD mice do develop the antipyruvate dehydrogenase Abs typical of human PBC. The NOD.ABD strain is a model of immune dysregulation affecting two organ systems, most likely by mechanisms that do not completely coincide.     

Presence of diabetes-inhibiting, glutamic acid decarboxylase-specific, IL-10-dependent, regulatory T cells in naive nonobese diabetic mice

Immunization of NOD mice with autoantigens such as glutamic acid decarboxylase (GAD) 221-235 peptide (p221) can induce Ag-specific CD4(+) T regulatory (Tr) cells. However, it is unclear whether these Tr cells acquire their regulatory capacity due to immunization or whether they are constitutively harbored in unimmunized naive mice. To address this question, we used an I-Ag7 tetramer to isolate p221-specific T cells from naive NOD mice (N221(+) cells) after peptide-specific in vitro expansion. The N221(+) T cells produced IFN-gamma and IL-10, but very little IL-4, in response to p221 stimulation. These T cells could function as regulatory cells and inhibit in vitro proliferation of diabetogenic BDC2.5 cells. This suppressive activity was cell contact-independent and was abrogated by Abs to IL-10 or IL-10R. Interestingly, IL-2 produced by other T cells present in the cell culture induced unactivated N221(+) T cells to exhibit regulatory activities involving production of IL-10. In vivo, N221(+) cells inhibited diabetes development when cotransferred with NOD splenocytes into NOD/scid recipients. Together, these results demonstrate that p221-specific IL-10-dependent Tr cells, including Tr type 1 cells, are present in naive NOD mice. The use of spontaneously arising populations of GAD peptide-specific Tr cells may represent a promising immunotherapeutic approach for preventing type 1 diabetes.