Autoimmune diabetes in HLA-DR3/DQ8 transgenic mice expressing the co-stimulatory molecule B7-1 in the ß cells of islets of Langerhans
Govindarajan Rajagopalan1,
Yogish C. Kudva2,
Lieping Chen1,
Li Wen3 and
Chella S. David1
1 Department of Immunology and 2 Division of Endocrinology, Mayo Clinic, Rochester, MN 55905, USA 3 Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
Correspondence to: C. S. David; E-mail: davic4{at}mayo.edu
Transmitting editor: C. Terhorst
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Abstract
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The major predisposing genetic component in type 1 diabetes (T1D) maps to the MHC locus in both mice and humans. To better understand the HLA class II association with disease pathogenesis, we bred mice expressing HLA-DQ8 and -DR3, either alone or in combination, to transgenic mice expressing the co-stimulatory molecule B7-1 in the ß cells of islets of Langerhans. Spontaneous diabetes occurred only in RIP-B7-1 transgenic mice expressing transgenic HLA-DR3 or -DQ8 molecules and the incidence of diabetes was comparable between the two (
30% in either sex up to 50 weeks of age). Presence of DR3 and DQ8 together only marginally elevated the overall incidence of spontaneous disease (38%). Non-specific activation of T cells by superantigen and provision of concomitant co-stimulation through 4-1BB (CD137) by an agonistic antibody did not accelerate the incidence of diabetes over a short period of time. Neither the antibody-mediated depletion of CD25+ T cells nor sublethal, whole-body irradiation of young, naive HLA transgenic mice expressing RIP-B7-1 resulted in diabetes. However, administration of only two doses of the ß cell toxin streptozotocin (STZ; 40 mg/kg) induced autoimmune diabetes in 85% of mice within 7 weeks after STZ treatment only when B7-1 was expressed on the pancreatic ß cells. This effect was HLA dependent as none of the STZ-treated RIP-B7-1 transgenic mice lacking HLA class II developed diabetes. In conclusion, this study confirmed the diabetogenic potential of HLA-DQ8 and established the role of HLA-DR3 in the pathogenesis of T1D.
Keywords: autoimmunity, diabetes, HLA-DQ8, HLA-DR3, transgenic/knockout
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Introduction
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Genetic studies carried out in humans suggest a polygenic causation of type 1 diabetes (T1D) in different pedigrees (1,2). The major predisposing region maps to the segment of the genome that encodes the MHC in humans (3,4). Even though several genes contained within this locus have been shown to be variably associated with T1D (57), the genes encoding the MHC class II molecules are thought to play a major role. Patients with T1D express certain HLA class II haplotypes such as DQ2/DR3 and DQ8/DR4 more frequently than the control population (3,8). Due to the tight linkage disequilibrium between certain DQ and DR molecules (9), elucidating the exact pathogenic role played by individual DQ or DR molecules in the pathogenesis of T1D as well as in other human autoimmune diseases has been very difficult (10,11).
The availability of transgenic mice expressing human class II molecules of interest either alone or in combination has helped in better understanding the HLA class II association with human autoimmune diseases (12). With respect to T1D, the epitopes on the putative autoantigens presented by the T1D-associated class II molecules were effectively identified only after the availability of HLA transgenic mice (13,14). Interestingly, transgenic expression of T1D-associated class II molecules per se resulted in breakdown of tolerance to certain islet autoantigens in these mice without progression to diabetes (15,16). Provision of local co-stimulation by transgenic expression of B7-1 (17) or the pro-inflammatory cytokine tumor necrosis factor-
(18) in the pancreatic ß cells under the control of the rat insulin promoter (RIP) resulted in spontaneous diabetes in HLA-DQ8 (DQB1*0302) transgenic mice. At the same time, RIP-B7-1 mice transgenic for HLA-DQ6 (DQB1*0601, the allele which is neutral with respect to incidence of spontaneous diabetes in humans) were diabetes-free (17). Interestingly, RIP-B7-1 transgenic mice expressing HLA-DR4 (DRB1*0401, another HLA class II molecule associated with predisposition to T1D in humans) had a low incidence of spontaneous diabetes when compared to HLA-DQ8.RIP-B7-1 mice. The incidence of diabetes in RIP-B7-1 mice expressing both DR4 and DQ8 was reduced to the levels seen in HLA-DR4 transgenic mice, indicating that DR4 might play a regulatory role (19). Thus, expression of different HLA class II molecules seems to have a differential effect on the pathogenesis of T1D. However, it is not known if the related class II molecule HLA-DR3 (DRB1*0301, which is also strongly associated with predisposition to T1D in humans) would have a regulatory role similar to that of DR4. The present study addresses the diabetogenic potential of HLA-DR3 on its own and also evaluates the modulatory effect of HLA-DR3 on the HLA-DQ8-dependent diabetes process using RIP-B7-1 transgenic mice. Using this model, we have also validated the in vivo effects of some of the potential immunological phenomena that have been implicated in the pathogenesis of T1D.
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Methods
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Generation of HLA transgenic RIP-B7-1 mice
C57BL/6 mice expressing the co-stimulatory molecule B7-1 in the ß cells of islets of Langerhans under the control of RIP have been described earlier (17,19). Aß0.DQ8 and Aß0.DR3 single-transgenic, and Aß0.DQ8.DR3 double-transgenic mice lacking any endogenous class II molecules have been described elsewhere (20). B6.RIP-B7-1 mice were mated with the above-mentioned HLA class II transgenic mice and the offspring were selectively bred to generate HLA transgenic mice expressing RIP-B7-1, but lacking endogenous class II molecules. Segregation of genes independently of each other results in offspring with several different genotypes (Table 1). The presence or absence of appropriate class II molecules was determined by PCR and flow cytometry as described elsewhere (20). The presence of RIP-B7-1 was confirmed by PCR. Mice were bred and maintained at the Immunogenetics mouse colony at Mayo Clinic (Rochester, MN).
Antibodies and flow cytometry
The following antibodies were used. The agonistic rat anti-mouse 4-1BB mAb 2A has been described elsewhere (21). Hybridomas producing anti-TCR Vß8.1, 2, 3 (F23.1), anti-TCR Vß7, anti-TCR Vß6 and anti-TCR Vß5 were obtained from ATCC (Rockville, MD). FITC-conjugated secondary antibodies were from Accurate Chemicals and Scientific Corporation (Westbury, NY). Anti-CD4, anti-CD8 and FITC-conjugated CD25 (7D4) were from PharMingen (San Diego, CA). Cytofluorimetric analysis was carried out using CellQuest software version 3.3 (Becton Dickinson, Mountain View, CA). The hybridoma-producing rat anti-mouse CD25 antibody (PC61) was procured from ATCC. Cells were grown in roller bottles in the Mayo Monoclonal Antibody Core Facility (Mayo Clinic, Rochester, MN) and the antibody from the supernatant was affinity-purified using Protein G columns.
In vivo depletion of CD25+ T cells
Normoglycemic Aß0.DQ8.RIP-B7-1 mice (6 weeks old) received one or two injections of 0.5 mg of the purified antibody per injection i.p. once in every 3 days. Spleens were collected 3 days after the last injection and analyzed for CD25+ T cells by flow cytometry. For the diabetes study, Aß0.DQ8.RIP-B7-1 mice (6 weeks old) received six injections of the purified anti-CD25 antibody once every 3 days at the rate of 0.5 mg/dose and their glycemic status was monitored weekly as described below.
Diabetes monitoring
Mice (46 weeks old) were moved from the barrier facility to the conventional facility after weaning and their glycemic status was monitored weekly using a glucometer (Bayer, Pittsburgh, PA). Diabetes was diagnosed when two consecutive random blood glucose levels were >13.9 mmol/l.
Intraperitoneal glucose tolerance test (IPGTT)
To identify impaired glucose tolerance, mice were fasted overnight for at least 14 h and were injected with glucose at a dose of 2 g/kg body wt, i.p. Venous plasma glucose was checked before, and 15, 30, 60 and 120 min after the injection (20).
Superantigen administration and treatment with agonistic anti-4-1BB antibody
Normoglycemic HLA-DQ8.RIP-B7-1 transgenic mice (8 weeks old) were injected i.p. with 10 µg of the bacterial superantigen staphylococcal enterotoxin B (SEB; Sigma, St Louis, MO) in 200 µl PBS. In some experiments, mice simultaneously received 100 µg of either the agonistic anti-4-1BB antibody or the control antibody (rat IgG; Sigma). Blood glucose levels were monitored every other day, and mice were sacrificed at 3 and 7 days post-SEB challenge to analyze T cell expansion.
Sublethal whole-body irradiation
Normoglycemic HLA-DR3/DQ8.RIP-B7-1 mice (6 weeks old) of either sex were
-irradiated from a 137Cs source at the dose of 600 rad/mouse and their glycemic status was monitored as described above.
Low-dose streptozotocin (STZ)-induced diabetes
Non-diabetic mice (812 weeks old) were injected with freshly prepared STZ dissolved in citrate buffer (pH 4.2) at the rate of 40 mg/kg, i.p., for 2 consecutive days. Glycemic status was monitored weekly as described and diabetes was diagnosed according to the criteria mentioned above. Mice were followed up to 7 weeks following STZ challenge.
Histopathology and immunohistochemistry of pancreas
Pancreata from euglycemic and hyperglycemic mice were collected at different time points in buffered formalin (Sigma). Thin sections were stained with hematoxylin & eosin and microscopically evaluated for insulitis. For immunofluorescence staining, pancreata were embedded in OCT compound (Tissue Tek, Sakura FineTek, CA), immersed in chilled isopentane and frozen immediately in liquid nitrogen. Thin sections made from frozen tissue were first air-dried, fixed in cold acetone and subsequently treated with 1% paraformaldehyde. Sections were subsequently incubated with rat anti-mouse primary antibodies CD4 (GK1.5), CD8 (Lyt 2.2), CD19 (PharMingen) and CD11b (PharMingen). A rat isotype control was also included. In the next step, sections were incubated with goat anti-rat secondary antibody (Jackson Immuno Research, West Grove, PA) followed by rabbit anti-goat IgGFITC conjugate (ICN, Costa Mesa, CA). Sections were examined under a fluorescent microscope.
Statistical analysis
The statistical analyses were performed using SAS software (version 4.0.4; SAS Institute, Cary, NC).
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Results
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Islet-specific expression of B7-1 does not affect lymphocyte development
As T1D is an autoimmune disease, interference with T and B cell development can significantly affect incidence/course of the disease. Therefore, single- and double-transgenic DR3 and DQ8 mice expressing RIP-B7-1 were evaluated for the distribution of mature T and B cells in the periphery, and also for the expression of the respective class II molecules. Distribution of CD4+ or CD8+ T cells and B cells was within the normal range, and was comparable between mice expressing or lacking RIP-B7-1 for a given HLA transgene (data not shown). Similarly, expression of transgenic class II molecules was also comparable between RIP-B7-1-positive or -negative littermates (data not shown). However, there were inherent differences between DR3, DQ8 and DR3/DQ8 double-transgenic mice. For yet unexplained reasons, expression of transgenic HLA class II molecules and the percentage of cells expressing transgenic class II molecules were slightly higher in DR3/DQ8 double-transgenic mice than the respective single-transgenic mice (Fig. 1a). Increased expression of class II molecules on double HLA transgenic mice was not an artifact due to the cross-reactivity of anti-DR (L227) or anti-DQ (IVD12) with DQ8 and DR3 respectively (data not shown). HLA-DQ8 transgenic mice, as reported earlier, harbored less CD4+ T cells than CD8+ T cells (20,22). However, DR3 and DR3/DQ8 double-transgenic mice harbored more CD4+ T cells than CD8+ T cells, as in conventional mice strains (Fig. 1b). The T cell repertoire (as assessed by TCR Vß usage) was, however, comparable between different HLA class II transgenic mice (data not shown).

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Fig. 1. Phenotype of HLA transgenic mice. (a) Splenic mononuclear cells from indicated age-matched mice were analyzed for cell-surface expression of DR3 and/or DQ8 using L227 and IVD12 respectively. Representative histograms are shown. (b) Distribution of CD4+, CD8+ T cells and B220+ cells in single or double HLA transgenic mice. Each bar represents mean ± SD from four mice.
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Islet-specific expression of B7-1 does not alter ß cell functions
To rule out the possibility that transgenic expression of the co-stimulatory molecule B7-1 might directly affect pancreatic ß cell function, 8- to 12-week-old normoglycemic DR3 and DQ8 mice of either sex expressing or lacking RIP-B7-1 were subjected to IPGTT. There was no significant difference in the glycemic status of fasted mice belonging to different groups in response to i.p. glucose challenge (data not shown).
Islet-specific expression of B7-1 results in spontaneous diabetes in DR3 and DQ8 transgenic mice
The glycemic status of HLA-DR3 or DQ8 single-transgenic mice expressing RIP-B7-1 was monitored for a maximum of 50 weeks beginning from 4 weeks of age. About 30% of mice belonging to either group became diabetic throughout the entire observation period (Fig. 2). The median onset of disease was earlier in DQ8 transgenic mice than in the DR3 group. Even though males had a higher incidence of disease, there was no significant difference in the incidence of diabetes between males and females (Table 2). Among the DR3/DQ8 double-transgenic mice, the overall incidence of diabetes and the median onset were not significantly different from the single-transgenic mice (Fig. 2 and Table 2). However, the incidence of disease in double-transgenic males was higher than that seen in single-transgenic mice. None of the RIP-B7-1 mice lacking transgenic HLA class II as well as the endogenous class II molecules (Aß0.RIP-B7-1) developed diabetes, implying that expression of class II is required for the occurrence of spontaneous diabetes in RIP-B7-1 transgenic mice (Table 2). Since previous studies have clearly shown that RIP-B7-1 mice expressing endogenous class II molecules seldom developed diabetes (17,2326), the results of the present study clearly indicate the pathogenic role of DR3 and DQ8 in precipitating the disease. Since none of the single or double DR3/DQ8 transgenic mice lacking RIP-B7-1 have ever developed spontaneous diabetes, expression of the co-stimulatory molecule B7-1 on the ß cells of islets of Langerhans somehow facilitates its autoimmune destruction in the presence of DR3 and/or DQ8.

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Fig. 2. Cumulative incidence of spontaneous diabetes. Mice belonging to different groups were monitored weekly to assess their glycemic status. Diabetes was diagnosed when two consecutive random blood glucose levels were >13.9 mmol/l. None of the single or double HLA-DR3/DQ8 transgenic mice lacking RIP-B7-1 nor any of the Aß0.RIP-B7-1 mice lacking endogenous as well transgenic class II molecules developed diabetes.
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Histopathology of spontaneous diabetes
Pancreata collected from diabetic and non-diabetic age- and sex-matched mice belonging to different groups were evaluated histopathologically for the presence of insulitis. Infiltration of islets with mononuclear cells was evident only in diabetic mice, but not in the non-diabetic littermates as demonstrated in earlier studies (17,19) (Fig. 3). There was no significant difference in the severity of islet infiltration between mice expressing single or double HLA transgenes, and also between males and females (data not shown). We also characterized the phenotype of the islet-infiltrating cells by immunofluorescence staining. As shown in Fig. 4, CD4+ T cells were the least abundant of all cell types. CD8+ T cells constituted the majority of the infiltrating cell types, followed by B cells and macrophages. A strikingly similar distribution of lymphocytes has also been described in human patients with recent onset T1D (27). In a previous study, salivary glands in the diabetic HLA-DQ8.RIP-B7-1 mice were infiltrated with T and B lymphocytes (17). However, in the present study, salivary glands from single or double HLA transgenic diabetic mice were devoid of any mononuclear infiltration (not shown).

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Fig. 3. Histopathology of islets during spontaneous diabetes. Pancreata from a hyperglycemic DQ8.RIP-B7-1 mouse and an age- and sex-matched euglycemic littermate were fixed in buffered formalin. Thin sections were stained with hematoxylin/eosin and evaluated microscopically. (a and b) Hyperglycemic DQ8.RIP-B7-1 mice. (c and d) Non-diabetic littermate. Magnification: a and b, x10; c and d, x 20.
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Fig. 4. In situ immunophenotyping of islet-infiltrating cells in spontaneously diabetic mice by immunofluorescence. Cryosections of pancreas from a hyperglycemic DQ8.RIP-B7-1 mouse were stained with hematoxylin & eosin (a), or with CD4 (b), CD8 (c), CD19 (d), CD11b (e) or control antibody (f) as described in Methods. Magnification: x100.
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Perturbations in lymphocyte homeostasis do not induce/accelerate diabetes in HLA transgenic RIP-B7-1 mice
Occurrence of spontaneous diabetes in RIP-B7-1 transgenic mice expressing HLA-class II indicated that autoreactive T cells are present in the periphery and are capable of inducing ß cell destruction culminating in diabetes. Taking into consideration the existing evidence that superantigens might be involved in the pathogenesis of T1D (2830), we intended to study whether non-specific activation of predominant populations of T cells by superantigens would induce/accelerate diabetes in these mice. Furthermore, the lymphoproliferative responses to SEB are far greater in HLA class II transgenic mice than in conventional mice (3134). Therefore, we challenged HLA-DQ8.RIP-B7-1 transgenic mice with the superantigen SEB. In spite of a profound T cell activation (data not shown), none of the SEB-treated mice became hyperglycemic during the first 3 days following SEB challenge. Rather there was evidence of hypoglycemia (data not shown) due to massive superantigen-induced T cell activation and the concomitant cytokine production (35).
The initial superantigen-induced T cell activation phase is followed by the deletion phase during which the activated T cells undergo rapid death (36). Co-stimulation through 4-1BB has been shown to protect activated T cells from this deletion process (37 and our personal observation). In vivo ligation of 4-1BB by the agonistic antibody has also been shown to augment the cytotoxic potential of T cells (38) and augment the responses of activated T cells (39,40). Therefore, we treated one group of HLA-DQ8.RIP-B7-1 mice with SEB and also with the agonistic anti-4-1BB antibody with the hypothesis that 4-1BB-dependent augmentation of T cell responses and persistence of activated T cells subsequent to superantigen stimulation might rapidly precipitate diabetes. In spite of the fact that anti-4-1BB treatment significantly increased the survival of CD8+ T cells bearing the SEB-reactive TCR (data not shown), none of the mice developed diabetes. These results indicate that even though HLA transgenic RIP-B7-1 mice harbor autoreactive T cells in the periphery, their non-specific activation does not rapidly precipitate autoimmune diabetes and the spontaneous diabetes occurring in these mice is a specific phenomenon occurring over a period of time (discussed later). The other possibilities include absence of autoreactive T cells in the SEB-reactive pool and/or activation of regulatory T cells by SEB that effectively controlled rapid progression to diabetes.
A single sublethal irradiation can increase the incidence of autoimmune diabetes in some murine models of diabetes, but not in non-obese diabetic (NOD) mice (D. Serreze, personal communication). Rapid expansion of effector T cell populations resulting from radiation-induced lymphopenia and/or irradiation-induced perturbation in the immune regulation are thought to be the operating mechanisms (41,42). However, sublethal irradiation of HLA-DR3/DQ8.RIP-B7-1 transgenic mice as described in Methods did not result in accelerated diabetes during the 12-week observation period.
Effect of in vivo depletion of CD25+ T cells
Recently, a population of naive CD4+ T cells expressing CD25 has emerged as a strong candidate for regulating the immune response (reviewed in 43). These CD4+CD25+ T regulatory cells have been shown to be capable of suppressing the effector functions of autoreactive T cells (44) as well as other T cells (45). In vivo depletion of these CD25+ T cells in naive mice by administration of anti-CD25 antibody has been shown to result in organ-specific autoimmunity (46). However, a recent study has shown that in vivo depletion of CD25+ T cells by itself rarely induced autoimmunity and concomitant immunization with autoantigens resulted in organ-specific autoimmunity only in CD25-depleted, but not normal, mice (47). This indicated that additional signals are required to trigger autoimmunity in the absence of CD25+ T cells (47). CD4+CD25+ T cells have also been implicated in the pathogenesis of T1D. For example, defective development of CD4+CD25+ T cells in NOD mice has been shown to accelerate the development of diabetes (48) and enhancing the development of CD4+CD25+ T cells has been shown to confer protection from T1D in this model (49,50). Co-transfer of purified CD4+CD25+ T cells could also confer significant protection from diabetes induced by adoptive transfer of islet-infiltrating cells (51). The protective role of CD4+CD25+ T cells was also elegantly established in an inducible model of murine T1D (52).
Existing evidence indicates the presence of a similar population of cells in humans (53,54) and a recent study has indicated that human patients with T1D have deficiency of CD4+CD25+ T cells (55). Therefore, we wanted to study whether depletion of CD25+ T cells in vivo in young naive normoglycemic HLA-DQ8.RIP-B7-1 transgenic mice would accelerate the onset/severity of T1D. To achieve this, young mice were treated with purified anti-CD25 antibody that has been shown to effectively deplete CD25+ T cells in vivo (47,56,57). The preliminary results showed that even one or two injections of the depleting antibody caused a significant reduction in CD25+ cells among the CD4+ T cells (Fig. 5). To study the effect of in vivo depletion on the course of T1D, mice received six injections of the antibody at 3-day intervals and were monitored for 18 weeks. However, only one of six mice treated with anti-CD25 antibody developed diabetes in this period, indicating that in vivo depletion of CD25+ T cells does not accelerate the incidence of T1D in the RIP-B7-1 model of diabetes in HLA-DQ8 transgenic mice. Depletion of activated, pathogenic, autoreactive T cells expressing CD25 by an anti-CD25 antibody treatment protocol is another possibility that could inhibit/delay diabetes progression.

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Fig. 5. Antibody-mediated in vivo depletion of CD25+ T cells. Naive, normoglycemic, HLA-DQ8.RIP-B7-1 mice were injected with one or two injections (3 days apart) of purified anti-CD25 antibody (PC61, 500 µg per injection). Mice were sacrificed 3 days after the last injection and the distribution of CD25+ T cells among the T cells in spleens was determined by flow cytometry. One representative histogram is shown. CD25+ expression on CD8+ T cells was negligible even in untreated mice.
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Effect of low doses of STZ in RIP-B7-1 mice
Existing evidence suggests that in situ death of pancreatic ß cells can lead to efficient priming of auto-specific T cells in the local draining lymph nodes and can result in diabetes under appropriate conditions (5860). STZ is one such chemical that can induce ß cell apoptosis. While a single dose of 200 mg/kg of STZ has been shown to result in diabetes due to direct toxicity to ß cells, five consecutive injections of low doses of STZ (4050 mg/kg/day) has been shown to result in immune-mediated diabetes in some mouse strains (61,62). In addition to this, previous studies have shown that RIP-B7-1 transgenic mice (but not non-RIP-B7-1 controls) expressing endogenous (murine) class II molecules were highly susceptible to very-low-dose STZ-induced diabetes following administration of five consecutive doses of STZ (2040 mg/kg/day), indicating that ß cell damage mediated by STZ can culminate in autoimmune diabetes if B7-1 is expressed on the target (ß) cells (25,26). As five doses of low-dose STZ can induce diabetes in mice expressing endogenous diabetes-resistant endogenous MHC class II molecules with (25,26) and without RIP-B7-1 (63), we hypothesized that minimal ß cell death induced by just two low doses of STZ would be able to activate autoreactive T cells present in the HLA class II transgenic mice, which in the presence of islet-specific B7-1 might precipitate the disease.
As hypothesized,
7085% of RIP-B7-1 transgenic mice expressing single or double HLA transgenes developed diabetes in
7 weeks time. At the same time, none of the Aß0.RIP-B7-1 transgenic mice (lacking any class II molecules), NOD mice or HLA-transgenic mice lacking RIP-B7-1 developed hyperglycemia at any time point (Table 3). Previous studies on RIP-B7-1 transgenic mice expressing murine endogenous class II molecules have also shown a delayed onset of autoimmune diabetes following low-dose STZ treatment. However, at least five consecutive injections of STZ have been shown to be mandatory in such instances (26). Histopathologically, islets from hyperglycemic, but not non-diabetic STZ-treated, mice showed insulitis (Fig. 6) indicating autoimmune etiology. The salivary glands were devoid of any inflammatory infiltrates (data not shown).

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Fig. 6. Histopathology of islets from low-dose STZ-treated mice. Pancreata from a non-diabetic HLA transgenic mouse lacking RIP-B7-1 (a and b) and a hyperglycemic DR3.RIP-B7-1 mouse at day 35 post-STZ treatment (c and d) were fixed in buffered formalin. Thin sections were stained with hematoxylin & eosin and evaluated microscopically. Magnification: a and c, x10; b and d, x20.
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In a previous study, Wen et al. (22) have shown that administration of two low doses of STZ (50 mg/kg/day) alone does not cause any islet pathology in HLA-DQ8 transgenic mice. (It should be noted that these mice do not express RIP-B7-1.) However, adoptive transfer of GAD-reactive, DQ8-restricted CD4+ T cell clones rapidly induced insulitis in such STZ-treated DQ8 mice, but not in STZ-untreated DQ8 mice. This indicated that priming with such small doses of STZ somehow facilitates autoimmune attack of islets.
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Discussion
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T1D is an organ-specific autoimmune disease resulting from the complex interactions of several genetic, environmental and immunological factors. MHC class II molecules (and CD4+ T cells) seem to play a central role in the pathogenesis of immune-mediated diabetes. Even in the RIP-B7-1 model, class II molecules (and CD4+ T cells) are important as mice lacking any class II molecules (endogenous as well as transgenic) seldom get diabetes (present study and 17). Nevertheless, expression of appropriate MHC class II molecules is mandatory for the occurrence of diabetes in this model. For example, only the RIP-B7-1 transgenic mice expressing diabetes-predisposing class II molecules such as I-Ag7 (64) and DQ8 (17) develop spontaneous autoimmune diabetes, while RIP-B7-1 mice expressing diabetes-resistant endogenous class II molecules such as H-2b (23,24), H-2q (26) and those expressing certain transgenic HLA class II molecules (such as DQ6) are diabetes-free (17).
Among the class II molecules linked to T1D in humans, HLA-DQ8 is the most studied due to its strong structural (65) and functional (66) similarity to the murine class II molecule I-Ag7 expressed in spontaneously diabetic NOD mice. The results of the present study strongly support the existing evidence regarding the diabetogenic potential of HLA-DQ8. However, elucidating the exact roles played by the DR molecules linked to T1D has been impossible due to their linkage disequilibrium with the DQ molecules (67). Further, among the DR molecules, expression of certain DR (such DR3 and DR4) is linked to susceptibility to T1D, while some (such as DR2) are associated with conferring resistance. Allelic variations within each DR also have differential associations (67,68). Existing evidence also indicates that there could be some inherent differences in the etiological roles of HLA-DR3 and -DR4 in the pathogenesis of T1D, and hence these are termed the DR3-associated pathway and DR4-associated pathway respectively (68,69). Furthermore, genetic studies in humans have shown that heterozygosity for DR3 and DR4 can significantly increase the risk of T1D (70).
HLA transgenic mice have again helped in resolving to a certain extent the complex association of DR molecules with the incidence of autoimmune diseases (7175). With respect to T1D, a recent study has shown that HLA-DR4 by itself does not strongly predispose to autoimmune diabetes in a transgenic mouse model. It rather down-regulated the diabetogenic potential of DQ8 (19). However, as evident from the present investigation, the related class II molecule DR3 seems to have no such regulatory potential and appears to be as permissible to T1D as DQ8. Our earlier observations that the response of lymphocytes from naive HLA-DR3 transgenic mice to certain islet autoantigens was comparable to that of DQ8 transgenic mice, and that lymphocytes from DR3/DQ8 double-transgenic mice also responded to human GAD65, support the hypothesis that DR3 is not inhibitory for the development of islet-specific autoimmunity (15). Taken together, it appears as though DR3 and DR4 might function differently. However, the underlying mechanisms are unclear at present.
Several hypotheses have been put forth to explain the events leading to the occurrence of autoimmunity (76). Non-specific or bystander activation of T cells by superantigens (28,77), defective immunoregulation (78) and perturbation of T cell homeostasis (79) are some of the suggested peripheral mechanisms. Nevertheless, as evident from the present study, the incidence of autoimmunity, at least in the RIP-B7-1 model of diabetes, is not greatly influenced by these factors. It is of interest to note that in a recent study, induction of ß cell apoptosis by a single low dose of STZ (4060 mg/kg) accelerated insulitis in NOD mice. However, the incidence of diabetes in such mice was significantly reduced due to induction of cross-tolerance (80). Nevertheless, as evident from the present study, induction of ß cell apoptosis by low doses of STZ seems to effectively prime T cells in the presence of permissible HLA class II and islet-specific expression of co-stimulatory molecule B7-1. Either the priming pathway or the effector pathway is more efficient when B7-1 is expressed on the ß cells and seems to overcome induction of cross-tolerance. Dissection of the molecular mechanisms of diabetes induced by two injections of low-dose STZ is currently ongoing. Collectively, the evidence generated from HLA transgenic mice models indicates that autoreactive T cells are present in the periphery of mice expressing diabetes-predisposing MHC molecules, which under appropriate conditions mediate ß cell destruction culminating in diabetes. In conclusion, the present study establishes the complex pathogenesis of T1D and underscores the value of these transgenic mice in studying the effect of multiple HLA class II molecules that are either neutral, or linked with susceptibility or protection from T1D.
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Acknowledgements
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We thank Julie Hanson and her crew for excellent mice husbandry, and Michelle Smart for characterizing the transgenic mice. We thank Thomas Beito (Mayo Monoclonal Antibody Core Facility, Mayo Clinic, Rochester, MN) for purification of anti-CD25 and anti-TCR antibodies. We also thank Tim Plummer, Karen Lien and J. L. Platt for immunostaining and fluorescence microscopy.
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Abbreviations
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IPGTTi.p. glucose tolerance test
NODnon-obese diabetic
RIPrat insulin promoter
SEBstaphylococcal enterotoxin B
STZstreptozotocin
T1Dtype 1 diabetes
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