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T-cell receptor-transgenic NOD mice: a reductionist approach to understand autoimmune diabetes
Journal of Autoimmunity 22 (2004) 121–129 www.elsevier.com/locate/issn/08968411
Forum on Transgenic autoimmune mice
T-cell receptor-transgenic NOD mice: a reductionist approach to understand autoimmune diabetes Yang Yang a,c, Pere Santamaria b,c* a
Department of Biochemistry & Molecular Biology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada b Department of Microbiology and Infectious Diseases, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada c Julia McFarlane Diabetes Research Centre, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada
1. Introduction Insulin-dependent diabetes mellitus (Type 1 Diabetes, T1D) in humans and nonobese diabetic (NOD) mice is the result of a chronic autoimmune process directed against the pancreatic beta cells [1,2]. There is extensive evidence in both humans and mice indicating that T1D is a T-cell mediated disease. Macrophages, dendritic cells (DCs) and B-cells also play an important role in the disease process, most likely as professional antigenpresenting cells (APC) responsible for driving the activation and recruitment of autoreactive T cells [1–6]. It is believed that development of diabetogenic T cells in diabetes-prone genetic backgrounds requires the presence of certain disease-associated Major Histocompatibility Complex (MHC) genes, and that diabetes susceptibility results in a breakdown of T-cell tolerance to beta cell autoantigens [1,7–10]. However, whether T1D resistance in non-diabetes prone individuals is the result of T-cell tolerance or of ignorance of beta cells by autoreactive T cells remains unclear. Studies of T-cell development in mice expressing transgenic neo-antigens in beta cells and transgenic neo-antigen-specific T-cell receptors (TCRs) resulted in different outcomes, possibly due to differences in the nature, amount and timing of transgene expression, and/or to genetic differences among the mice that were studied [11–27]. These pioneering works provided seminal information towards our understanding of the mechanisms that control the development of neo-autoantigen-specific T lymphocytes in
* Corresponding author. Tel.: +1-403-220-8735; fax: +1-403-270-8520 E-mail address: [email protected] (P. Santamaria). 0896-8411/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.10.003
genetic backgrounds that are naturally resistant to diabetes. However, since neither the antigens nor the TCRs that were studied in these models are involved in spontaneous T1D, it is difficult to determine which, if any, of the outcomes that were observed in these models is T1D-relevant. This is not a trivial issue, as the fate of autoreactive T cells in vivo is clearly a function of the nature, levels and expression anatomy of naturally occurring autoantigens. During the last decade or so, a number of transgenic mice expressing TCRs targeting naturally occurring, non-transgenic beta cell autoantigens have become available [8,28–33]. These mice are powerful tools with which to address some of the limitations of previous studies, and have enabled detailed investigations of the mechanisms that control the development, regulation, activation, differentiation and effector function of diabetogenic T cells, in both T1D-prone and T1D-resistant genetic backgrounds. Some of these models are ideal to study how anti-diabetogenic genes afford diabetes resistance, or to uncover and test new avenues for therapeutic intervention. Other models afford the opportunity to study immunoregulatory processes capable of keeping autoreactive T cells in check. This reductionist approach to diabetes research affords the opportunity to discover some of the not-so-obvious pieces that make the “big puzzle”, and allows a stepby-step reconstruction of a very complex process that, in our view, is very difficult to dissect otherwise. This review summarizes some of the lessons learned through studies of these mice as seen through the authors’ eyes. Other reviews in this series will cover equally important aspects that, for space limitations, are not dealt with in detail here.
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2. CD4+ versus CD8+ T cells in the initiation phases of T1D Any discussion on the usefulness of TCR-transgenic models to study autoimmunity warrants a summary of what has been learned in previous models. T-cell transfer studies have shown that diabetogenesis requires both CD4+ and CD8+ T cells [34–45]. Since beta cells do not express major histocompatibility complex (MHC) class II molecules [46,47], autoreactive CD4+ T cells must engage beta cell antigens (shed by a prior insult) on local antigen-presenting cells (APCs) [48,49]. Studies of CD8+ T-cell deficient NOD mice have suggested that the initial insult that sheds beta cell antigens is mediated by CD8+ cytotoxic T lymphocytes (CTL) [50–53], which invariably infiltrate NOD islets [29,49,54–60]. This hypothesis, however, is in conflict with observations that support the view that T1D is initiated by CD4+ T cells. For example, splenic CD4+ T cells from NOD mice can transfer islet inflammation (insulitis) into NOD.scid mice, but splenic CD8+ T cells cannot [34,43]. Furthermore, beta cells do not express costimulatory molecules [61] and thus naı¨ve autoreactive CD8+ T cells are unlikely to differentiate into CTL unless their target antigens are shed from beta cells by a prior insult, and later presented to them by professional APCs. Finally, genetic susceptibility and resistance to T1D are profoundly affected by polymorphisms of MHC class II genes [1,62], which control the development and function of CD4+, but not CD8+ T cells. As described at some length below, studies in TCR-transgenic NOD mice have provided valuable insights towards resolution of these apparently paradoxical observations.
3. Autoreactive CD8+ T cells in T1D: fishing antigenic ligands with TCR-transgenic T-cell probes The antigenic specificity(ies) of the CD8+ T cells that are involved in the early stages of the diabetogenic process is (are) unknown. Nevertheless, several lines of evidence suggested that the antigenic repertoire of these T cells is quite restricted. For example, a large fraction of the CD8+ T cells that can be isolated from islets of diabetic NOD mice are cytotoxic to beta cells in the context of the class I molecule Kd, and use TCR chains with homologous CDR3 sequences [58]. Furthermore, the majority of the islet-associated CD8+ T cells of transgenic NOD mice expressing the TCR chain of the CD8+ clone NY8.3 (which uses a representative CDR3 sequence) express an endogenously derived TCR chain that is identical to the one employed by the clonotype donating the TCR transgene [30]. Strikingly, a significant percentage of the CD8+ T cells that can be propagated from the earliest insulitic lesions of NOD mice use TCR chains that are identical or nearly identical to the
one used by 8.3-CD8+ T cells (V17 and J42 elements joined by the N-region sequence MRD/E) [63]. This TCR heterodimer is highly pathogenic, as demonstrated by the fact that 8.3-TCR-transgenic NOD mice (8.3-NOD) develop overt diabetes shortly after the onset of insulitis [30]. Availability of 8.3-NOD mice afforded a unique opportunity to search for antigenic ligands of early insulitic T cells. CTL derived from islets of NOD mice are notoriously unstable: they either do not survive repeated antigenic stimulation in vitro, or lose cytotoxic activity within days or weeks in culture [58]. We now know that this is in part due to the fact that repeated antigenic stimulation of TCR-transgenic 8.3-CTL with antigen-pulsed DCs induces transient re-expression of the recombination-activating genes (RAGs) and TCR revision, leading to loss in antigenic reactivity [64]. Screens of combinatorial peptide libraries with 8.3-CTL (freshly derived in vitro from naı¨ve 8.3-CD8+ splenocytes) led to the identification of two major peptide ligands for CTL expressing the prevalent V17-MRDJ42 TCR chain: NRP and NRP-A7, an alanine mutant analog of NRP with superior agonistic properties [65]. Additional experimentation confirmed what earlier TCR repertoire studies had suggested, namely that these two peptides are recognized by a large fraction of the islet-associated CD8+ T cells from wild-type NOD mice [65,66]. Hypermutation of the NRP sequence eventually led to the identification of a higher affinity ligand of the 8.3-TCR, as compared to NRP or NRP-A7 (NRP-V7) [67]. The availability of this mimotope was key to the discovery that progression of insulitis to overt diabetes in NOD mice can be predicted, with high degrees of specificity and sensitivity, by measuring the cumulative percentage of peripheral blood CD8+ T cells that bind NRP-V7/Kd tetramers [68]. More recently, Lieberman et al. have discovered the identity of the naturally occurring ligand of NRP-A7/V7-reactive CD8+ T cells, a beta cell-specific protein of unknown function [123]. We would like to emphasize here that these exciting results should not be taken to imply that the CD8+ T-cell response in T1D is exclusively directed against NRP-like peptides. Wong et al., for example, have reported that insulitic CD8+ T cells in young NOD mice recognize an insulin-derived peptide [69]. Although in our hands these cells only represent a very small fraction of all insulitic CD8+ T cells, even in young animals [66,68], they may still play an important role in the disease process.
4. CD4+ T-cell assisted recruitment of NRP-A7/NRP-V7-reactive CD8+ T cells in T1D We do not yet know for sure whether NRP-A7reactive CD8+ T cells are required for the initiation of
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diabetes. However, there is evidence suggesting that they are not sufficient. Studies of RAG-2-deficient 8.3-NOD mice, for example, support the view that efficient accumulation of 8.3-CD8+ pre-CTL into islets requires CD4+ T cell help [30,70]. The insulitogenic and diabetogenic activities of 8.3-CD8+ T cells are significantly enhanced by the presence of endogenous, non-transgenic CD4+ T cells [30,70], such that adoptive transfer of splenic CD4+ T cells from wild-type, non-diabetic NOD mice into RAG-2-deficient 8.3-NOD hosts results in a significantly increased incidence of diabetes [30,70]. In this model, CD4+ T cells potentiate the recruitment and/or accumulation of 8.3-CD8+ T cells in islets, rather than their differentiation into CTL [30]. These results do not necessarily imply that recruitment of all beta cell autoreactive CD8+ T-cell specificities in T1D is T-helper dependent. CD8+ T cells expressing the AI4-TCR, which is not NRP-A7/NRPV7-reactive (D. Serreze and T. DiLorenzo, personal communication), are as diabetogenic in the absence of endogenous CD4+ T cells as they are in their presence [31]. The factors that determine the T-helper dependency of a given clonotype are not known, but the affinity of its TCR for peptide/MHC is an attractive candidate. This hypothesis, currently under investigation in our laboratory, predicts that clonotypes recognizing peptide/MHC with high affinity will trigger diabetes in a T-helper cell-independent manner. It would be therefore reasonable to suspect that initiation of diabetes is triggered by high avidity CD8+ clonotypes. None the less, since the initial CD8+ T-cell response in T1D involves predominantly low avidity clonotypes [66], we favor the idea that diabetes initiation involves both CD4+ and CD8+ T cells acting in concert.
5. The anatomy of T-cell priming in T1D It has been recently shown that the pancreatic lymph nodes (PLN) are absolutely essential for diabetogenesis [71]. Experiments employing carboxyfluorescein diacetate succinidimyl ester (CFSE)-labeled T cells expressing MHC class I (Kd)- or class II-restricted (I-Ag7), beta cell autoreactive TCRs (8.3-CD8+; and BDC2.5 or 4.1-CD4+, respectively) have shown that beta cellreactive T cells first encounter cognate peptide/MHC complexes in the PLNs, on the surface of autoantigenloaded, bone marrow-derived APCs [70,72,73]. These experiments have also shown that priming does not occur in neonatal mice, suggesting that beta cell autoantigens do not become available until later on in life [72,73]. In the case of 8.3-CD8+ T cells, for example, priming in the PLNs is undetectable before 3 weeks, and its magnitude increases progressively with age. An increased incidence of beta cell apoptosis following the administration of low doses of the -cell toxin
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streptozotocin augmented the magnitude of 8.3-CD8+ T-cell priming and allowed it to occur at 2 weeks of age, when it is not observed to occur spontaneously. These results strongly implicate in situ -cell death in the facilitation of autoantigen specific CD8+ T-cell priming in T1D. Interestingly, the timing of 8.3-CD8+ T-cell priming in NOD mice is preceded by a period of remodeling and physiologic apoptosis of pancreatic islets that peaks at 2 weeks of age [74,75]. Regardless of whether this “wave” of apoptosis is a key event in T1D, these results demonstrate that in situ -cell death induced by CTLs can perpetuate the priming of their naı¨ve precursors in the PLNs.
6. T–T collaboration: a role for CD154 in the activation of autoreactive CD4+ T cells and in the T-helper assisted differentiation of CD8+ CTL Productive collaboration between CD4+ T-helper cells and pre-cytotoxic CD8+ T cells requires the presentation of different epitopes by the same APC, usually a DC, in a CD40/CD154-dependent manner [76–83]. CD40 ligation on DCs induces the upregulation of T-cell costimulatory molecules, elicits the production of proinflammatory cytokines, and endows DCs with the ability to differentiate CD8+ T cells. Accordingly, blockade of the CD40–CD154 pathway completely prevents the development of insulitis and diabetes in NOD mice [84,85]. Experiments in TCR-transgenic NOD mice have shown that, whereas disruption of CD154 completely prevents the development of insulitis and diabetes in monoclonal 4.1-TCR-transgenic NOD mice, which express a highly diabetogenic, I-Ag7-restricted TCR, it has no effect on the T-helper independent diabetogenic activity of 8.3-CD8+ T cells [70]. CD154/ 8.3-CD8+ T cells proliferated as well as their CD154+ conterparts in the PLNs of wild-type NOD hosts, and the 8.3-CD8+ T cells maturing in both types of mice were equally diabetogenic, indicating that 8.3-CD8+ T cells do not need CD154 to undergo antigen-driven activation in vivo [70]. Notwithstanding this, the ability of T-helper cells to enhance the diabetogenic activity of 8.3-CD8+ T cells (see above) is CD154-dependent, because only CD154+, but not CD154/, CD4+ T cells were able to do this [70]. CD154 here seems to function by ligating CD40 on DCs, as well as by inducing costimulatory activity on DCs. In agreement with this, systemic activation of DCs (and macrophages) with an agonistic anti-CD40 mAb or with CpG DNA dramatically increased the incidence of T1D in monoclonal 8.3-NOD mice. When expressed locally, TNF can also overcome the need for CD154 in another (non-TCR-transgenic) model of CD8+ T-cell induced diabetes [85]. However, this interpretation of the data (the pro-diabetogenic role of CD154 is mediated via DCs),
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cannot account for the observation that CD154/ 4.1-NOD mice remain resistant to both insulitis and diabetes upon systemic activation of their DCs with anti-CD40 mAb or CpG DNA [70]. Accordingly, CD154 does not merely act by ligating CD40 on DCs. Since ligation of CD154 can costimulate CD4+ T-cell responses in vitro [86,87], these results suggest that CD154 transduces key intracellular signals to 4.1-like CD4+ T cells, and that activation of DCs in the absence of these signals leads to abortive immune responses. Another important consideration is that, since the development and regulatory activity of CD4+CD25+ T cells in NOD mice is independent of CD154 (Santamaria P, unpublished data), CD154 blockade interferes with the diabetogenic process both by blocking CD4+ T cell help and by allowing CD4+CD25+ T-cell mediated suppression.
7. Progression of islet inflammation to overt diabetes: T-cell avidity maturation NOD mice begin to develop insulitis at 3 weeks, but do not begin to develop T1D until at least 9 weeks later. Since many of the CD8+ CTLs derived from islets of diabetic NOD mice express V17-MRD/E-J42 rearrangements and recognize NRP-A7, we hypothesized that progression of insulitis to diabetes might be driven by the accumulation of NRP/NRP-A7-reactive CTL in islets. Studies of the fate of the NRP/NRP-A7-reactive T-cell subpopulation in non-transgenic NOD mice using peptide/MHC tetramers revealed that progression of islet inflammation to overt diabetes in the NOD mouse is driven, at least in part, by “avidity maturation” of CD8+ T cells, including NRP-A7-reactive CTL [66]. As prediabetic NOD mice age, their islet-associated CD8+ T cells contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/Kd tetramers with increasing avidity. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunted the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by deleting those clonotypes expressing TCRs with the highest affinity and lowest dissociation rates for peptide/MHC binding, and by expanding clonotypes expressing low affinity TCRs. This inhibited the production of CTL and halted the progression of insulitis to diabetes. These observations led to the conclusion that avidity maturation of pathogenic T-cell populations is a key event in the progression of benign inflammation to overt disease in autoimmunity. We suspect that this process also occurs with other autoreactive T-cell specificities, but this remains to be determined. Recent studies have uncovered a molecular basis for avidity maturation of the NRP-A7-reactive CD8+ T-cell subpopulation: clonal competition for MHC (Han et al., manuscript in preparation). When the
developmental fates of TCR-transgenic CD8+ T cells expressing high and low affinity NRP-A7/NRP-V7reactive TCRs were compared, we also discovered that whereas central and peripheral tolerance selectively decrease the size of the high-avidity T-cell pool, autoimmune inflammation fuels its expansion (Han et al., unpublished observations). On the basis of these observations, we have proposed that diabetogenesis is initiated by low avidity T-cell clonotypes, and executed by the few high avidity clonotypes that manage to evade mechanisms of central and peripheral tolerance by localizing into pancreatic islets. 8. CD4+ and CD8+ T cells as beta cell killers There is now ample evidence indicating that CD8+ CTL function as major effectors of beta cell lysis in T1D, along with CD4+ T cells. CD8+ CTL are consistently present in islets of NOD mice [29,49,54–60], can transfer diabetes into NOD.scid mice [49,54], and can kill beta cells of T1D-resistant mice in vivo [88]. Our study of NOD mice expressing the 8.3-TCR transgene provided in vivo evidence for a contribution of CD8+ CTL to beta cell loss in spontaneous T1D: these mice have a minor (but selective) increase in the frequency of beta cell-reactive CD8+ T cells and develop accelerated T1D, owing to accelerated recruitment of CD8+ (but not CD4+) T cells into islets [29]. Although 8.3-CD8+ CTL kill beta cells via Fas exclusively [89], studies of perforindeficient NOD mice have shown that CD8+ CTL clonotypes recruited to islets later on in the disease process need to express perforin to kill beta cells [90]. T-cell transfer studies using beta cell-specific CD4+ T-cell clones have shown that some clonotypes can also kill beta cells in vivo [45]. In agreement with this, TCR-transgenic NOD mice expressing the I-Ag7restricted BDC2.5- or 4.1-TCRs develop diabetes in the absence of other T cells [30,91]. Whereas BDC2.5-CD4+ T cells kill target beta cells through a TNF-receptordependent pathway [92], 4.1-CD4+ CTL kill beta cells via Fas [93]. It is important to point out here that the fact that T1D in “monoclonal” T-cell NOD mice bypasses the need for CD4+ or CD8+ T cells does not imply that beta cell damage in T1D is exclusively effected by CD8+ or CD4+ T-cell subsets, respectively. The high frequency of autoreactive CD4+ or CD8+ T cells in these TCR-transgenic animals likely overwhelms the mechanisms that, in non-transgenic mice, would prevent these cells from reaching an insulitogenic mass upon activation. 9. The MHC class II-associated resistance to T1D TCR-transgenic mice are also excellent tools with which to probe the mechanisms through which antidiabetogenic genes, such as certain MHC class II alleles,
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afford diabetes resistance. Genetic susceptibility and resistance to most autoimmune disorders, including T1D, are associated with the MHC and, to a lesser extent, with polygenic modifiers on other chromosomes. In humans, the MHC-linked T1D susceptibility and resistance are primarily associated with the HLA-DQB1 locus. Alleles encoding DQ chains with Ser, Ala or Val at position 57 provide risk, whereas those encoding DQ chains with Asp at this position provide different degrees of protection [1,62]. In mice, T1D susceptibility and resistance are also linked to the MHC. The NOD mouse is homozygous for a unique H-2 haplotype (H-2g7). This haplotype carries a non-productive I-E gene and encodes an I-Ad/I-Ag7 heterodimer in which the His and Asp found at positions 56 and 57 in most I-A chains are replaced by Pro and Ser, respectively [94,95]. Studies of congenic NOD mice expressing non-NOD MHC haplotypes, and of NOD mice expressing I-Ed, I-Ek, modified I-Ag7, I-Ak/I-Ak or I-Ad transgenes have proven that class II molecules play a direct role in providing susceptibility or resistance to T1D [96–108]. It is also known that the factors responsible for the MHClinked T1D susceptibility and resistance reside in the marrow [107,109–114]. Since MHC molecules play a pivotal role in T-cell development [88–99], some authors hypothesized that protective MHC molecules provided T1D resistance by tolerizing autoreactive T cells [98,101,115]. As studies in MHC-congenic/TCRtransgenic NOD mice did not find evidence of T-cell tolerance (the mice were resistant to autoimmune disease despite exporting autoreactive T cells to the periphery), it was later proposed that the MHC-induced resistance to T1D is mediated by immunoregulation [28,100,103,106,108,109]. This postulate, however, assumed that mechanisms of tolerance target all autoreactive T cells, regardless of pathogenicity. Our studies with 4.1-NOD mice have revealed the existence of a relationship between deletion of certain highly diabetogenic CD4+ T-cells and the MHC-linked resistance to T1D [8]. We found, quite serendipitously, that the 4.1-TCR undergoes central deletion in T1Dresistant H-2g7/b, H-2g7/k, H-2g7/q and H-2g7/nb1 NOD mice, by engaging anti-diabetogenic MHC class II molecules on bone marrow-derived APCs, independently of endogenous superantigens. We also discovered that, unlike I-Ag7 molecules, deleting class II molecules (i.e. I-Ab) could restrict neither the positive selection of 4.1-thymocytes nor the presentation of their target autoantigen [116]. We also found that deletion of 4.1-CD4+ thymocytes by I-Ab was a peptide-specific process, since it could only be triggered by hematopoietic cells expressing heterogeneous (as opposed to single) peptide/I-Ab complexes [116]. These studies led us to hypothesize that this form of MHC-induced protection from diabetes is based on the presentation of a specific peptide to a putative group of 4.1-like, MHC
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promiscuous, beta cell-reactive CD4+ T-cells that play a critical role in the initiation of T1D. This hypothesis predicted that the ability of certain protective MHC class II molecules to delete 4.1thymocytes should be shared by other (even structurally dissimilar) class II molecules with anti-diabetogenic potential in non-TCR-transgenic NOD mice. We have tested this recently, by investigating the mechanism of diabetes protection afforded by three additional class II molecules: I-Ek/I-Eg7, I-Ad and I-Ag7PD [117]. I-Ek or I-Ad molecules tolerized 4.1-CD4+ thymocytes in MHCtransgenic 4.1-NOD mice and protected the mice from insulitis and T1D. Expression of the I-Ag7PD transgene (mutated I-Ag7 encoding Pro and Asp at positions 56 and 57) in 4.1-NOD mice did not cause 4.1-thymocyte deletion but rendered 4.1-CD4+ T-cells partially unresponsive to antigen. Hence, when taken together, the available data are compatible with our hypothesis that protective MHC class II alleles afford T1D resistance (in non-TCR-transgenic NOD mice) by tolerizing a group of 4.1-like, highly pathogenic, MHC-promiscuous CD4+ T-cells that play a critical role in diabetogenesis. 4.1-CD4+ T-cells would be representative of this T-cell subpopulation. This hypothesis is consistent with the observation that the tolerogenic ability of class II molecules maps to residues around I-A and I-E chain position 57 [117], which are clearly implicated in the DQB1-linked susceptibility and resistance to diabetes in humans [1,62]. It is important to note, however, that this hypothesis is not incompatible with the alternative view that protective MHC class II molecules afford diabetes resistance by selecting regulatory T cells.
10. Immunoregulation Not all the islet-autoreactive T cells that escape central tolerance in NOD mice differentiate into diabetogenic T cells. For example, transgenic NOD mice expressing a TCR specific for a Glutamic Acid Decarboxylase-derived peptide (G286-NOD) contained significant numbers of peptide/I-Ag7 tetramer positive cells, yet the mice did not develop insulitis or diabetes [32]. In fact, the TCR-transgenic T cells of these mice displayed anti-diabetogenic activity in adoptive T-cell transfer experiments, suggesting that some autoreactive T cells acquire immunoregulatory properties during development. BDC2.5 TCR-transgenic NOD mice provide another model with which to study regulatory T-cell function in vivo. Although BDC2.5-NOD mice develop massive insulitis from a very young age [72,118], their intra-islet BDC2.5 CD4+ T cells do not differentiate into diabetogenic T cells. As a result, these mice develop a very low incidence of diabetes [91]. Four lines of evidence suggest that the diabetes resistance of these mice is
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mediated by recruitment of NKT cells. First, in vitroactivated BDC2.5 CD4+ T cells readily transfer diabetes into NOD.scid recipients, which lack both B- and T cells [91]. Second, expression of the BDC2.5-TCR in TCRC- or RAG deficient and NOD.scid backgrounds (all T-cell deficient), result in a very accelerated form of diabetes [89,118]. Third, introduction of a CD1d deficiency into these mice abrogates their diabetes resistance [119]. And fourth, NOD splenic DX5+ NKT cells actively suppress diabetes development in young BDC2.5-NOD.RAG-2/ hosts [118]. These results, albeit compelling, are somewhat paradoxical because development of NKT cells in NOD mice is known to be defective [120–122]. They raise the possibility that expression of the transgenic TCR somehow rescues NKT cell development and/or function.
11. Concluding remarks T1D results from a T-cell dependent autoimmune process directed against the pancreatic beta cells that develop in genetically susceptible individuals. The effector mechanisms of beta cell destruction in T1D and the mechanisms underlying the genetic susceptibility/ resistance to this disease, which are under complex polygenic control, are issues of fundamental importance that remain poorly understood. During the last decade, and with these questions in mind, we and others have generated transgenic mouse strains expressing the TCR gene rearrangements of beta cell-specific CD4+ and CD8+ T-cell clones isolated from NOD mice. These mice, expressing TCRs that target disease-relevant autoantigens (that is, targeted during spontaneous disease in wild-type NOD mice) represent very useful tools with which to probe, from a reductionist point of view, the development and function of autoreactive T-lymphocytes in diabetes-prone and diabetes-resistant genetic backgrounds. Studies of these different TCR transgenic animals have helped uncover new immunological paradigms of relevance not only to diabetes but also other autoimmune diseases alike.
Acknowledgements We thank all the members of our laboratories for technical assistance and scientific discussions, and Ms Valerie Crosbie for editorial assistance. We apologize to authors whose important work was not cited in this review owing to space limitations. This paper was not meant to be an in-depth review of all the work done in this area, but rather a forum to present the authors’ views on the diabetogenic process. This work was supported by the Diabetes Foothills Association and Julia McFarlane Diabetes Research Centre Funds (to
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Forum on Transgenic autoimmune mice
T-cell receptor-transgenic NOD mice: a reductionist approach to understand autoimmune diabetes Yang Yang a,c, Pere Santamaria b,c* a
Department of Biochemistry & Molecular Biology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada b Department of Microbiology and Infectious Diseases, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada c Julia McFarlane Diabetes Research Centre, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada
1. Introduction Insulin-dependent diabetes mellitus (Type 1 Diabetes, T1D) in humans and nonobese diabetic (NOD) mice is the result of a chronic autoimmune process directed against the pancreatic beta cells [1,2]. There is extensive evidence in both humans and mice indicating that T1D is a T-cell mediated disease. Macrophages, dendritic cells (DCs) and B-cells also play an important role in the disease process, most likely as professional antigenpresenting cells (APC) responsible for driving the activation and recruitment of autoreactive T cells [1–6]. It is believed that development of diabetogenic T cells in diabetes-prone genetic backgrounds requires the presence of certain disease-associated Major Histocompatibility Complex (MHC) genes, and that diabetes susceptibility results in a breakdown of T-cell tolerance to beta cell autoantigens [1,7–10]. However, whether T1D resistance in non-diabetes prone individuals is the result of T-cell tolerance or of ignorance of beta cells by autoreactive T cells remains unclear. Studies of T-cell development in mice expressing transgenic neo-antigens in beta cells and transgenic neo-antigen-specific T-cell receptors (TCRs) resulted in different outcomes, possibly due to differences in the nature, amount and timing of transgene expression, and/or to genetic differences among the mice that were studied [11–27]. These pioneering works provided seminal information towards our understanding of the mechanisms that control the development of neo-autoantigen-specific T lymphocytes in
* Corresponding author. Tel.: +1-403-220-8735; fax: +1-403-270-8520 E-mail address: [email protected] (P. Santamaria). 0896-8411/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2003.10.003
genetic backgrounds that are naturally resistant to diabetes. However, since neither the antigens nor the TCRs that were studied in these models are involved in spontaneous T1D, it is difficult to determine which, if any, of the outcomes that were observed in these models is T1D-relevant. This is not a trivial issue, as the fate of autoreactive T cells in vivo is clearly a function of the nature, levels and expression anatomy of naturally occurring autoantigens. During the last decade or so, a number of transgenic mice expressing TCRs targeting naturally occurring, non-transgenic beta cell autoantigens have become available [8,28–33]. These mice are powerful tools with which to address some of the limitations of previous studies, and have enabled detailed investigations of the mechanisms that control the development, regulation, activation, differentiation and effector function of diabetogenic T cells, in both T1D-prone and T1D-resistant genetic backgrounds. Some of these models are ideal to study how anti-diabetogenic genes afford diabetes resistance, or to uncover and test new avenues for therapeutic intervention. Other models afford the opportunity to study immunoregulatory processes capable of keeping autoreactive T cells in check. This reductionist approach to diabetes research affords the opportunity to discover some of the not-so-obvious pieces that make the “big puzzle”, and allows a stepby-step reconstruction of a very complex process that, in our view, is very difficult to dissect otherwise. This review summarizes some of the lessons learned through studies of these mice as seen through the authors’ eyes. Other reviews in this series will cover equally important aspects that, for space limitations, are not dealt with in detail here.
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2. CD4+ versus CD8+ T cells in the initiation phases of T1D Any discussion on the usefulness of TCR-transgenic models to study autoimmunity warrants a summary of what has been learned in previous models. T-cell transfer studies have shown that diabetogenesis requires both CD4+ and CD8+ T cells [34–45]. Since beta cells do not express major histocompatibility complex (MHC) class II molecules [46,47], autoreactive CD4+ T cells must engage beta cell antigens (shed by a prior insult) on local antigen-presenting cells (APCs) [48,49]. Studies of CD8+ T-cell deficient NOD mice have suggested that the initial insult that sheds beta cell antigens is mediated by CD8+ cytotoxic T lymphocytes (CTL) [50–53], which invariably infiltrate NOD islets [29,49,54–60]. This hypothesis, however, is in conflict with observations that support the view that T1D is initiated by CD4+ T cells. For example, splenic CD4+ T cells from NOD mice can transfer islet inflammation (insulitis) into NOD.scid mice, but splenic CD8+ T cells cannot [34,43]. Furthermore, beta cells do not express costimulatory molecules [61] and thus naı¨ve autoreactive CD8+ T cells are unlikely to differentiate into CTL unless their target antigens are shed from beta cells by a prior insult, and later presented to them by professional APCs. Finally, genetic susceptibility and resistance to T1D are profoundly affected by polymorphisms of MHC class II genes [1,62], which control the development and function of CD4+, but not CD8+ T cells. As described at some length below, studies in TCR-transgenic NOD mice have provided valuable insights towards resolution of these apparently paradoxical observations.
3. Autoreactive CD8+ T cells in T1D: fishing antigenic ligands with TCR-transgenic T-cell probes The antigenic specificity(ies) of the CD8+ T cells that are involved in the early stages of the diabetogenic process is (are) unknown. Nevertheless, several lines of evidence suggested that the antigenic repertoire of these T cells is quite restricted. For example, a large fraction of the CD8+ T cells that can be isolated from islets of diabetic NOD mice are cytotoxic to beta cells in the context of the class I molecule Kd, and use TCR chains with homologous CDR3 sequences [58]. Furthermore, the majority of the islet-associated CD8+ T cells of transgenic NOD mice expressing the TCR chain of the CD8+ clone NY8.3 (which uses a representative CDR3 sequence) express an endogenously derived TCR chain that is identical to the one employed by the clonotype donating the TCR transgene [30]. Strikingly, a significant percentage of the CD8+ T cells that can be propagated from the earliest insulitic lesions of NOD mice use TCR chains that are identical or nearly identical to the
one used by 8.3-CD8+ T cells (V17 and J42 elements joined by the N-region sequence MRD/E) [63]. This TCR heterodimer is highly pathogenic, as demonstrated by the fact that 8.3-TCR-transgenic NOD mice (8.3-NOD) develop overt diabetes shortly after the onset of insulitis [30]. Availability of 8.3-NOD mice afforded a unique opportunity to search for antigenic ligands of early insulitic T cells. CTL derived from islets of NOD mice are notoriously unstable: they either do not survive repeated antigenic stimulation in vitro, or lose cytotoxic activity within days or weeks in culture [58]. We now know that this is in part due to the fact that repeated antigenic stimulation of TCR-transgenic 8.3-CTL with antigen-pulsed DCs induces transient re-expression of the recombination-activating genes (RAGs) and TCR revision, leading to loss in antigenic reactivity [64]. Screens of combinatorial peptide libraries with 8.3-CTL (freshly derived in vitro from naı¨ve 8.3-CD8+ splenocytes) led to the identification of two major peptide ligands for CTL expressing the prevalent V17-MRDJ42 TCR chain: NRP and NRP-A7, an alanine mutant analog of NRP with superior agonistic properties [65]. Additional experimentation confirmed what earlier TCR repertoire studies had suggested, namely that these two peptides are recognized by a large fraction of the islet-associated CD8+ T cells from wild-type NOD mice [65,66]. Hypermutation of the NRP sequence eventually led to the identification of a higher affinity ligand of the 8.3-TCR, as compared to NRP or NRP-A7 (NRP-V7) [67]. The availability of this mimotope was key to the discovery that progression of insulitis to overt diabetes in NOD mice can be predicted, with high degrees of specificity and sensitivity, by measuring the cumulative percentage of peripheral blood CD8+ T cells that bind NRP-V7/Kd tetramers [68]. More recently, Lieberman et al. have discovered the identity of the naturally occurring ligand of NRP-A7/V7-reactive CD8+ T cells, a beta cell-specific protein of unknown function [123]. We would like to emphasize here that these exciting results should not be taken to imply that the CD8+ T-cell response in T1D is exclusively directed against NRP-like peptides. Wong et al., for example, have reported that insulitic CD8+ T cells in young NOD mice recognize an insulin-derived peptide [69]. Although in our hands these cells only represent a very small fraction of all insulitic CD8+ T cells, even in young animals [66,68], they may still play an important role in the disease process.
4. CD4+ T-cell assisted recruitment of NRP-A7/NRP-V7-reactive CD8+ T cells in T1D We do not yet know for sure whether NRP-A7reactive CD8+ T cells are required for the initiation of
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diabetes. However, there is evidence suggesting that they are not sufficient. Studies of RAG-2-deficient 8.3-NOD mice, for example, support the view that efficient accumulation of 8.3-CD8+ pre-CTL into islets requires CD4+ T cell help [30,70]. The insulitogenic and diabetogenic activities of 8.3-CD8+ T cells are significantly enhanced by the presence of endogenous, non-transgenic CD4+ T cells [30,70], such that adoptive transfer of splenic CD4+ T cells from wild-type, non-diabetic NOD mice into RAG-2-deficient 8.3-NOD hosts results in a significantly increased incidence of diabetes [30,70]. In this model, CD4+ T cells potentiate the recruitment and/or accumulation of 8.3-CD8+ T cells in islets, rather than their differentiation into CTL [30]. These results do not necessarily imply that recruitment of all beta cell autoreactive CD8+ T-cell specificities in T1D is T-helper dependent. CD8+ T cells expressing the AI4-TCR, which is not NRP-A7/NRPV7-reactive (D. Serreze and T. DiLorenzo, personal communication), are as diabetogenic in the absence of endogenous CD4+ T cells as they are in their presence [31]. The factors that determine the T-helper dependency of a given clonotype are not known, but the affinity of its TCR for peptide/MHC is an attractive candidate. This hypothesis, currently under investigation in our laboratory, predicts that clonotypes recognizing peptide/MHC with high affinity will trigger diabetes in a T-helper cell-independent manner. It would be therefore reasonable to suspect that initiation of diabetes is triggered by high avidity CD8+ clonotypes. None the less, since the initial CD8+ T-cell response in T1D involves predominantly low avidity clonotypes [66], we favor the idea that diabetes initiation involves both CD4+ and CD8+ T cells acting in concert.
5. The anatomy of T-cell priming in T1D It has been recently shown that the pancreatic lymph nodes (PLN) are absolutely essential for diabetogenesis [71]. Experiments employing carboxyfluorescein diacetate succinidimyl ester (CFSE)-labeled T cells expressing MHC class I (Kd)- or class II-restricted (I-Ag7), beta cell autoreactive TCRs (8.3-CD8+; and BDC2.5 or 4.1-CD4+, respectively) have shown that beta cellreactive T cells first encounter cognate peptide/MHC complexes in the PLNs, on the surface of autoantigenloaded, bone marrow-derived APCs [70,72,73]. These experiments have also shown that priming does not occur in neonatal mice, suggesting that beta cell autoantigens do not become available until later on in life [72,73]. In the case of 8.3-CD8+ T cells, for example, priming in the PLNs is undetectable before 3 weeks, and its magnitude increases progressively with age. An increased incidence of beta cell apoptosis following the administration of low doses of the -cell toxin
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streptozotocin augmented the magnitude of 8.3-CD8+ T-cell priming and allowed it to occur at 2 weeks of age, when it is not observed to occur spontaneously. These results strongly implicate in situ -cell death in the facilitation of autoantigen specific CD8+ T-cell priming in T1D. Interestingly, the timing of 8.3-CD8+ T-cell priming in NOD mice is preceded by a period of remodeling and physiologic apoptosis of pancreatic islets that peaks at 2 weeks of age [74,75]. Regardless of whether this “wave” of apoptosis is a key event in T1D, these results demonstrate that in situ -cell death induced by CTLs can perpetuate the priming of their naı¨ve precursors in the PLNs.
6. T–T collaboration: a role for CD154 in the activation of autoreactive CD4+ T cells and in the T-helper assisted differentiation of CD8+ CTL Productive collaboration between CD4+ T-helper cells and pre-cytotoxic CD8+ T cells requires the presentation of different epitopes by the same APC, usually a DC, in a CD40/CD154-dependent manner [76–83]. CD40 ligation on DCs induces the upregulation of T-cell costimulatory molecules, elicits the production of proinflammatory cytokines, and endows DCs with the ability to differentiate CD8+ T cells. Accordingly, blockade of the CD40–CD154 pathway completely prevents the development of insulitis and diabetes in NOD mice [84,85]. Experiments in TCR-transgenic NOD mice have shown that, whereas disruption of CD154 completely prevents the development of insulitis and diabetes in monoclonal 4.1-TCR-transgenic NOD mice, which express a highly diabetogenic, I-Ag7-restricted TCR, it has no effect on the T-helper independent diabetogenic activity of 8.3-CD8+ T cells [70]. CD154/ 8.3-CD8+ T cells proliferated as well as their CD154+ conterparts in the PLNs of wild-type NOD hosts, and the 8.3-CD8+ T cells maturing in both types of mice were equally diabetogenic, indicating that 8.3-CD8+ T cells do not need CD154 to undergo antigen-driven activation in vivo [70]. Notwithstanding this, the ability of T-helper cells to enhance the diabetogenic activity of 8.3-CD8+ T cells (see above) is CD154-dependent, because only CD154+, but not CD154/, CD4+ T cells were able to do this [70]. CD154 here seems to function by ligating CD40 on DCs, as well as by inducing costimulatory activity on DCs. In agreement with this, systemic activation of DCs (and macrophages) with an agonistic anti-CD40 mAb or with CpG DNA dramatically increased the incidence of T1D in monoclonal 8.3-NOD mice. When expressed locally, TNF can also overcome the need for CD154 in another (non-TCR-transgenic) model of CD8+ T-cell induced diabetes [85]. However, this interpretation of the data (the pro-diabetogenic role of CD154 is mediated via DCs),
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cannot account for the observation that CD154/ 4.1-NOD mice remain resistant to both insulitis and diabetes upon systemic activation of their DCs with anti-CD40 mAb or CpG DNA [70]. Accordingly, CD154 does not merely act by ligating CD40 on DCs. Since ligation of CD154 can costimulate CD4+ T-cell responses in vitro [86,87], these results suggest that CD154 transduces key intracellular signals to 4.1-like CD4+ T cells, and that activation of DCs in the absence of these signals leads to abortive immune responses. Another important consideration is that, since the development and regulatory activity of CD4+CD25+ T cells in NOD mice is independent of CD154 (Santamaria P, unpublished data), CD154 blockade interferes with the diabetogenic process both by blocking CD4+ T cell help and by allowing CD4+CD25+ T-cell mediated suppression.
7. Progression of islet inflammation to overt diabetes: T-cell avidity maturation NOD mice begin to develop insulitis at 3 weeks, but do not begin to develop T1D until at least 9 weeks later. Since many of the CD8+ CTLs derived from islets of diabetic NOD mice express V17-MRD/E-J42 rearrangements and recognize NRP-A7, we hypothesized that progression of insulitis to diabetes might be driven by the accumulation of NRP/NRP-A7-reactive CTL in islets. Studies of the fate of the NRP/NRP-A7-reactive T-cell subpopulation in non-transgenic NOD mice using peptide/MHC tetramers revealed that progression of islet inflammation to overt diabetes in the NOD mouse is driven, at least in part, by “avidity maturation” of CD8+ T cells, including NRP-A7-reactive CTL [66]. As prediabetic NOD mice age, their islet-associated CD8+ T cells contain increasing numbers of NRP-A7-reactive cells, and these cells bind NRP-A7/Kd tetramers with increasing avidity. Repeated treatment of pre-diabetic NOD mice with soluble NRP-A7 peptide blunted the avidity maturation of the NRP-A7-reactive CD8+ T-cell population by deleting those clonotypes expressing TCRs with the highest affinity and lowest dissociation rates for peptide/MHC binding, and by expanding clonotypes expressing low affinity TCRs. This inhibited the production of CTL and halted the progression of insulitis to diabetes. These observations led to the conclusion that avidity maturation of pathogenic T-cell populations is a key event in the progression of benign inflammation to overt disease in autoimmunity. We suspect that this process also occurs with other autoreactive T-cell specificities, but this remains to be determined. Recent studies have uncovered a molecular basis for avidity maturation of the NRP-A7-reactive CD8+ T-cell subpopulation: clonal competition for MHC (Han et al., manuscript in preparation). When the
developmental fates of TCR-transgenic CD8+ T cells expressing high and low affinity NRP-A7/NRP-V7reactive TCRs were compared, we also discovered that whereas central and peripheral tolerance selectively decrease the size of the high-avidity T-cell pool, autoimmune inflammation fuels its expansion (Han et al., unpublished observations). On the basis of these observations, we have proposed that diabetogenesis is initiated by low avidity T-cell clonotypes, and executed by the few high avidity clonotypes that manage to evade mechanisms of central and peripheral tolerance by localizing into pancreatic islets. 8. CD4+ and CD8+ T cells as beta cell killers There is now ample evidence indicating that CD8+ CTL function as major effectors of beta cell lysis in T1D, along with CD4+ T cells. CD8+ CTL are consistently present in islets of NOD mice [29,49,54–60], can transfer diabetes into NOD.scid mice [49,54], and can kill beta cells of T1D-resistant mice in vivo [88]. Our study of NOD mice expressing the 8.3-TCR transgene provided in vivo evidence for a contribution of CD8+ CTL to beta cell loss in spontaneous T1D: these mice have a minor (but selective) increase in the frequency of beta cell-reactive CD8+ T cells and develop accelerated T1D, owing to accelerated recruitment of CD8+ (but not CD4+) T cells into islets [29]. Although 8.3-CD8+ CTL kill beta cells via Fas exclusively [89], studies of perforindeficient NOD mice have shown that CD8+ CTL clonotypes recruited to islets later on in the disease process need to express perforin to kill beta cells [90]. T-cell transfer studies using beta cell-specific CD4+ T-cell clones have shown that some clonotypes can also kill beta cells in vivo [45]. In agreement with this, TCR-transgenic NOD mice expressing the I-Ag7restricted BDC2.5- or 4.1-TCRs develop diabetes in the absence of other T cells [30,91]. Whereas BDC2.5-CD4+ T cells kill target beta cells through a TNF-receptordependent pathway [92], 4.1-CD4+ CTL kill beta cells via Fas [93]. It is important to point out here that the fact that T1D in “monoclonal” T-cell NOD mice bypasses the need for CD4+ or CD8+ T cells does not imply that beta cell damage in T1D is exclusively effected by CD8+ or CD4+ T-cell subsets, respectively. The high frequency of autoreactive CD4+ or CD8+ T cells in these TCR-transgenic animals likely overwhelms the mechanisms that, in non-transgenic mice, would prevent these cells from reaching an insulitogenic mass upon activation. 9. The MHC class II-associated resistance to T1D TCR-transgenic mice are also excellent tools with which to probe the mechanisms through which antidiabetogenic genes, such as certain MHC class II alleles,
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afford diabetes resistance. Genetic susceptibility and resistance to most autoimmune disorders, including T1D, are associated with the MHC and, to a lesser extent, with polygenic modifiers on other chromosomes. In humans, the MHC-linked T1D susceptibility and resistance are primarily associated with the HLA-DQB1 locus. Alleles encoding DQ chains with Ser, Ala or Val at position 57 provide risk, whereas those encoding DQ chains with Asp at this position provide different degrees of protection [1,62]. In mice, T1D susceptibility and resistance are also linked to the MHC. The NOD mouse is homozygous for a unique H-2 haplotype (H-2g7). This haplotype carries a non-productive I-E gene and encodes an I-Ad/I-Ag7 heterodimer in which the His and Asp found at positions 56 and 57 in most I-A chains are replaced by Pro and Ser, respectively [94,95]. Studies of congenic NOD mice expressing non-NOD MHC haplotypes, and of NOD mice expressing I-Ed, I-Ek, modified I-Ag7, I-Ak/I-Ak or I-Ad transgenes have proven that class II molecules play a direct role in providing susceptibility or resistance to T1D [96–108]. It is also known that the factors responsible for the MHClinked T1D susceptibility and resistance reside in the marrow [107,109–114]. Since MHC molecules play a pivotal role in T-cell development [88–99], some authors hypothesized that protective MHC molecules provided T1D resistance by tolerizing autoreactive T cells [98,101,115]. As studies in MHC-congenic/TCRtransgenic NOD mice did not find evidence of T-cell tolerance (the mice were resistant to autoimmune disease despite exporting autoreactive T cells to the periphery), it was later proposed that the MHC-induced resistance to T1D is mediated by immunoregulation [28,100,103,106,108,109]. This postulate, however, assumed that mechanisms of tolerance target all autoreactive T cells, regardless of pathogenicity. Our studies with 4.1-NOD mice have revealed the existence of a relationship between deletion of certain highly diabetogenic CD4+ T-cells and the MHC-linked resistance to T1D [8]. We found, quite serendipitously, that the 4.1-TCR undergoes central deletion in T1Dresistant H-2g7/b, H-2g7/k, H-2g7/q and H-2g7/nb1 NOD mice, by engaging anti-diabetogenic MHC class II molecules on bone marrow-derived APCs, independently of endogenous superantigens. We also discovered that, unlike I-Ag7 molecules, deleting class II molecules (i.e. I-Ab) could restrict neither the positive selection of 4.1-thymocytes nor the presentation of their target autoantigen [116]. We also found that deletion of 4.1-CD4+ thymocytes by I-Ab was a peptide-specific process, since it could only be triggered by hematopoietic cells expressing heterogeneous (as opposed to single) peptide/I-Ab complexes [116]. These studies led us to hypothesize that this form of MHC-induced protection from diabetes is based on the presentation of a specific peptide to a putative group of 4.1-like, MHC
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promiscuous, beta cell-reactive CD4+ T-cells that play a critical role in the initiation of T1D. This hypothesis predicted that the ability of certain protective MHC class II molecules to delete 4.1thymocytes should be shared by other (even structurally dissimilar) class II molecules with anti-diabetogenic potential in non-TCR-transgenic NOD mice. We have tested this recently, by investigating the mechanism of diabetes protection afforded by three additional class II molecules: I-Ek/I-Eg7, I-Ad and I-Ag7PD [117]. I-Ek or I-Ad molecules tolerized 4.1-CD4+ thymocytes in MHCtransgenic 4.1-NOD mice and protected the mice from insulitis and T1D. Expression of the I-Ag7PD transgene (mutated I-Ag7 encoding Pro and Asp at positions 56 and 57) in 4.1-NOD mice did not cause 4.1-thymocyte deletion but rendered 4.1-CD4+ T-cells partially unresponsive to antigen. Hence, when taken together, the available data are compatible with our hypothesis that protective MHC class II alleles afford T1D resistance (in non-TCR-transgenic NOD mice) by tolerizing a group of 4.1-like, highly pathogenic, MHC-promiscuous CD4+ T-cells that play a critical role in diabetogenesis. 4.1-CD4+ T-cells would be representative of this T-cell subpopulation. This hypothesis is consistent with the observation that the tolerogenic ability of class II molecules maps to residues around I-A and I-E chain position 57 [117], which are clearly implicated in the DQB1-linked susceptibility and resistance to diabetes in humans [1,62]. It is important to note, however, that this hypothesis is not incompatible with the alternative view that protective MHC class II molecules afford diabetes resistance by selecting regulatory T cells.
10. Immunoregulation Not all the islet-autoreactive T cells that escape central tolerance in NOD mice differentiate into diabetogenic T cells. For example, transgenic NOD mice expressing a TCR specific for a Glutamic Acid Decarboxylase-derived peptide (G286-NOD) contained significant numbers of peptide/I-Ag7 tetramer positive cells, yet the mice did not develop insulitis or diabetes [32]. In fact, the TCR-transgenic T cells of these mice displayed anti-diabetogenic activity in adoptive T-cell transfer experiments, suggesting that some autoreactive T cells acquire immunoregulatory properties during development. BDC2.5 TCR-transgenic NOD mice provide another model with which to study regulatory T-cell function in vivo. Although BDC2.5-NOD mice develop massive insulitis from a very young age [72,118], their intra-islet BDC2.5 CD4+ T cells do not differentiate into diabetogenic T cells. As a result, these mice develop a very low incidence of diabetes [91]. Four lines of evidence suggest that the diabetes resistance of these mice is
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mediated by recruitment of NKT cells. First, in vitroactivated BDC2.5 CD4+ T cells readily transfer diabetes into NOD.scid recipients, which lack both B- and T cells [91]. Second, expression of the BDC2.5-TCR in TCRC- or RAG deficient and NOD.scid backgrounds (all T-cell deficient), result in a very accelerated form of diabetes [89,118]. Third, introduction of a CD1d deficiency into these mice abrogates their diabetes resistance [119]. And fourth, NOD splenic DX5+ NKT cells actively suppress diabetes development in young BDC2.5-NOD.RAG-2/ hosts [118]. These results, albeit compelling, are somewhat paradoxical because development of NKT cells in NOD mice is known to be defective [120–122]. They raise the possibility that expression of the transgenic TCR somehow rescues NKT cell development and/or function.
11. Concluding remarks T1D results from a T-cell dependent autoimmune process directed against the pancreatic beta cells that develop in genetically susceptible individuals. The effector mechanisms of beta cell destruction in T1D and the mechanisms underlying the genetic susceptibility/ resistance to this disease, which are under complex polygenic control, are issues of fundamental importance that remain poorly understood. During the last decade, and with these questions in mind, we and others have generated transgenic mouse strains expressing the TCR gene rearrangements of beta cell-specific CD4+ and CD8+ T-cell clones isolated from NOD mice. These mice, expressing TCRs that target disease-relevant autoantigens (that is, targeted during spontaneous disease in wild-type NOD mice) represent very useful tools with which to probe, from a reductionist point of view, the development and function of autoreactive T-lymphocytes in diabetes-prone and diabetes-resistant genetic backgrounds. Studies of these different TCR transgenic animals have helped uncover new immunological paradigms of relevance not only to diabetes but also other autoimmune diseases alike.
Acknowledgements We thank all the members of our laboratories for technical assistance and scientific discussions, and Ms Valerie Crosbie for editorial assistance. We apologize to authors whose important work was not cited in this review owing to space limitations. This paper was not meant to be an in-depth review of all the work done in this area, but rather a forum to present the authors’ views on the diabetogenic process. This work was supported by the Diabetes Foothills Association and Julia McFarlane Diabetes Research Centre Funds (to
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