T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8+ T cells specific for IGRP206–214 (NOD8.3 mice) develop accelerated diabetes that requires CD4+ T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP206–214-specific CD8+ T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP206–214-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4+ T cell help to activate IGRP206–214-specific T cells.

Type 1 diabetes (T1D)3 is a T cell-mediated organ-specific autoimmune disease that results from selective destruction of the β cells in the islets of the pancreas (1). T cells specific for a number of islet Ags can be detected in humans and nonobese diabetic (NOD) mice (2). Initiation of autoimmunity in NOD mice may be dependent on an immune response against insulin alone rather than an immune response against many β cell Ags. We and others have independently shown that transgenic overexpression of proinsulin 2 in APCs of NOD mice prevents insulitis and diabetes (3, 4). Knocking out both insulin genes and introduction of mutated insulin with alanine rather than tyrosine at position 16 of the insulin B chain prevents insulitis and diabetes (5). Eliminating immune responses to insulin not only blocks development of diabetes and insulitis but also immune responses to downstream autoantigens, such as the islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) (6). In both humans and animal models, the major determinants of T1D are genes within the MHC [HLA in humans] (7). The remarkable homogeneity of high-risk genotypes across different populations and the conservation of aspects of the Ag-binding groove suggest that there may be specific peptides that for most individuals represent the initiating targets of autoimmunity (8). In NOD mice (and probably also in humans), the primary autoantigen for diabetes appears to be insulin, specifically the insulin B chain peptide B9-23 (5).

Identification of target autoantigens for T cells is particularly important because manipulation of immune responses to these Ags offers the hope of specifically removing the cells that damage β cells. We and others have proposed that autoreactivity to insulin is necessary and precedes autoreactivity to IGRP and other autoantigens (3, 5, 6). If insulin is the primary Ag, a strategy that induces tolerance to insulin should prevent diabetes. Although this prevented diabetes in NOD mice (9), major preventive trials with either oral or i.v. insulin have thus far failed to prevent diabetes in humans (10, 11, 12). Human subjects enrolled in prevention trials have multiple autoantibodies, suggesting advanced preclinical diabetes. By the time the intervention occurs, tissue inflammation has already developed, the immune response has already expanded beyond the initial “triggering” Ag, and the frequency of diverse array of Ag-specific T cells may have expanded substantially. Therefore, a clinically important question is whether induction of tolerance to the primary Ag will be effective when there is already an expanded pool of Ag-specific T cells. We wished to know whether inducing tolerance to insulin would have any effect in NOD mice that already have an expanded number of T cells specific for IGRP. To address this question, we crossed TCR transgenic mice that have >90% of their CD8+ T cells specific for IGRP206–214 (NOD8.3 mice) (13) with NOD mice tolerant to proinsulin (NOD-PI mice) that express proinsulin 2 in APCs, making them tolerant to proinsulin (3). These mice have dramatically reduced diabetes, indicating that the requirement for immunity to proinsulin is even present in the majority of NOD8.3 mice with a pre-existing repertoire of IGRP206–214-specific cells.

NOD mice expressing mouse proinsulin 2 under the control of an MHC class II (I-Eακ) promoter (NOD-PI) and NOD8.3 mice expressing the TCRαβ rearrangements of the H-2Kd-restricted CD8+ T cell clone NY8.3 have been described (3, 5, 6). Perforin knockout NOD mice were obtained from The Jackson Laboratory T1D repository. All animal studies were conducted at St. Vincent’s Institute (Melbourne, Australia) and were approved by the institutional animal ethics committee.

The peptides IGRP206–214 (VYLKTNVFL) and TUM (KYQAVTTTL) were synthesized by Auspep. H-2Kd tetramers were made by ImmunoID. Tetramer function was validated by staining NOD8.3 splenocytes.

Islet infiltrating T cells were stained with IGRP206–214 or TUM H-2Kd tetramer as previously described (6).

CD8+ T cells from NOD8.3 mouse were labeled with CFSE as previously described (6). In cotransfer experiments, mice receiving CFSE-labeled 8.3 CD8+ T cells were injected i.v. (a) on day 0 with 100 μg agonistic anti-CD40 mAb FGK45 or 100 μg isotype control Ab (GL117) in 200 μl PBS and (b) on day −1 with 80 mg/kg streptozotocin (STZ) in 200 ml citrate buffer (pH 4.5) or citrate buffer alone. Hosts were sacrificed on day 3 and their pancreatic and inguinal lymph nodes examined for CFSE+ cells.

Splenic 8.3-CD8+ T cells (2 × 104 cells/well) were cultured, in triplicate, either alone or with 2 × 104 cells/well (1:1), 5× 103 cells/well (1:4), or 2 × 103 cells/well (1:10) CD4+CD25 or CD4+CD25+ T cells from NOD or NOD-PI mice in the presence of IGRP206–214 (0.1 μM)-pulsed NOD splenic irradiated DCs (104 cells/well). The cells were cultured in 96-well round-bottom plates in 200 μl of RPMI 1640 (Invitrogen Life Technologies) supplemented with antibiotics, 2 mM glutamine, nonessential amino acids, 50 μM mercaptoethanol, and 10% FCS for 3 days at 37°C in 5% CO2. Wells were pulsed with 1 μCi [3H]thymidine during the last 18 h of culture.

Islets of Langerhans were isolated from mice according to methods previously described (14). For MHC class I staining, antisera used were anti-mouse H-2Db (28–14-8; BD Pharmingen) followed by allophycocyanin-conjugated streptavidin (Caltag Laboratories). Leukocytes were excluded from analysis by staining with anti-CD45 conjugated to PerCp-Cy5.5 (3F11; BD Pharmingen), and β cells were identified based on their high autofluorescence (14).

Five 7-wk-old NOD-PI/NOD8.3 mice were injected with anti-CD4 mAb (GK1.5) or isotype control (GL121). The dose was 1 mg followed by 0.5 mg after 3 days. Thereafter 0.5 mg was injected every week until the mouse became diabetic or for 8 wk. Mice were monitored for success of depletion using noncompeting anti-CD4 (clone RM4-4; BD Pharmingen).

Immunohistochemical staining and scoring of frozen pancreas sections was performed as described (15, 16). Mice were monitored for diabetes as described (6).

IAA were measured with a 96-well filtration plate micro IAA assay as described. (6) We have participated in all the Diabetes Autoantibody Standardization Program workshops. In the murine IAA workshop (2002), the sensitivity and specificity for IAA were 69 and 83%, respectively.

Insulin autoantibody levels and insulitis scores were analyzed by Student’s t test. Survival curves were analyzed with the log-rank test. Statistical tests used PRISM software (version 3.02; GraphPad). Values of p < 0.05 was considered significant.

We have shown that tolerance to proinsulin prevents expansion of IGRP206–214-specific T cells (6). To study the effect of tolerance to insulin on an expanded pool of autoreactive T cells, we crossed NOD8.3 TCR transgenic mice to NOD-PI mice.

The proportions of CD4+ or CD8+ T cells in NOD-PI/NOD8.3 mice were similar to NOD8.3 mice, and there was no difference in numbers of IGRP206–214-specific T cells, suggesting normal development of CD8+ T cells in NOD-PI/NOD8.3 mice (Fig. 1, A and B). IGRP206–214-specific naive T cells from the spleens of NOD-PI/NOD8.3 mice proliferated in response to the peptide both in vitro and in vivo similarly to IGRP206–214-specific T cells from NOD8.3 mice (Fig. 1, C and D). Moreover, IGRP206–214-specific splenic T cells from NOD-PI/NOD8.3 and NOD8.3 mice secreted IFN-γ in response to the peptide to a similar extent, and splenocytes from these mice transfer diabetes into irradiated NOD mice to a similar extent, suggesting normal cytotoxic potential (Fig. 1, E and F).

FIGURE 1.

IGRP206–214-specific CD8+ T cells develop normally in NOD-PI/NOD8.3 mice and are cytotoxic. A and B, CD4 vs CD8 plots of cell suspensions from spleen (A) or thymus (B) of NOD-PI/NOD8.3 and NOD8.3 mice (top panel). Lower panels, IGRP206–214/H-2Kd tetramer+ CD8+ T cells after gating on CD8 subset (A) or CD8 single positive subset (B). Numbers indicate the average percentage of live cells (top) or the percentage of tetramer+ cells in CD8 subset. C, Proliferation of CFSE-labeled splenocytes isolated from 6- to 8-wk-old NOD-PI/NOD8.3 or NOD8.3 mice when injected into 8–12-wk-old NOD mice. D, Proliferation of CFSE-labeled CD8+ T cells from NOD-PI/NOD8.3 or NOD8.3 when cultured with IGRP206–214 or TUM peptide-pulsed (0.1 mM) irradiated NOD DCs. E, Intracellular IFN-γ staining of CD8+ T splenocytes from NOD-PI/NOD8.3 mice or NOD8.3 mice after stimulation with 0.1 μM of IGRP206–214 or TUM peptide. A–E, Data correspond to 4–8 mice/group. F, Incidence of diabetes following transfer of splenocytes from either NOD-PI/NOD8.3 or NOD8.3 mice into 10-wk-old irradiated NOD mice (n = 5 per group).

FIGURE 1.

IGRP206–214-specific CD8+ T cells develop normally in NOD-PI/NOD8.3 mice and are cytotoxic. A and B, CD4 vs CD8 plots of cell suspensions from spleen (A) or thymus (B) of NOD-PI/NOD8.3 and NOD8.3 mice (top panel). Lower panels, IGRP206–214/H-2Kd tetramer+ CD8+ T cells after gating on CD8 subset (A) or CD8 single positive subset (B). Numbers indicate the average percentage of live cells (top) or the percentage of tetramer+ cells in CD8 subset. C, Proliferation of CFSE-labeled splenocytes isolated from 6- to 8-wk-old NOD-PI/NOD8.3 or NOD8.3 mice when injected into 8–12-wk-old NOD mice. D, Proliferation of CFSE-labeled CD8+ T cells from NOD-PI/NOD8.3 or NOD8.3 when cultured with IGRP206–214 or TUM peptide-pulsed (0.1 mM) irradiated NOD DCs. E, Intracellular IFN-γ staining of CD8+ T splenocytes from NOD-PI/NOD8.3 mice or NOD8.3 mice after stimulation with 0.1 μM of IGRP206–214 or TUM peptide. A–E, Data correspond to 4–8 mice/group. F, Incidence of diabetes following transfer of splenocytes from either NOD-PI/NOD8.3 or NOD8.3 mice into 10-wk-old irradiated NOD mice (n = 5 per group).

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Despite normal number and function of IGRP206–214-specific T cells, NOD-PI/NOD8.3 mice have reduced insulitis and are markedly protected from diabetes (Fig. 2, A and B). Most of the CD8+ T cells infiltrating islets of NOD-PI/NOD8.3 mice are specific for IGRP (Fig. 2,E). The important difference between NOD-PI/NOD8.3 mice and NOD8.3 mice is the ability to mount an immune response against insulin. A total of 40% of female NOD8.3 mice (4/10) had high titer IAA, whereas only 9.4% NOD-PI/NOD8.3 mice (3/32) had a borderline positive IAA (Fig. 2 C). Recently, it has been shown that insulin primed CD4+ T cells are required for expression of IAA (16). IGRP206–214-specific T cells therefore depend on proinsulin-specific immune responses for their full activation and ability to mediate β cell destruction.

FIGURE 2.

NOD-PI/NOD8.3 mice are protected from diabetes. A, Insulitis scores at 40, 60, and 100 days of age. B, Incidence of diabetes in NOD8.3 (n = 20) and NOD-PI/NOD8.3 (n = 20) mice. Values of p < 0.0005. C, Insulin autoantibodies in 8–12-wk-old female NOD-PI/NOD8.3 (n = 32) and NOD8.3 (n = 10) mice. Horizontal line indicates upper limit of normal. Values of p < 0.01. D, H-2Db expression on islet cells from 6- to 8-wk-old NOD-PI/NOD8.3 or NOD8.3 mice. E, Islet infiltrating T cells from 8- to 10-wk-old NOD-PI/NOD8.3 and NOD8.3 mice were cultured and stained with IGRP or TUM H-2Kd tetramers.

FIGURE 2.

NOD-PI/NOD8.3 mice are protected from diabetes. A, Insulitis scores at 40, 60, and 100 days of age. B, Incidence of diabetes in NOD8.3 (n = 20) and NOD-PI/NOD8.3 (n = 20) mice. Values of p < 0.0005. C, Insulin autoantibodies in 8–12-wk-old female NOD-PI/NOD8.3 (n = 32) and NOD8.3 (n = 10) mice. Horizontal line indicates upper limit of normal. Values of p < 0.01. D, H-2Db expression on islet cells from 6- to 8-wk-old NOD-PI/NOD8.3 or NOD8.3 mice. E, Islet infiltrating T cells from 8- to 10-wk-old NOD-PI/NOD8.3 and NOD8.3 mice were cultured and stained with IGRP or TUM H-2Kd tetramers.

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Regulatory T cells (Tregs) could account for diabetes protection in NOD-PI/NOD8.3 mice but we previously did not find evidence for this mechanism in NOD-PI mice (6). Also, there was no difference in number of FoxP3+ CD4+ T cells between NOD and NOD-PI mice and between NOD8.3 and NOD-PI/NOD8.3 mice (Fig. 3,A). However, Tregs could have different suppressive potential. No difference in suppressive activity of CD4+CD25+ from NOD or NOD-PI mice on proliferation of IGRP-specific CD8+ T cell was observed in vivo or in vitro (Fig. 3, B and C). Moreover, IGRP206–214- specific T cells from NOD-PI/NOD8.3 mice had similar proliferative and IFN-γ responses to IGRP206–214 compared with cells from NOD8.3 mice (Fig. 1, C–E). Tregs could also act indirectly by preventing maturation of DC, as seen in NOD8.3 mice lacking CD154 (NOD8.3/CD154−/−) (17). CD4+CD25+ T cells from NOD8.3/CD154−/− mice prevented maturation of DC and made them hypoimmunogenic. DCs showed an immature phenotype (low MHC class II and CD86 expression) and 8.3 T cells showed decreased proliferation in response to Ag-loaded DCs from these mice (17). However, we did not see any phenotypic difference in DCs from pancreatic lymph nodes of NOD and NOD-PI mice (data not shown). Moreover, Ag-loaded splenic and bone marrow-derived DCs from NOD and NOD-PI mice stimulated 8.3 T cells similarly (16). Finally, treating NOD-PI/NOD8.3 mice with anti-CD4 GK1.5 Ab did not change the level of MHC class I expression on the β cells or incidence of diabetes in NOD-PI/NOD8.3 mice, whereas it might have been expected to increase diabetes if FoxP3+ CD4+ Tregs were depleted (Fig. 3, D and E).

FIGURE 3.

No evidence of dominant tolerance to account for protection from diabetes in NOD-PI/NOD8.3 mice. A, Flow cytometric analysis of intracellular Foxp3 (anti-Foxp3 mAb FJK-16s) expression in CD4+CD8 cells. The percentages of CD4+CD8 cells in each region is shown. B, Proliferation of CFSE-labeled 8.3 T cells when injected i.v. (4–6 × 106 cells/mouse) with CD4+CD25+ or CD4+CD25 cells (4–6 × 106 cells/mouse) from NOD-PI mice into 8-wk-old NOD mice (n = 4 per group). C, Proliferation of 8.3 T cells in response to IGRP206–214-peptide pulsed irradiated NOD DC when cultured with CD4+CD25 or CD4+CD25+ T cells from NOD or NOD-PI mice. D, Diabetes incidence in NOD-PI/NOD8.3 mice treated with GK1.5 (anti-CD4) mAb (n = 5) or isotype control Ab (n = 5). Arrows indicate the time of administration of Ab P = ns. E, Islets from anti-CD4 mAb or isotype control Ab-treated NOD-PI/NOD8.3 or untreated NOD8.3 mice were analyzed for expression of class I MHC expression using anti-mouse H-2Db.

FIGURE 3.

No evidence of dominant tolerance to account for protection from diabetes in NOD-PI/NOD8.3 mice. A, Flow cytometric analysis of intracellular Foxp3 (anti-Foxp3 mAb FJK-16s) expression in CD4+CD8 cells. The percentages of CD4+CD8 cells in each region is shown. B, Proliferation of CFSE-labeled 8.3 T cells when injected i.v. (4–6 × 106 cells/mouse) with CD4+CD25+ or CD4+CD25 cells (4–6 × 106 cells/mouse) from NOD-PI mice into 8-wk-old NOD mice (n = 4 per group). C, Proliferation of 8.3 T cells in response to IGRP206–214-peptide pulsed irradiated NOD DC when cultured with CD4+CD25 or CD4+CD25+ T cells from NOD or NOD-PI mice. D, Diabetes incidence in NOD-PI/NOD8.3 mice treated with GK1.5 (anti-CD4) mAb (n = 5) or isotype control Ab (n = 5). Arrows indicate the time of administration of Ab P = ns. E, Islets from anti-CD4 mAb or isotype control Ab-treated NOD-PI/NOD8.3 or untreated NOD8.3 mice were analyzed for expression of class I MHC expression using anti-mouse H-2Db.

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It is surprising that despite being present in such a large number, IGRP-specific T cells are tolerant to IGRP in the absence of immune responses to insulin. IGRP206–214-specific T cells remain ignorant of their Ag in NOD-PI hosts (6). We therefore reasoned that IGRP206–214-specific T cells, despite being present in large numbers, might remain ignorant of their Ag, meaning that tolerance should be broken by increasing T cell activation signals. Indeed, FACS analysis of β cells from NOD-PI/NOD8.3 mice showed decreased MHC class I expression, which could reduce their targeting by CD8+ T cells (Fig. 2 D).

We questioned whether decreased cross-priming of IGRP206–214- specific T cells in NOD-PI hosts could be overcome in response to β cell death or islet inflammation. In the absence of infection, DC maturation occurs following uptake of Ags from apoptotic cells or by ligation of CD40 by CD154 on CD4+ T cells (18, 19, 20, 21). We assessed proliferation of transferred IGRP206–214-specific T cells following CD40 agonist Ab treatment or induction of β cell apoptosis by low dose STZ. A single dose of 80 mg/kg of STZ or 100 μg of CD40 agonist Ab was used based on published reports (22, 23, 24). Each of these increased proliferation of NOD8.3 T cells (Fig. 4, A–C).

FIGURE 4.

Induction of DC maturation or increase in available Ag in NOD-PI mice increases cross-presentation of IGRP206–214-specific CD8+ T cells. A, Proliferation of CFSE-labeled 8.3 T cells when injected into anti-CD40 mAb or isotype control Ab treated NOD-PI (n = 6 in each group) and NOD (n = 6 in each group) mice. B, Proliferation of CFSE-labeled 8.3 T cells when injected into STZ- or citrate buffer-treated NOD-PI (n = 8 in each group) and NOD (n = 8 in each group) mice. C, NOD and NOD-PI mice were treated i.v. either with STZ (80 mg/kg) (n = 2 in each group) or citrate buffer (n = 2 in each group). Islets were harvested 3 h later, incubated for 12 h in CMRL medium 1066, and dispersed into single cells. Dispersed cells were incubated in a PI-containing hypotonic solution, and nuclei were analyzed by flow cytometry. Data show typical profiles and the percentage of fragmented nuclei. D, Proliferation of CFSE-labeled 8.3 T cells when injected into NOD (n = 4) and NOD perforin−/− (n = 4) mice. Representative data from 2 to 4 independent experiments is shown.

FIGURE 4.

Induction of DC maturation or increase in available Ag in NOD-PI mice increases cross-presentation of IGRP206–214-specific CD8+ T cells. A, Proliferation of CFSE-labeled 8.3 T cells when injected into anti-CD40 mAb or isotype control Ab treated NOD-PI (n = 6 in each group) and NOD (n = 6 in each group) mice. B, Proliferation of CFSE-labeled 8.3 T cells when injected into STZ- or citrate buffer-treated NOD-PI (n = 8 in each group) and NOD (n = 8 in each group) mice. C, NOD and NOD-PI mice were treated i.v. either with STZ (80 mg/kg) (n = 2 in each group) or citrate buffer (n = 2 in each group). Islets were harvested 3 h later, incubated for 12 h in CMRL medium 1066, and dispersed into single cells. Dispersed cells were incubated in a PI-containing hypotonic solution, and nuclei were analyzed by flow cytometry. Data show typical profiles and the percentage of fragmented nuclei. D, Proliferation of CFSE-labeled 8.3 T cells when injected into NOD (n = 4) and NOD perforin−/− (n = 4) mice. Representative data from 2 to 4 independent experiments is shown.

Close modal

In addition, we also analyzed IGRP206–214-specific T cells transferred into perforin knockout NOD mice (NOD perforin−/−). These mice have insulitis but very little β cell destruction and reduced and delayed diabetes development (25). Transferred IGRP206–214-specific T cells proliferated to a similar extent as in NOD mice (Fig. 4 D), indicating that the reduced β cell destruction in these mice does not decrease Ag presentation. Together, these experiments suggested that protection by induction of tolerance to proinsulin could be bypassed.

We next examined whether breaking tolerance by STZ or CD40 Ab treatment in NOD-PI/NOD8.3 could induce diabetes. Administration of a single dose of STZ or CD40 Ab treatment increased MHC class I expression on β cells within 3 days (Fig. 5,A) and resulted in diabetes within 1–4 wk in NOD-PI/NOD8.3 mice (Fig. 5, C and D). The islet infiltrating cells were predominantly IGRP206–214 CD8+ cells in these mice (Fig. 5 B). This suggests that even with many Ag-specific T cells, immune responses to insulin are required for full diabetes development. However, this requirement can be bypassed by stimuli that induce β cell apoptosis or promote inflammation. In contrast, this treatment had no effect on the incidence of diabetes in NOD or NOD-PI mice indicating (a) the treatment is stimulating T cells to mediate diabetes and not directly inducing β cell death and (b) mere stimulation of β cell apoptosis or promotion of islet inflammation is not sufficient, without a large number of Ag-specific T cells.

FIGURE 5.

Inducing islet inflammation in NOD-PI/NOD8.3 mice promotes development of diabetes. A, NOD-PI/NOD8.3 mice (6–8 wk old) were treated with either a single dose of STZ (80 mg/kg body weight)/citrate buffer (n = 2 in each group) or anti-CD40 Ab (100 μg/mouse)/isotype control Ab (n = 2 in each group). Three days later, islets were isolated, dispersed into single cells, and analyzed for expression of class I MHC using anti-mouse H-2Db. B, Islets from STZ or anti-CD40 Ab-treated diabetic NOD-PI/NOD8.3 were analyzed for expression of class I MHC as described above (top panels). Islet infiltrating T cells were stained with IGRP H-2Kd tetramers and analyzed by flow cytometry (bottom panels). C, Incidence of diabetes in NOD-PI/NOD8.3 mice (n = 8 in each group) and NOD (n = 5 in each group) treated with a single dose of either STZ (80 mg/kg body weight) or citrate buffer. Values of p < 0.001. D, Incidence of diabetes in NOD-PI/NOD8.3 mice (n = 5 in each group) and NOD (n = 5 in each group) treated with a single dose of either anti-CD40 Ab or isotype control Ab (100 μg/mouse). Values of p < 0.001.

FIGURE 5.

Inducing islet inflammation in NOD-PI/NOD8.3 mice promotes development of diabetes. A, NOD-PI/NOD8.3 mice (6–8 wk old) were treated with either a single dose of STZ (80 mg/kg body weight)/citrate buffer (n = 2 in each group) or anti-CD40 Ab (100 μg/mouse)/isotype control Ab (n = 2 in each group). Three days later, islets were isolated, dispersed into single cells, and analyzed for expression of class I MHC using anti-mouse H-2Db. B, Islets from STZ or anti-CD40 Ab-treated diabetic NOD-PI/NOD8.3 were analyzed for expression of class I MHC as described above (top panels). Islet infiltrating T cells were stained with IGRP H-2Kd tetramers and analyzed by flow cytometry (bottom panels). C, Incidence of diabetes in NOD-PI/NOD8.3 mice (n = 8 in each group) and NOD (n = 5 in each group) treated with a single dose of either STZ (80 mg/kg body weight) or citrate buffer. Values of p < 0.001. D, Incidence of diabetes in NOD-PI/NOD8.3 mice (n = 5 in each group) and NOD (n = 5 in each group) treated with a single dose of either anti-CD40 Ab or isotype control Ab (100 μg/mouse). Values of p < 0.001.

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This study showed that immune responses against proinsulin, that could be eliminated by expression of proinsulin in APCs, are required in the majority of NOD8.3 TCR transgenic mice for their IGRP-specific T cells to become activated and kill β cells. Diabetes was reduced from nearly 100 to 20%, indicating that little T cell activation and progression occurred in mice with proinsulin immune tolerance (confirmed by absent IAA). That an immune response to one Ag is required for an effective immune response by TCR transgenic T cells specific for a different Ag has not previously been reported. However, aspects of previous studies are consistent with the current finding.

Some previous CD8+ TCR transgenic mice specific for certain Ags that have been transgenically expressed in the β cell have not shown immune reactivity against that Ag (26). An activation step is required, either infection with virus, for example LCMV when LCMV glycoprotein is expressed in β cells (26), or cotransfer of Ag-specific CD4+ T cells when a model pathogen or nonpathogen-derived Ag, such as influenza hemagglutinin or OVA, is expressed in β cells (27, 28). Therefore exposure to pathogens with activation of the innate immune system or “help” from CD4+ T cells can promote progression to diabetes. Further, it was already known that NOD8.3 CD8+ T cells need CD4+ “help” for activation since NOD8.3 mice on a recombination activating genes knock out (Rag−/−) background also reduced frequency of diabetes (13). NOD8.3 mice have high titer IAA, indicating that the mice can generate immune responses against islet Ags other than IGRP despite their biased repertoire. Lastly, we previously showed that IGRP responses in nontransgenic NOD mice are dependent on responses to proinsulin (6).

In contrast to NOD-PI mice, the protection from diabetes is not complete in NOD-PI/NOD8.3 mice. Approximately 20% of the mice develop delayed diabetes. Reduced and delayed diabetes also occurs in NOD8.3 mice on Rag−/− background, in which insulin-specific T cells are not expected to be present (13). Because of the unnaturally high frequency of β cell-specific T cells in NOD8.3 mice, some may undergo activation without insulin-specific T cells either in response to cross-reactive Ags or IGRP occasionally shed from β cells and presented in the pancreatic lymph node even in the absence of any autoimmune-mediated β cell damage (13).

The current study shows that requirements for T cell help, suggested by artificial transgenic models of diabetes, apply also to NOD8.3 mice with T cells that recognize IGRP, a natural β cell Ag and, in the NOD mouse, a spontaneous model of diabetes. Surprisingly, it appears that this help can come from T cells specific for proinsulin, spontaneously generated as a result of the NOD MHC and other NOD genes, rather than a more general requirement for CD4+ T cells of broad specificity. Although there is evidence that induction of tolerance to insulin can induce dominant tolerance to other Ags (29), to our knowledge, this is the first study to show recessive tolerance to insulin can induce tolerance to other Ags in a TCR transgenic mouse. We could reproduce the effect of insulin-specific T cells in vivo by activating APCs with agonistic anti-CD40 Ab or by STZ-induced β cell apoptosis, that may act through TLR-2 (30). Therefore, IGRP-specific CD8+ T cells, even when present in large numbers, need an insulin-specific response, or something to replace it, to become fully active effector T cells and cause diabetes.

There may be significant clinical implications for this study. Subjects with preclinical diabetes enrolled in autoantigen intervention studies usually have responses to several autoantigens, indicating existing expanded T and B cell autoreactive populations. Our study suggests that depletion of proinsulin (primary autoantigen)-specific T cells may have a beneficial effect on other autoreactive T cells despite their prior expansion. There may be indirect evidence in humans for this. There was a significant effect of oral insulin in individuals who began the DPT-1 trial with high levels of insulin autoantibodies, indicating insulin autoimmunity. Tolerance induction with proinsulin in such subjects might, however, be bypassed if stimuli that enhance Ag presentation occur. Expansion of Ag-specific T cells in NOD8.3 mice is a result of expression of the TCR transgene in T cell precursors and thymic positive selection and is independent of T cell help. This has allowed us to show that NOD8.3 T cells need T cell help for effector function independent of proliferation. In a nontransgenic T cell repertoire, expansion and activation of Ag-specific T cells are not separated in this way. The current study suggests there might be beneficial effects of terminating insulin-specific CD4+ T cell help in NOD mice or humans with established preclinical diabetes and autoimmunity to multiple β cell Ags.

We thank Jie Lin for help with tetramer production and Rochelle Ayala for technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

P.S. is a scientist of the Alberta Heritage Foundation for Medical Research and is supported by the Canadian Institutes of Health Research, the Canadian Diabetes Association, and the Juvenile Diabetes Research Foundation. The Julia McFarlane Diabetes Research Centre is supported by the Diabetes Association (Foothills). The work was supported by a Program Grant; Career Development Award (to H.T.) and Clinical Centre for Research Excellence from the National Health and Medical Research Council of Australia; a Program-Project Grant and a post-doctoral fellowship (to B.K.) from the Juvenile Diabetes Research Foundation; and a Millennium Research Grant from Diabetes Australia (to T.K.).

3

Abbreviations used in this paper: T1D, type 1 diabetes; NOD, nonobese diabetic; IGRP, islet-specific glucose-6-phosphatase catalytic subunit related protein; DC, dendritic cell; NOD-PI, NOD mice tolerant to proinsulin; STZ, streptozotocin; IAA, insulin autoantibody assay; Treg, regulatory T cell.

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