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The Journal of Immunology, 2008, 180, 58-63
Copyright © 2008 by The American Association of Immunologists, Inc.
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A Th1-Recognized Peptide P277, When Tandemly Repeated, Enhances a Th2 Immune Response toward Effective Vaccines against Autoimmune Diabetes in Nonobese Diabetic Mice1

Liang Jin*, Aihua Zhu{dagger}, Yu Wang*, Qingmei Chen*, Qiyan Xiong*, Jianping Li*, Yunxiao Sun*, Taiming Li*, Rongyue Cao*, Jie Wu* and Jingjing Liu2,*

* Minigene Pharmacy Laboratory, Biopharmaceutical College, China Pharmaceutical University, Nanjing, People’s Republic of China; and {dagger} Key Laboratory of Biotechnology for Medicinal Plants of Jinangsu Province, Xuzhou Normal University, People’s Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Subunit immunogens containing tandemly repeated copies of T and B cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. To investigate whether the increased copies of the Th2-activated peptide sequence will enhance the Th2-like immune response, we compared the cytokine secreted in mice that inoculated with two immunogens containing one or six tandemly repeated copies of a Th2-activated peptide sequence P277. Immunization of mice with a 6xP277 fusion protein elicited much higher levels of Th2-type cytokines and lower Th1-type cytokines than with a fusion protein with one P277 peptide. The data of tandemly repeated peptide P277 potentiate the anti-inflammatory in NOD mice, most likely associated with a Th1 to Th2 cytokine shift specific for the autoimmune T cells, which suggested that application of multiple tandem repeats of a Th2-activated epitope is an efficient method to enhance the anti-inflammatory immune response by shifting the immune response from Th1-like to Th2-like. The subunit immunogens containing tandemly repeated copies of peptide P277 might be effective vaccines against autoimmune diabetes in NOD mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 diabetes is caused by an autoimmune process in which autoreactive T lymphocytes invade the pancreatic islet and destroy the insulin-producing β cells (1). Although the autoimmune T cells of both CD4+ and CD8+ subsets are all involved in the disease process (2), CD4+ T cells on their own are able to trigger diabetes (3). It is known that the autoimmune CD4+ T cells can be divided into two functional groups by the cytokines they secrete when activated: Th1 and Th2 (4). Th1 cells secrete IL-2, IFN-{gamma}, and TNF. IL-2 can induce T cell proliferation and proinflammatory cytokines; IFN-{gamma} can mediate tissue inflammation. In contrast, Th2 cells secrete anti-inflammatory cytokines such as IL-4 and IL-10 that can inhibit Th1 activation by suppressing the production of proinflammatory cytokines (5) and affecting Ag presentation (6). Adoptive transfer experiments have led to the conclusion that Th1-type, but not Th2-type T cell clones can transfer diabetes (7, 8), which suggested that the diabetogenic autoimmune process might be aborted by inducing a shift in the relevant autoimmune T cell activity from the Th1 type to the Th2 type (9, 10).

Recent studies of the factors important in the pathogenesis of the autoimmune diabetes have implicated certain islet proteins as early targets of the autoreactive T cells (11), one of which is the 60-kDa heat shock protein (HSP60)3 (12). Peptide P277 is a 24-aa fragment of the HSP60 molecule, first discovered to be an Ag for diabetogenic T cell clones in NOD mice (13). Maryam et al. (14) showed that the peptide P277 recognized by Th1 cell clone. Therapeutic vaccination with P277 can arrest the spontaneous diabetogenic process associated with a Th1 to Th2 cytokine shift specific for the autoimmune T cells (15, 16). The results suggest that the peptide P277 leads to activation of T cells specific for Th2 clone.

In recent years, it has been established that immunogens containing tandemly repeated B or T cell epitopes increased immunogenicity associated with increased copies of the target sequence (17, 18). We have been interested in whether the increased Th2 immune response is also attributable to copies of the Th2-activated peptide sequence. Therefore, two immunogens were constructed containing either a single or six tandemly repeated copies of the Th2-activated peptide P277 (19). The hypotheses have been examined by comparing the cytokine secreted in the mice that inoculated with the two immunogens. It is shown that the effectiveness of anti-autoimmune diabetes of the tandem repeat construct is superior to that of the single copy construct. This effect appears to result from an enhanced recognition of the tandemly repeated P277 sequence by the Th1 cells, leading secondarily to more efficient inhibition of the Th1 cells and the consequent increased recruitment of Th2 cells. These results demonstrated an important role of the multiple tandem repeats of a Th2-activated peptide in enhancing the Th2 immune response.


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

Four-week-old female NOD/Lt mice, with average weight of 18.8 g, were purchased from Slaccas Experimental Animal (SCXK 2003-0003) and maintained under pathogen-free conditions at the Animal Center of our institute. The onset of clinical insulitis begins at ~3 mo of age and reaches a cumulative incidence of 80% or greater by 8 mo of age in this colony for females.

Antigens

The fusion proteins HSP65-6 x P277, HSP65-P277, and HSP65 were prepared, as described (19). Briefly, a single or six tandemly repeated copies of the peptide were fused to mycobacterial HSP65 to construct the fusion protein HSP65-6 x P277 and HSP65-P277. The resulted recombinant proteins were expressed in Escherichia coli and subsequently purified as immunogens in the studies. All proteins were dissolved in sterilized PBS (pH 7.4). The sequence of P277 residues (437–460 in the H-HSP65 sequence) is VLGGGCALLRCIPALDSLTPANED. Purified human rVEGF-P277 was supplied by Z. Aihua (Shanghai, China) in our laboratory. OVA, Con A, and BSA were purchased from Sigma-Aldrich.

Vaccination

Four-week-old female NOD mice were divided into 4 groups of 10 animals each (n = 10 per group). Three groups of mice received three in trans inoculations of immunogen containing 100 µg of purified HSP65-6 x P277, HSP65-P277, and HSP65, respectively, at 4, 7, and 10 wk of the age; the control mice received three in trans inoculations of PBS (pH 7.4). The serum samples were collected before every inoculation. After the third administration, serum samples were collected at monthly interval for 5 mo. All collected samples were stored at –20°C until use.

Hyperglycemia

The mice blood glucose level was measured monthly by Hitachi automatic analyzer (model 7150). A mouse was considered to be diabetic if the blood glucose level was >11 mM on two consecutive examinations.

Pancreas histopathology

Mice from each treatment group were analyzed at the age of 8 mo, when almost all the control NOD mice receiving phosphate buffer were sick. The pancreata were fixed with 10% formalin solution. Formalin-fixed paraffin blocks of pancreas tissue were sectioned with a microtome and stained with H&E (Sangon). The degree of insulitis was evaluated by a pathologist in a blinded fashion. The average degree of insulitis was assessed over 20 islets scored per pancreas. Each islet was classified as follows: clear, if no infiltrate was detected; mildly infiltrated, if peri-insulitis or an intraislet infiltrate occupied <25% of the islet; infiltrated or heavily infiltrated, if 25–50% or >50% of the islet was occupied by inflammatory cells.

T cell proliferation

Four weeks after the last adminstration, the mice spleens were removed and the T cell proliferative responses were assayed in vitro. Briefly, microtiter wells contained 2 x 105 cells in 0.1 ml of culture medium in the presence of one kind of Ag as follows: Con A, HSP65-6 x P277, HSP65-P277, and HSP65, respectively. Dose-response curves were made to establish optimal doses (data not shown). The concentration of 10 µg/ml was chosen for HSP65-6 x P277, HSP65-P277, and HSP65, and 1.25 µg/ml was chosen for Con A. Cultures were incubated for 72 h at 37°C in a humidified atmosphere with 7.5% CO2. T cell responses were detected by MTT method. Briefly, 0.02 ml of MTT (Sigma-Aldrich) solution (5 mg/ml in PBS) was added to each well, and the microplates were further incubated at 37°C for 4 h in a humidified atmosphere with 7.5% CO2. Supernatants were then discarded, and 0.2 ml of acidified 20% SDS (0.04 N HCl in 20% SDS) was added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan for 24 h. Formazan quantification was measured by Multiskan spectrum microplate spectrophotometer (Thermo) with a 570-nm test wavelength and a 690-nm reference wavelength. Data were expressed as mean stimulation index of triplicate samples ± SEM.

Cytokine assays

Supernatants were collected after 72 h of stimulation with Ags of HSP65-6 x P277, HSP65-P277, Con A, and medium alone, respectively. Murine IL-10 and IFN-{gamma} were quantitated in culture supernatants by ELISA (BioSource International), according to the manufacturer’s instructions. BioSource recombinant mouse cytokines were used as standards for calibration curves. Briefly, 0.1-ml culture of supernatant or recombinant cytokine was incubated for 2 h at 37°C. After the plates were washed, 0.1 ml of biotinylated detection Abs was added for 1 h at 37°C, then extensively washed and incubated with streptavidin conjugated with alkaline phosphatase for 1 h at 37°C. The plates were washed, and alkaline phosphatase substrate was added and incubated at 37°C for 10 min in dark room. The reaction was stopped by 1 drop of 2 M H2SO4, and the samples were read at 492 nm by a microplate spectrophotometer (Multiskan; Thermo) at room temperature. Cytokine levels are calculated as picograms per milliliter based on standard curves. The detection limit for the experiments described in this study was 15 pg/ml for IL-10 and IFN-{gamma}.

Statistical significance

Data generated from animals immunized with HSP65-6 x P277 were compared with animals that received HSP65-P277 and PBS. Student’s t test was conducted to assay significant differences between the different experimental groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of HSP65-6 x P277 treatment on type 1 diabetes in NOD mice

Fig. 1A shows the progression of hyperglycemia development recorded in real time. At the beginning of treatment, all the 4-wk-old female NOD/Lt mice were healthy and had normal blood glucose. Approximately 80% of the mice either developed hyperglycemia or were dead in control group within 6~8-mo period. Of the total of 10 mice that received HSP65-P277, 4 were dead from severe diabetes by 8 mo. By contrast, none of the 10 mice treated with HSP65-6 x P277 at 8 mo of age died (Fig. 1, B and C). Fig. 1E shows the concentration of each mouse and the cumulative incidence of each group at the age of 8 mo. In control group, 8 of the 10 mice eventually developed type 1 diabetes and only 2 escaped the disease; the incidence of HSP65-P277 group was 50%; but none of the mice in group HSP65-6 x P277 developed type 1 diabetes disease (p < 0.05).


Figure 1
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  		FIGURE 1. Effect of HSP65-6 x P277 treatment on type 1 diabetes in NOD mice. To test whether inoculation with HSP65-6 x P277 might indeed protect these mice from developing diabetes and whether the level of protection is higher than in mice inoculated with HSP65-P277, we immunized 4-wk-old female NOD mice with HSP65-6 x P277, HSP65-P277, HSP65, and PBS three times at 4, 7, and 10 wk of age, respectively, and followed their glucose levels. A, The actual progression of hyperglycemia recorded in real time. B, The mortality of the mice immunized with HSP65-6 x P277 (represented by beeline, n = 10) in each month comparing with PBS (arrow). C, The mortality of the mice immunized with HSP65-P277 (beeline) in each month comparing with PBS (arrow). D, The mortality of the mice immunized with HSP65 (beeline) in each month comparing with PBS (arrow). E, The concentration of blood glucose and cumulative incidence of diabetes at the age of 8 mo. In control group, eight of the mice eventually developed type 1 diabetes and only two escaped the disease, and the highest blood glucose level arrived at 24.28 mmol/L; in the HSP65-P277 group, five of the mice eventually developed type 1 diabetes. However, none the mice of group HSP65-6 x P277 eventually developed type 1 diabetes, and all escaped the disease. The level of protection was higher than in mice inoculated with HSP65-P277 (p < 0.05).

 
Effect of HSP65-6 x P277 treatment on insulitis

At the end of the observation period, when the mice were 8 mo old, the pancreata from all mice were obtained for histological examination. The predicament of the pancreas in mice treated at 20 wk showed a difference between the HSP65-6 x P277-treated and the HSP65-P277-treated mice as follows: ~80% of islets in HSP65-6 x P277-treated mice, but 30% of those in HSP65-P277-treated mice were free of insuitis (p < 0.05) (Fig. 2A). Fig. 2B demonstrated the results obtained on histological examination of the pancreas showed that there was a significant increase in the number of islets free of insulitis, fewer necrosis areas formed in the pancreas tissue, and a few lymphocytes filtrated around the islets of pancreas in the mice treated with HSP65-6 x P277. There were a few necrosis areas formed in the pancreas tissue and a few lymphocytes filtrated around the islets of pancreas from HSP65-P277-vaccinated mice (Fig. 2C). In contrast, much necrosis and marked atrophy of pancreas islets and many lymphocytes filtrated around the islets were observed in PBS-treated mice (Fig. 2D).


Figure 2
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  		FIGURE 2. Effect of HSP65-6 x P277 treatment on insulitis. A, Reduction of insulitis by HSP65-6 x P277 vaccination. At the end of the observation period, when the mice were 8 mo old, pancreata were obtained for histological examination. The average degree of insulitis was assessed over 20 islets scored per pancreas. B, From HSP65-6 x P277-vaccinated mice: a significant increase in the number of islets free of insulitis, fewer necrosis areas formed in the pancreas tissue, and a few lymphocytes filtrated around the islets of pancreas (original magnification, x200). C, From HSP65-P277-vaccinated mice: a few necrosis areas formed in the pancreas tissue and a few lymphocytes filtrated around the islets of pancreas (original magnification, x200). D, From control mice: much necrosis and marked atrophy of pancreas islets showed and many lymphocytes filtrated around the islets (original magnification, x200).

 
Effect of HSP65-6 x P277 treatment on T cell proliferation

Increasing spontaneous T cell reactivity to HSP60 has been previously related to the progression of diabetes (20), and modulation of the HSP60-specific immune response was associated with the control of the diabetogenic process (21). We therefore analyzed the splenocytes isolated from HSP65-6 x P277-treated and HSP65-P277-treated animals to check their proliferative response to HSP65-6 x P277 and HSP65-P277. As shown in Fig. 3, PBS-treated NOD mice manifested spontaneous reactivities to HSP65-6 x P277 and HSP65-P277. In contrast, splenocytes from the mice vaccinated with HSP65-6 x P277 showed diminished reactivates to HSP65-6 x P277, and the ability of down-regulation of spontaneous proliferative T cell responses much stronger than HSP65-P277 (p < 0.05). However, T cells from both treated and nontreated mice showed similar reactivates to Con A and indicated that there was no general inhibition of T cell reactivity induced by HSP65-6 x P277 vaccination. This result suggested that prevention of diabetes was associated with down-regulation of spontaneous proliferative T cell responses to the fusion protein HSP65-6 x P277. No responses were detected by the same preparation of E. coli contained an expression vector harbors no HSP65-6 x P277 gene; this result excluded the possibility that the above observed phenomenon was due to E. coli-derived artifacts.


Figure 3
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  		FIGURE 3. Effect of HSP65-6 x P277 treatment on T cell proliferation. The mice immunized with HSP65-6 x P277, HSP65-P277, HSP65, P277, and PBS. Four weeks after the last dose, the spleens were removed and the T cell proliferative responses were assayed in vitro after 72 h of stimulation with 10 µg/ml HSP65-6 x P277 and HSP65-P277 or 1.25 µg/ml T cell mitogen Con A. Data were expressed as mean stimulation index (SI) of triplicate samples ± SEM. *, p < 0.01 compared with Con A-treated mice.

 
Effect of HSP65-6 x P277 treatment on cytokines

To test whether the increased copies of the Th2-activated peptide P277 would enhance the Th2-like immune response, we compared the levels of cytokine secreted in mice that inoculated with two immunogens, HSP65-6 x P277 and HSP65-P277. The amount of IL-10 and IFN-{gamma} secreted by spleen cells after HSP65-6 x P277 and HSP65-P277 stimulation in vitro was analyzed. Because different cytokines are secreted in different physiological amount, we incubated the control mice of spleen cells with a prototypic T cell mitogen of Con A. As shown in Fig. 4, the different experimental groups did not differ in their response to Con A, and immunization of mice with the fusion protein HSP65-6 x P277 elicited much higher levels of Th2-type cytokines (Fig. 4A) and lower Th1-type cytokines (Fig. 4B) than with HSP65-P277 (p < 0.05). However, compared with the amount of cytokine released in response to Con A stimulation, IFN-{gamma} secretion on stimulation with HSP65-6 x P277 was lower in the HSP65-6 x P277-treated animals. This down-regulation of IFN-{gamma} secretion was associated with an increase in IL-10 secretion in response to stimulation with HSP65-6 x P277.


Figure 4
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  		FIGURE 4. Effect of HSP65-6 x P277 treatment on cytokines. Supernatants were collected after 72 h of stimulation with test Ags, Con A, or medium alone. The concentration of 10 µg/ml was chosen for the HSP65-6 x P277 and HSP65-P277, and 1.25 µg/ml was chosen for Con A. Murine IL-10 (A) and IFN-{gamma} (B) were quantitated in culture supernatants by ELISA kit purchased from BioSource International, according to the manufacturer’s instructions. Data are shown as the mean ± SD of triplicates. *, p < 0.01 compared with Con A-treated mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The major findings of the present study were the increased effectiveness of prevention of autoimmune diabetes in NOD mice associated with the copies of the peptide P277 in the immunogen. It is possible that the Th2-activated peptide P277, when tandem repeated, enhanced the Th2 immune response. We examined this hypothesis by comparing the cytokine secreted in mice that inoculated with two immunogens containing one or six tandemly repeated copies of a Th2-activated peptide sequence P277. Immunization of mice with the fusion protein HSP65-6 x P277 elicited much higher levels of Th2-type cytokines and lower Th1-type cytokines than with HSP65-P277 (p < 0.05).

More and more evidence supported the idea that the peptide P277 was a bidirectional bomb. Peptide P277 has been found to serve as a functionally important target in mouse type 1 diabetes, but a single s.c. administration of peptide P277 (100 µg in oil), even late in the autoimmune process, can arrest β cell damage in most NOD mice (22). Thus, specific Abs to P277 have been found to mark the arrest of spontaneous type 1 diabetes induced by treatment of NOD mice with peptide P277 itself (21, 23). To overcome the potential poor immunogenicity of the peptide P277, we have constructed the fusion protein HSP65-6 x P277, as described (19). Oscherwitz et al., as well as Richard et al. (17, 18) reported that the immunogens containing tandemly repeated B or T cell epitopes increased immunogenicity associated with increased copies of the target sequence. Our data confirmed their results. The anti-P277 Ab level induced by the tandem repeat construct is superior to that of the single copy construct (p < 0.01). We may not elucidate the mechanisms leading to the results of that tandem repeat epitope can enhance immunogenicity of the target sequence; however, Ab-producing cells, B cells, might be, very possibly, recruited by the immunogens containing tandemly repeated cell epitopes in vivo. B cells are not only central to the production and amplification of humoral immune response, they also play an important role as APCs in the generation of T cell-mediated immune responses (24, 25). In addition, recent studies indicated that human HSP60 induced naive mouse B cells to proliferate and to secrete IL-10 (26) and the activated B cells inhibited spontaneous Th1 autoimmunity (27, 28). But we cannot confirm whether or not the activated B cells play a primary role in enhancing the Th2-type immune response in our studies, because the possibilities have not been tested experimentally in depth.

Peptide P277 was recognized by Th1 cell clone (14) and identified as containing a target epitope for diabetogenic T cells (13). However, administration of P277 can arrest the diabetogenic process (22). Peptide treatment was marked by down-regulation of T cell proliferation to P277 and by up-regulation of anti-P277 Abs of the IgG1 and IgG2b isotypes. These changes in autoimmune reactivity were accompanied by a shift of the autoimmune process from a proinflammatory Th1-like response to an anti-inflammatory Th2-like response (21). The results suggest that the peptide P277 leads to activation of T cells specific for Th2 clone. It is conceivable that the increased copies of the Th2-activated peptide P277 will enhance the immune response specific for Th2 type. Interestingly, in our studies, we found that the mice inoculated with the fusion protein HSP65-6 x P277 elicited much higher levels of Th2-type cytokines and lower Th1-type cytokines than with HSP65-P277 (p < 0.05). At present, we do not know how the Th2-activated peptide P277, when tandem repeated, inhibited the Th1 cells and recruited Th2, but the results reported in this study and elsewhere suggest the following possibility: 1) mice vaccinated with HSP65-6 x P277 elicit high level Ab formation, which suggested that Ab-producing cells, B cells, must be activated, and the activated B cells secreted IL-10 and inhibited spontaneous Th1-like response (26, 27, 28); 2) successful treatment of NOD mice with peptide P277 is associated with the recognition of Th1 clone to P277 (13) along with a decrease in the proliferation of T cells in response to HSP60 and P277 (21). Peptide P277, when tandem repeated, may be facile recognized by diabetogenic T cells, leading secondarily to more efficient inhibition of the Th1 cells and the consequent increased recruitment of Th2 cells. Irrespective of how HSP65-6 x P277 might function as a immunogen in mouse type 1 diabetes, the present results show that a Th1-recognized peptide P277, when tandem repeated, inhibited the proliferation of T cells in response to HSP65-6 x P277 and elicited much higher levels of Th2-type cytokines (such as IL-10) and lower Th1-type cytokines (such as IFN-{gamma}). HSP65-6 x P277 might be used as a preventive agent to arrest the autoimmune process in NOD mice.

It is also worth noting that we inoculated the mice by mucosal administration. Mucosal administration not only has provided numerous advantages over conventional s.c. injection, such as convenience, safety, and acceptability by more people, but also has proven effective as a treatment for a large number of animal models of autoimmune disease (29). Now, mucosal administration has been a novel therapeutic approach for treating processes not classically considered to be autoimmune, but involving an inflammatory component. For example, mucosal administration of HSP in mice lacking the receptor for low density lipoprotein can cause significant decrease in the size of atherosclerotic plaques, and suppress inflammation and atherosclerosis development (30). Weimer et al. (31) showed that nasal administration of amyloid A-β peptide limits decreased amyloid plaque deposition in a transgenic animal model of Alzheimer’s disease. The results of the present study suggest similar consideration for the treatment of type 1 diabetes. On the basis of our results, we postulate that mucosal treatment with HSP65-6 x P277 stimulates the development of adaptive immune cells that secret anti-inflammatory cytokines such as IL-10. However, whether mucosal administration plays a key role in inducing anti-inflammatory response to prevent the β-cell destruction needs to be tested experimentally in depth, because we did not adopt any other approach of inoculation in this study.

Our data, taken together with those cited above, can support the following possible model: an enhanced recognition of the tandemly repeated P277 sequence by the Th1 cells, leading secondarily to more efficient inhibition of the Th1 cells and the consequent increased recruitment of Th2 cells. Certainly, the evidence we have provided in this study that the mechanism contributing to the increased copies of the Th2-activated peptide P277 will enhance the immune response specific for Th2 type is plausible. Nevertheless, the beneficial effects of HSP65-6 x P277 on the type 1 diabetes are clear. We conclude that the behavior of the mouse immune system is susceptible to specific modification by the immunogens harboring tandem repeat peptide P277. These observations support the idea that the immune system is a cognitive system in which behavior can be self-correcting if the system is given suitable information (32, 33).


   Acknowledgments
 
We thank Dr. Z. Y. Shi for helping us to evaluate the degree of insulitis, Dr. Y. S. Wang for testing the purity of T cells, and Dr. Z. H. Fang for measuring the concentration of blood glucose.


   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 the National High Technology "863" Programs of China (2002 AA217031-2) and China National Natural Science Fund Committee (Grants 30701023, 30672464, and 30500458). Back

2 Address correspondence and reprint requests to Dr. Jingjing Liu, Minigene Pharmacy Laboratory, Biopharmaceutical College, China Pharmaceutical University, Tongjia Xiang 24, Nanjing, 210009, People’s Republic of China. E-mail address: minigene1{at}yahoo.com.cn Back

3 Abbreviation used in this paper: HSP, heat shock protein. Back

Received for publication April 6, 2007. Accepted for publication October 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Daneman, D.. 2006. Type 1 diabetes. Lancet 367: 847-858. [Medline]
  2. Miller, B. J., M. C. Appel, J. J. O’Neil, L. S. Wicker. 1988. Both the Lyt-2+ and L3T4+ T cell subsets are required for the transfer of diabetes in nonobese diabetic mice. J. Immunol. 140: 52-58. [Abstract]
  3. Christianson, S. W., L. D. Shultz, E. H. Leiter. 1993. Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice: relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NOD-Thy-la donors. Diabetes 42: 44-55. [Abstract]
  4. Mosmann, T. R., R. L. Coffiman. 1989. TH1 and TH2 cells, different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7: 145-173. [Medline]
  5. Banchereau, J., M. E. Rybak. 1994. Interleukin 4. A. Thompson, ed. The Cytokines Handbook 2nd Ed.99-126. Academic Press, London.
  6. Moore, K. V., A. O’Garra, M. R. de Waal, P. Vieira, T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11: 165-190. [Medline]
  7. Katz, J. D., C. Benoist, D. Mathis. 1995. T helper cell subsets in insulin-dependent diabetes. Science 268: 1185-1188. [Abstract/Free Full Text]
  8. Healey, D., P. Ozegbe, S. Arden, P. Chandler, J. Hutton, A. Cooke. 1995. In vivo activity and in vitro specificity of CD4+ Th1 and Th2 cells derived from the spleens of diabetic NOD mice. J. Clin. Invest. 95: 2979-2985. [Medline]
  9. Tisch, R., B. Wang, D. V. Serreze. 1999. Induction of glutamic acid decarboxylase 65-specific Th2 cells and suppression of autoimmune diabetes at late stages of disease is epitope dependent. J. Immunol. 163: 1178-1187. [Abstract/Free Full Text]
  10. Quintana, F. J., P. Carmi, I. R. Cohen. 2002. DNA vaccination with heat shock protein 60 inhibits cyclophosphamide-accelerated diabetes. J. Immunol. 169: 6030-6035. [Abstract/Free Full Text]
  11. Roep, B. O.. 1996. Perspective in diabetes: T-cell responses to autoantigens in IDDM, the search for the Holy Grail. Diabetes 45: 1147-1156. [Abstract]
  12. Elias, D., D. Markovits, T. Reshef, Z. R. Van der, I. R. Cohen. 1990. Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc. Natl. Acad. Sci. USA 87: 1576-1580. [Abstract/Free Full Text]
  13. Elias, D., T. Reshef, O. S. Birk, Z. R. Van der, M. D. Walker, I. R. Cohen. 1991. Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc. Natl. Acad. Sci. USA 88: 3088-3091. [Abstract/Free Full Text]
  14. Maryam, F. H., M. O. Frantz, P. A. Morel. 2000. Prevention of diabetes in the NOD mouse by a Th1 clone specific for a hsp60 peptide. J. Autoimmun. 14: 133-142. [Medline]
  15. Jana, B., D. Elias, I. R. Cohen. 1997. Treatment of NOD diabetes with a novel peptide of the hsp60 molecule induces Th2-type antibodies. J. Autoimmun. 10: 323-329. [Medline]
  16. Francisco, J. Q., A. Rotem, P. Carmi, I. R. Cohen. 2000. Vaccination with empty plasmid DNA or CpG oligonucleotide inhibits diabetes in nonobese diabetic mice: modulation of spontaneous 60-kDa heat shock protein autoimmunity. J. Immunol. 165: 6148-6155. [Abstract/Free Full Text]
  17. Oscherwitz, J., M. E. Zeigler, C. J. Loh, T. E. Gribbin, K. B. Cease. 1997. The enhanced immunogenicity of a recombinant immunogen containing a tandemly repeated HIV-1 T helper epitope is an intrinsic immunological property unrelated to epitope dose. Vaccine Res. 6: 15-25.
  18. Richard, L. M., P. Martineau, M. Hofnung, C. Leclerc. 1993. Induction of T-cell responses by chimeric bacterial proteins expressing several copies of a viral T-cell epitope. Eur. J. Immunol. 23: 2998-3002. [Medline]
  19. Jin, L., Y. Wang, Q. Y. Xiong, Q. M. Chen, J. P. Li, A. H. Zhu, R. Y. Cao, J. Wu, J. J. Liu. 2007. Long-lasting specific antibodies against P277 induced by mucosal administration of P277 repeat sequences carried by Hsp65 in the absence of adjuvants. Vaccine 25: 2043-2050. [Medline]
  20. Birk, O. S., D. Elias, A. S. Weiss, A. Rosen, R. van-der Zee, M. D. Walker, I. R. Cohen. 1996. NOD mouse diabetes: the ubiquitous mouse hsp60 is a β-cell target antigen of autoimmune T cells. J. Autoimmun. 9: 159-166. [Medline]
  21. Elias, D., A. Meilin, V. Ablamunits, O. S. Birk, P. Carmi, S. Konen-Waisman, I. R. Cohen. 1997. Hsp60 peptide therapy of NOD mouse diabetes induces a Th2 cytokine burst and down-regulates autoimmunity to various β-cell antigens. Diabetes 46: 758-764. [Abstract]
  22. Elias, D., I. R. Cohen. 1994. Peptide therapy for diabetes in NOD mice. Lancet 343: 704-706. [Medline]
  23. Elias, D., I. R. Cohen. 1995. Treatment of autoimmune diabetes and insulitis in NOD mice with heat shock protein 60 peptide p277. Diabetes 44: 1132-1138. [Abstract]
  24. Mamula, M. J., C. A. Janeway. 1993. Do B cells drive the diversification of immune responses?. Immunol. Today 14: 151-153. [Medline]
  25. Kurt-Jones, E. A., D. Liano, K. A. HayGlass, B. Benacerraf, M. S. Sy, A. K. Abbas. 1988. The role of antigen-presenting B cells in T cell priming in vivo: studies of B cell-deficient mice. J. Immunol. 140: 3773-3778. [Abstract]
  26. Michal, C. S., N. Gabriel, P. F. Meirav, M. Felix, C. Pnina, Z. Z. Alexandra, L. Ofer, I. R. Cohen. 2005. Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway. J. Immunol. 175: 3594-3602. [Abstract/Free Full Text]
  27. Jide, T., Z. Dan, H. Lorraine, L. Yuxin, O. Angelica, L. K. Daniel. 2001. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice 1. J. Immunol. 167: 1081-1089. [Abstract/Free Full Text]
  28. Emiko, M., M. Atsushi, I. P. Frederic, K. B. Atul. 2000. Regulatory role of mature B cells in a murine model of inflammatory bowel disease. Int. Immunol. 12: 597-605. [Abstract/Free Full Text]
  29. Faria, A. M. C., H. L. Weiner. 1999. Oral tolerance: mechanisms and therapeutic applications. Adv. Immunol. 73: 153-264. [Medline]
  30. Maron, R., G. Sukhova, A. M. Faria, E. Hoffmann, F. Mach, P. Libby, H. L. Weiner. 2002. Mucosal administration of hsp65 decreases atherosclerosis and inflammation in Aortic Arch of low-density lipoprotein receptor-deficient mice. Circulation 106: 1708-1715. [Abstract/Free Full Text]
  31. Weimer, H. L., C. A. Lemere, R. Maron. 2000. Nasal administration of amyloid-β peptide decreases cerebral amyloid burden in mouse model of Alzheimer’s disease. Ann. Neurol. 48: 567-579. [Medline]
  32. Cohen, I. R.. 1992. The cognitive principle challenges clonal selection. Immunol. Today 13: 441-444. [Medline]
  33. Cohen, I. R.. 1992. The cognitive paradigm and the immunological homunculus. Immunol. Today 13: 490-494. [Medline]



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