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The Journal of Immunology, 2007, 179, 2082 -2088
Copyright © 2007 by The American Association of Immunologists, Inc.
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Altered B:9–23 Insulin, When Administered Intranasally with Cholera Toxin Adjuvant, Suppresses the Expression of Insulin Autoantibodies and Prevents Diabetes1

Masakazu Kobayashi2,*,{dagger}, Norio Abiru3,*, Takeshi Arakawa{ddagger}, Keiko Fukushima*, Hongbo Zhou*, Eiji Kawasaki{dagger}, Hironori Yamasaki*, Edwin Liu§, Dongmei Miao§, F. Susan Wong, George S. Eisenbarth§ and Katsumi Eguchi*

* First Department of Internal Medicine and {dagger} Department of Metabolism/Diabetes and Clinical Nutrition, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan; {ddagger} Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus, Okinawa, Japan; § Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO 80262; and Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Insulin peptide B:9–23 is a major autoantigen in type 1 diabetes that contains two distinct CD4 epitopes (B:9–16 and B:13–23). One of the two epitopes, B:13–23, overlaps with a CTL epitope (B:15–23). In this study, we report that the elimination of the CTL epitope from the B:9–23 peptide by amino acid substitution (with alanine) at positions B:16 and 19 (A16,19 altered peptide ligand) or truncation of the C-terminal amino acids from the peptide (B:9–21), neither of which stimulated the proliferation of insulin B:15–23 reactive CD8 T cells, provided significant intranasally induced suppression of diabetes when coadministered with a potent mucosal adjuvant cholera toxin (CT). Intranasal treatment with A16,19 resulted in the elimination of spontaneous insulin autoantibodies, significant inhibition of insulitis and remission from hyperglycemia, and prevented the progression to diabetes. Intranasal administration of native B:9–23/CT or B:11–23/CT resulted in a significant enhancement of insulin autoantibody expression and severity of insulitis and failed to prevent diabetes. Our present study indicates that elimination of the CTL epitope from the B:9–23 peptide was critically important for mucosally induced diabetes prevention. The A16,19 altered peptide ligand, but not other native insulin peptides, suppresses insulin autoantibodies associated with protection from and remission of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peptide immunotherapy targeting autoreactive T cells is a potential strategy for the treatment of autoimmune diseases where an autoantigen has been identified. However, it has been suggested that mucosal administration of an autoantigenic peptide may act as a double-edged sword with the potential to induce not only protective but also pathogenic immunity, especially if a CTL epitope is present within the peptide. Martinez and coworkers suggested that the induction of CTL immunity needs to be avoided for efficient and safe diabetes prevention in mucosal peptide immunotherapy with an autoantigen, which can be accomplished by disabling the integral CTL epitopes (1). Insulin is a major target in type 1 diabetes, and the insulin B chain peptide with amino acids 9–23 (B:9–23)4 has been suggested as a primary antigenic epitope in the pathogenesis of type 1 diabetes in NOD mice (2, 3). The B:9–23 peptide is capable of preventing diabetes in NOD mice when given s.c. (4) or as an altered peptide ligand (APL) of insulin. The peptide contains two distinct CD4 epitopes (B:9–16 and B:13–23), and B:13–23 overlaps with a CTL epitope (B:15–23) (5, 6). Insulin B:15–23 binds the Kd MHC class I molecule, with the B chain tyrosine at position 16 (binding in p2) providing the primary anchor and the glycine at position 23 (p9) providing the secondary anchor. Substitution of the tyrosine residue at position 16 with alanine or truncation from the C-terminal of B:9–23, i.e., B:9–21/22 peptide, abrogates binding of the peptide to the Kd molecule (7). An APL of B:9–23 that contains an alanine substitution at positions B:16 and B:19 (A16,19 APL) has been shown to prevent spontaneous diabetes in NOD mice when administered s.c. (8). In this study, we test the hypothesis that replacing the CTL epitope of B:9–23 with B:9–21 or A16,19 APL is superior to the native B:9–23 peptide in preventing diabetes.

We have previously demonstrated that s.c. administration of ~50 µg of B:9–23 peptide without the use of IFA induces high levels of insulin autoantibodies (IAA) in normal BALB/c mice that specifically react with intact insulin and are not absorbed by the peptide (9). However, our preliminary study showed that intranasal administration of native B:9–23 peptide (10–80 µg) failed to induce IAA in BALB/c mice. Based on our previous study that cholera toxin (CT) was able to strongly augment Th2-type humoral immunity when intranasally coadministered with a vaccine Ag (10), we used CT (2 µg) as a mucosal adjuvant with the peptide and successfully induced high levels of IAA in the BALB/c mice. In this study, we evaluated the effects of disabling the CTL epitope within the B:9–23 peptide (i.e., B:9–23 derived truncated peptide and A16,19 APL) in mucosal immunization regimens on IAA induction and the development of insulitis and diabetes in NOD mice. Our results indicate that elimination of the CTL epitope from the insulin peptide B:9–23 conferred efficient suppression of insulitis and diabetes in NOD mice when the peptide was intranasally coadministered with CT. In addition, prevention of diabetes by A16,19 APL/CT was associated with the elimination of IAA expression and significant inhibition of insulitis, while treatment with the native peptide that contains the CTL epitope enhanced IAA expression without providing any protection from the disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and antigens

Female NOD mice were purchased from Clea Japan and maintained in the Laboratory Animal Center for Biomedical Research at Nagasaki University (Nagasaki, Japan) under specific pathogen-free condition. All animal experiments described in this study were approved by the institutional animal experimentation committee and were conducted in accordance with the committee’s guidelines for animal experimentation.

Mouse proinsulin II B chain-derived peptides B:9–23, B:9–21, B:11–23, A16,19 APL (Table I), and tetanus toxin (TT) peptide:830–843 (QYIKANSKFIGITE) were chemically synthesized and purified by HPLC to >95% homogeneity (purchased from Sigma-Aldrich). Insulin-related peptides and the TT peptide were dissolved in sterile saline and adjusted to a neutral pH at a concentration of 4 mg/ml. CT was purchased from List Biological Laboratories and dissolved in sterile saline at a concentration of 400 µg/ml until use.


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  		Table I. Summary of the amino acid sequences of the insulin derived peptides studied and known epitopes for NOD islet derived T cell clones

 
Intranasal administration of synthetic peptides to mice

For diabetes prevention studies, mice were intranasally immunized starting at 4 wk of age (days 1–5 and 8 and then weekly until 10 wk of age) with 20 µg of peptide alone or together with 2 µg of CT. For remission studies, NOD mice at age 16–24 wk were treated with intranasal peptide administration once they were confirmed to have blood glucose levels between 200 and 249 mg/dl. The first day of intranasal treatment was counted as day 1 for this immunization regimen. Ten micrograms of the peptide was administered on days 1–5 and then twice a week for 40 days. Mice were anesthetized with ether, and 10 µl of a mixture containing the peptide and the adjuvant was slowly introduced into each nostril with a micropipette.

Measurement of antipeptide-specific Abs and IAA

Mice were bled and serum were obtained at 4, 6, 8, and 12 wk of age and stored at –20°C until the Ab assay was done. Abs against insulin peptide B:9–23 and A16,19 APL were measured by ELISA as previously described (11). Briefly, high binding Costar EIA/RIA 96-well microplates (catalog no. 3369; Corning) were coated overnight at room temperature with 1 µg of B:9–23 peptide dissolved in 100 µl of PBS per well. Wells were thoroughly washed three times with PBS and blocked with PBS containing 2% BSA and 0.01% sodium azide for a minimum of 2 h at room temperature. Wells were washed and 100 µl of 1/500 buffer-diluted serum samples were incubated for 2 h at room temperature. Wells were washed and biotin-conjugated rat anti-mouse IgG1 mAb (catalog no. 553441; BD Pharmingen) diluted 1/5000 in PBS was added to each well (100 µl/well) and incubated for 30 min. Five microliters of europium-labeled streptavidin (Wallac) was diluted in 10 ml and 100 µl was added to each well for 15 min. Wells were washed and 200 µl of enhancement solution (Wallac) was added per well and gently shaken for 10 min. Plates were analyzed by using the Victor2 V 1420 multilabel counter (Wallac). Specific Ab levels were expressed as the index defined as follows: (sample cpm – negative control cpm)/(positive control cpm – negative control cpm).

The levels of IAA in serum were evaluated prospectively at 4, 6, 8, and 12 wk of age by using a 96-well filtration plate micro-IAA assay as previously described (12). The 125I-labeled insulin Ag (Amersham Biosciences) at 20,000 cpm was incubated with 5 µl of serum with and without cold human insulin, respectively, for 3 days at 4°C in buffer A (20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1% BSA, 0.15% Tween 20, and 0.1% sodium azide). Fifty microliters of 50% protein A with 8% protein G-Sepharose (Amersham Biosciences) was added to the serum/insulin mixture solution in a MultiScreen NOB 96-well filtration plate (Corning) that was precoated with buffer A. The plate was shaken for 45 min at 4°C followed by two cycles of four washes with cold buffer B (identical to the buffer A except for 0.1% BSA). After washing the plates, 40 µl of scintillation liquid (Microscint-20; Packard Instrument) was added to each well and radioactivity was measured by using a TopCount scintillation counter (96-well plate beta counter; Packard). The result was calculated based on the differences in counts per minute ({Delta}cpm) between wells with and without cold insulin and expressed as an index defined as follows: (sample {Delta}cpm – human negative control {Delta}cpm)/(human positive control {Delta}cpm – human negative control {Delta}cpm). The index value of 0.01 was chosen as the cutoff limit of the normal serum level of IAA in a nondiabetic mouse strain.

Monitoring diabetes by blood glucose levels

Blood glucose levels were monitored by using Glutest-Ace (Sanwa Kagaku Kenkyusho) every other week starting at 12 wk of age for diabetes prevention studies. Mice with blood glucose levels >250 mg/dl for two consecutive measurements were considered diabetic. For remission studies, blood glucose levels were monitored twice a week for 40 days and mice having blood glucose levels >600 mg/dl or >400 mg/dl for four consecutive measurements were considered to have reached the study endpoint.

Insulin-reactive CD8 T Cells

Cloned CD8 T cells, derived from the islets of young NOD mice and designated G9C8, recognize the peptide insulin B:15–23 complex (6). A TCR transgenic mouse was generated from the TCR of the G9C8 clone on the NOD background (designated G9 transgenic mouse) and crossed to TCRC{alpha}–/– mice to ensure that all of the TCR transgenic cells are monoclonal and express the transgenic receptor. These mice are designated G9C{alpha}–/– NOD. The mice express CD8 T cells that respond to insulin and insulin peptide in an identical manner to that of the original clone (our unpublished observations). CD8 T cells were purified from G9C{alpha}–/– NOD splenocytes by using positive selection beads (Miltenyi Biotec) with >95% purity for proliferation and cytotoxic assays.

Proliferation assays

Purified insulin-reactive CD8 T cells (105) were incubated with 105 irradiated NOD spleen cells together with different concentrations of peptide, in triplicate, in a 96-well round-bottom plate for 72 h. Tritiated (0.5 µCl) thymidine was added for a further 14-h incubation. The plates were harvested and thymidine uptake was measured in counts per minute using a beta plate counter.

51Cr-release cytotoxicity assay

P815 cells (1 x 106) were incubated with 0.1 µCi of 51Cr-labeled sodium chromate in 100 µl for 1 h, washed, and resuspended at 104 cells per 50 µl. The cells were incubated, in triplicate, at 25°C with different peptide concentrations in round-bottom 96-well plates for 1 h. Purified insulin-reactive CD8 T cells were added to the plate at an E:T ratio of 10:1. The cell mixture was incubated for a further 16 h at 37°C. Fifty microliters of supernatant was assayed for 51Cr-release in a gamma counter. Specific lysis was determined as follows: ((cytotoxic release – minimum release)/(maximum – minimum)) x 100.

Histology

Pancreatic sections of mice immunized with insulin-related peptides were histologically analyzed at 12 wk of age by fixing tissues in 10% formalin and staining the paraffin-embedded samples with H&E. A minimum of 20 islets from each mouse were microscopically observed by two different observers for the presence of insulitis, and the levels of insulitis were scored according to the following criteria: 0, no lymphocyte infiltration; 1, islets with lymphocyte infiltration in <25% of the area; 2, 25–50% of the islet infiltrated; 3, 50–75% the islet infiltrated; 4, >75% infiltrated or small retracted islets.

Statistics analysis

Group differences were analyzed with the Tukey honestly significant difference (HSD) test and differences between Kaplan-Meier survival curves were estimated by the long rank test, with the use of Dr. SPSS II for Windows software (SPSS). Values of p < 0.05 were considered statistically significant. Insulitis levels were analyzed by Ridit analysis, and levels of t > 1.96 or < –1.96 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CT functions as an effective mucosal adjuvant for intranasally administered insulin B:9–23 peptide

Intranasal administration of 10 µg of the insulin B:9–23 peptide failed to induce anti-B:9–23-specific Abs (Fig. 1A). However, when the peptide was coadministered with a small amount of CT the Ab responses were significantly augmented, particularly at 6 wk of age (p < 0.0001 vs PBS group). The small amount of CT did not induce any obvious toxic effects. Mice given TT peptide with CT had no effect on B:9–23 peptide-specific responses, confirming that the response was insulin peptide-specific and that CT critically influenced the level of the specific immune response.


Figure 1
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  		FIGURE 1. Anti-B:9–23 peptide Ab expression (A), IAA expression (B), and life table analysis for the development of diabetes (C) following intranasal administration of B:9–23 either alone or together with CT in NOD mice are depicted. Peptides were intranasally administered starting at 4 wk of age (days 1–5 and 8, and then weekly until 10 wk of age). {square}, B:9–23 alone (n = 10); {blacksquare}, B:9–23/CT (n = 10); {triangleup}, TT/CT (n = 10); x, PBS, (n = 10); ***, p < 0.0001 vs PBS.

 
We next examined the effects of intranasal immunization with insulin peptides on IAA. IAA can be spontaneously expressed in NOD mice as early as 4 wk of age (13). Intranasal administration of B:9–23/CT, but not B:9–23 alone or TT/CT, significantly enhanced IAA expression at 6 wk of age (Fig. 1B and Table II) as compared with a group given PBS (p < 0.0001). Based on previously reported findings that the s.c. administration of B:9–23 peptide alone induced high levels of IAA and effectively prevented diabetes in the NOD mice (14), we expected that intranasal immunization with B:9–23/CT would also prevent the development of diabetes. However, in contrast to our expectation, intranasal administration of B:9–23/CT completely failed to prevent diabetes (Fig. 1C and Table II). As expected, the B:9–23 peptide alone or TT/CT had no effect on the development of diabetes.


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  		Table II. Summary of peptide/CT vaccination

 
CTL epitope elimination

As described in the Introduction, the failure of disease prevention by intranasal B:9–23/CT immunization could be partly due to the induction of peptide-specific CTL immunity by the CD8 insulin B:15–23 T cell epitope. Thus, we decided to evaluate the effects of CTL epitope elimination from the B:9–23 peptide by amino acid substitutions at the B:16 and 19 positions (i.e., A16,19 APL) or by truncation of the C-terminal amino acids (i.e., B:9–21 peptide) on nasally induced Ab responses and disease prevention. To confirm whether the substitution and truncation of a CTL epitope disabled B:15–23 specific CTL immunity, proliferation and 51Cr-release cytotoxicity assays of cloned insulin B:15–23 reactive CD8 T cells were performed. A comparison was made of insulin B:9–23 derived peptides with the B:15–23 peptide. As we expected, neither the A16,19 APL nor the B:9–21 peptide stimulated the proliferation of insulin-reactive CD8 T cells, while all other peptides containing the CTL epitope stimulated the CD8 T cells (Fig. 2A). In the proliferation, the larger peptides containing the B:15–23 epitope (B:9–23 and B:11–23) can be further processed to generate the epitope that can stimulate the CD8 T cells. In addition, as expected no cytotoxicity was stimulated by the A16,19 APL and the B:9–21 peptide. However unlike the proliferation assay, there was relatively low cytotoxicity toward targets coated with B:9–23 and B:11–23 (Fig. 2B). In the cytotoxicity assay, which is set up for peptides that will bind well into the Kd groove, the larger peptides are unlikely to bind appropriately to the MHC and, therefore, the stimulation of cytotoxicity using these peptides was limited.


Figure 2
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  		FIGURE 2. Functional responses of insulin-reactive CD8 T cells to peptides. A, Proliferation of insulin-reactive CD8 T cells to increasing concentrations of insulin B chain-derived peptides. The results are shown as a stimulation index (proliferation with peptide/proliferation without peptide). Background counts without peptide were 6786 + 657 cpm. B, 51Cr-release cytotoxicity assay showing lysis of P815 targets by the insulin-reactive CD8 T cells with increasing concentrations of peptides. The horizontal line shown is the cytotoxicity in the absence of peptide. •, B:9–23; {blacksquare}, B:9–21; {diamondsuit}, B:11–23,; {blacktriangleup}, A16,19 APL; x, B:15–23.

 
We evaluated both Abs to the peptide B:9–23 (Fig. 3A) and Abs to insulin (Fig. 3B). Although intranasal administration of A16,19 APL/CT or B:11–23/CT induced significant levels of anti-B:9–23 Abs as compared with control, B:9–21/CT was almost completely unable to induce anti-B:9–23 Abs (Fig. 3A and Table II). Analyses of serum IAA revealed that significantly higher IAA was induced at 8 and 12 wk of age in mice given the B:11–23/CT group (p < 0.05), but not in the B:9–21/CT-immunized mice as compared with spontaneously expressed levels of IAA in the control mice. Interestingly, mice intranasally treated with A16,19 APL/CT never expressed IAA at 6, 8, and 12 wk of age except for one mouse that was IAA positive at 4 wk of age before peptide immunization (Fig. 3B and Table II). To test whether A16,19 APL modulated the autoimmune response to native B:9–23 peptide, we coadministered A16,19 APL and native peptide. The addition of different doses of A16,19 APL did not suppress the IAA induced by the 20-µg dose of B:9–23 peptide/CT, indicating that A16,19 APL/CT suppressed spontaneous, but not induced, autoantibody formation (Fig. 3C).


Figure 3
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  		FIGURE 3. A and B, Anti-B:9–23 peptide Ab expression (A) and IAA expression (B) following intranasal administration of truncated B:9–23 and A16,19 APL together with CT in NOD mice. {blacksquare}, B:9–21/CT (n = 10); {diamondsuit}, B:11–23/CT (n = 9); {blacktriangleup}, A16,19 APL/CT (n = 8); •, control (CT/TT and PBS) (n = 14 and 15, respectively); *, p < 0.05; **, p < 0.001; ***, p < 0.0001 significant vs control. C, IAA expression following intranasal administration of 20 µg of B:9–23 peptide/CT supplemented with different doses (0–40 µg) of A16,19 APL in NOD mice. {diamondsuit}, B:9–23 peptide/CT alone (n = 10); {blacksquare}, B:9–23/CT with 10 µg of APL/CT (n = 10); {blacktriangleup}, B:9–23/CT with 20 µg of APL/CT (n = 10); x, B:9–23/CT with 40 µg of APL/CT (n = 10).

 
Elimination of CTL epitope critically influenced diabetes prevention efficacy

As expected, intranasal administration of A16,19 APL/CT, or B:9–21/CT strongly suppressed the development of diabetes as compared with the control group (p < 0.05) (Fig. 4 and Table II). The disease prevention efficacy attained by intranasal B:11–23/CT immunization was comparable with that achieved by the A16,19 APL/CT- and B:9–21/CT-immunized groups until 34 wk of age; however, the B:11–23/CT group started to develop the disease at 40 wk of age. We also found that the blood glucose levels in most of the A16,19 APL/CT immunized mice were <100 mg/dl until 40 wk of age, whereas animals of the other groups showed blood glucose levels higher than 100 mg/dl, suggesting that the A16,19 APL was able to more effectively suppress subclinical beta cell damage compared with the C-terminally truncated form of the peptide (data not shown).


Figure 4
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  		FIGURE 4. Life table analysis for the development of diabetes following intranasal administration of native B:9–23, truncated B:9–23, and A16,19 APL together with CT in NOD mice. Both intranasal-A16,19 APL/CT and intranasal-B:9–21/CT significantly suppressed the development of diabetes.

 
Prevention of diabetes was associated with the degree of insulitis inhibition

Histological analysis of pancreatic islets of mice intranasally immunized with insulin-derived peptides revealed that B:9–23/CT and B:11–23/CT accelerated the development of insulitis as compared with the control group (T = 2.43 and 2.81, respectively) (Fig. 5 and Table II). In contrast, we found that administration of B:9–21/CT or A16,19 APL/CT significantly suppressed the development of insulitis (T = –1.98 and –4.55, respectively), and all islets of mice immunized with the A16,19 APL/CT had no insulitis or only minimal peri-insulitis (Fig. 5 and Table II).


Figure 5
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  		FIGURE 5. Insulitis levels at 12 wk determined by Ridit analysis. A level of T > 1.96 was regarded as significant increase. A level of T < –1.96 was regarded as significant suppression. Insulitis levels were significantly increased in the B:9–23/CT (T = 2.43) and B:11–23/CT (T = 2.81) compared with the CT alone in contrast to being suppressed slightly in the B:9–21/CT (T = –1.98) and strongly in the A16,19 APL/CT (T = –4.55).

 
Reversal of hyperglycemia and prevention of progression to diabetes was more often achieved by intranasal administration of the A16,19 APL than the native B:9–23 peptide

Finally, we evaluated the efficacy of insulin-derived peptides to reverse diabetes after the onset of hyperglycemia (Fig. 6). We found that 85% of the control NOD mice that became hyperglycemic (glucose levels reaching 200–249 mg/dl) eventually developed the endpoint of sustained severe hyperglycemia (>400 mg/dl) within 40 days of initial hyperglycemia. In contrast, intranasal immunization with A16,19 APL/CT resulted in remission from hyperglycemia in 65% of mice, more often than the control mice given PBS (p < 0.05). Native B:9–23 peptide/CT-treated mice showed a 36% remission.


Figure 6
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  		FIGURE 6. Blood glucose levels in the NOD mice intranasally treated with A16,19 APL/CT (top left), B:9–23/CT (top right), and control (bottom left) starting on the onset of diabetes with glucose levels between 200 mg/dl and 249 mg/dl. The peptide was intranasally immunized on days 1–5 and then twice a week until 40 days. Blood glucose levels were monitored twice a week until 40 days and we stopped the experiment at the endpoint when blood glucose levels were >600 mg/dl or 400 mg/dl four times in continuity. The mice that developed endpoint are shown as open circles ({circ}) and others are shown as filled squares ({blacksquare}).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Multiple studies indicate that insulin/proinsulin is a key target in the pathogenesis of type 1 diabetes (2, 15). Several proinsulin-derived peptides, including insulin A and B chain peptides and a B-C junction peptide, have been reported to be recognized by T cells from humans at risk for type 1 diabetes (16, 17) and from NOD mice (15, 18). B:9–23-specific autoreactive T cell clones are found in islet infiltrates (19) and adoptively transfer diabetes into NOD SCID mice (18). The B:9–23 peptide protects NOD mice from diabetes when given subcutaneously (4, 14). With this "vaccine" NOD mice express significantly higher and long-lasting IAA compared with the levels of spontaneous IAA expression (9). Using insulin gene knockouts we have recently reported that the insulin peptide B:9–23 might be an essential target of immune destruction in the NOD mouse (3).

Mucosally induced immune regulation has been harnessed to prevent experimental autoimmune diseases, and therapeutic responses have been reported after nasal, but not oral, administration of Ags (20, 21). In this study, we found that intranasal immunization with native B:9–23 peptide in combination with CT induced significant levels of IAA without protecting against the development of diabetes in NOD mice (Fig. 1). Martinez and coworkers have reported that intranasal administration of proinsulin B24–C36 induces regulatory CD4+ T cells that, however, could not inhibit the development of spontaneous diabetes in the NOD mice (1). They found that B25–C34 was a CTL epitope and that disabling it (truncation at its C terminus) inhibited diabetes (1). In this study, we have altered the B:15–23 CTL Kd epitope, previously identified (6), by truncation at the B:9–23 C terminus or amino acid substitution (alanine for tyrosine) at position B:16. Intranasal immunization with B:11–23/CT that retains the B:15–23 CD8 epitope, but not with B:9–21/CT, induced significant levels of IAA (Fig. 3B). This is consistent with our previous finding that one of the identified minimum epitopes, B:13–23, but not the B:9–16 epitope, induces IAA expression in normal BALB/c mice (9). Interestingly, intranasal B:11–23/CT as well as B:9–23/CT accelerated the development of insulitis but did not accelerate or inhibit diabetes development (Fig. 4). Immunization of young NOD mice with islet autoantigens such as the insulin B chain peptide prevents autoimmune diabetes (4); however, it can accelerate the development of insulitis, especially when peptides are coadministered with polyinosinic-polycytidylic acid in NOD mice (K. Fukushima, N. Abiru, and M. Kobayashi, unpublished observations). The observations made in this study and by others suggest that the B:9–23-derived peptide containing a functional CTL epitope can expand both regulatory and pathogenic T cells. In contrast to the native peptide, intranasal B:9–21/CT immunization provided significant prevention of spontaneous diabetes development with a suppressive effect on insulitis (Figs. 4 and 5) and without any effect on the spontaneous expression of IAA (Fig. 3B).

APLs derived from T cell-reactive self-Ags have been shown to function as protective therapeutic agents in animal models of autoimmunity. The mechanisms by which APLs may modulate immune response include antagonism (22, 23), anergy (24, 25), and immune deviation (26). Many of the analogues to date were agonistic peptides rather than antagonistic ones. B16 tyrosine is a critical amino acid for peptides that activate both NOD islet-derived CD4+ and CD8+ T cell clones. We previously reported that alteration of the tyrosine residue to alanine at this position abrogated the proliferative responses of anti-B:9–23 CD4+ T cell clones (5). The substitution of tyrosine with alanine at the B:16 position results in the failure of the B:15–23 peptide to bind to the Kd molecule (7). Female NOD mice with a single amino acid alteration at residue 16 (from tyrosine to alanine) in the insulin B chain are dramatically protected from diabetes (3). Alleva and coworkers reported that the B:9–23-altered peptide ligand that contains alanine substitutions at residues 16 and 19, A16,19 APL (namely NBI-6024), substantially delays the onset and reduces the incidence of diabetes when given s.c. without adjuvant (8). We previously found that the induction of insulin autoantibodies is specific for the B:9–23 peptide among proinsulin-derived peptides. The Abs induced by the B:9–23 peptide react with intact insulin but are not absorbed with the peptide itself, suggesting that the B:9–23 peptide is not a B cell epitope in intact insulin and that spontaneously occurring anti-insulin B cells are activated with the help of activated T cells specific to the B:9–23 peptide (9). In our study, intranasal administration of A16,19 APL coadministered with a potent mucosal immune potentiator efficiently prevented diabetes and induced Abs reacting not only with A16,19 APL but also with the native B:9–23 peptide. Contrary to our expectation that the nasally administered A16,19 APL/CT would be associated with the enhancement of IAA expression and the acceleration of insulitis, similar to the native peptide, intranasal immunization with this peptide combined with CT completely eliminated IAA expression and strongly suppressed the development of insulitis. The inhibition of insulitis with A16,19 APL/CT should not be considered as an antagonist of the native insulin peptide binding to the Kd molecule, because A16,19 APL by itself is unable to bind MHC class I (7).

The presence of small amounts of CT was critically important for the induction of the Abs and disease prevention. Alternative adjuvants that might be safer for human use, such as a nontoxic mutant of CT or a related heat-labile toxin from enterotoxigenic Escherichia coli have been developed and their efficacy as mucosal adjuvants has been demonstrated (27, 28).

These studies in NOD mice demonstrate unique (native B:9–23 vs A16,19 APL) differential immunologic effects (induction vs suppression of IAA) and enhanced protection by combining the APL with specific adjuvant. Such a potent combined therapy we believe provides initial proof of concept that, with an appropriate adjuvant and a peptide, potent disease and autoantibody suppression can be achieved.


   Acknowledgment
 
We thank Marcella Li at Barbara Davis Center for Childhood Diabetes (Denver, CO) for her technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by research grants from Japan Society for the Promotion of Science (17590940), the Japan Diabetes Foundation (No. 14–55), and in part by a Grant-in-Aid for Scientific Research from Nagasaki University, Nagasaki, Japan. Back

2 Current address: Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO 80262. Back

3 Address correspondence and reprint requests to Dr. Norio Abiru, First Department of Internal Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki, Japan. E-mail address: f1931{at}cc.nagasaki-u.ac.jp Back

4 Abbreviations used in this paper: B:9–23, insulin B chain peptide with aa 9–23; APL, altered peptide ligand; A16,19 APL, APL with alanine substitutions at positions 16 and 19; CT, cholera toxin; IAA, insulin autoantibody; TT, tetanus toxin. Back

Received for publication February 25, 2006. Accepted for publication May 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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