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The Journal of Immunology, 2002, 169: 2436-2443.
Copyright © 2002 by The American Association of Immunologists

T Cell Response to Preproinsulin I and II in the Nonobese Diabetic Mouse1

Philippe Halbout*, Jean-Paul Briand{dagger}, Chantal Bécourt*, Sylviane Muller{dagger} and Christian Boitard2,*

* Institut National de la Santé et de la Recherche Médicale Unité 561, Hôpital Cochin-Saint Vincent de Paul, Paris, France; and {dagger} Centre National de la Recherche Scientifique-Unité Propre de Recherche 9021, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunization against insulin, insulin B chain, or B chain peptide B9–23 (preproinsulin peptide II33–47) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II33–47 is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Various autoantigens have been identified as targets of autoantibodies and T cells during the development of type 1 diabetes in both humans and the nonobese diabetic (NOD)3 mouse (1). Evidence that supports a critical role for candidate autoantigens includes preventing or accelerating diabetes in NOD mice, by inducing immune tolerance against autoantigens (1, 2, 3), by passively transferring autoantigen-specific T cell clones (4), or by immunizing with plasmid DNAs encoding autoantigens (5, 6, 7, 8). Further evidence has been sought by inducing (9, 10, 11) an autoimmune response to islets in normal mice immunized against candidate autoantigens (9, 12). In addition, targeting autoantigen expression as transgenes (13, 14, 15, 16) in APCs, pituitary cells, or pancreatic {beta}-cells has been shown to prevent NOD mice from developing diabetes. A main challenge, however, remains in deciphering the role of individual autoantigens and characterizing extensively, within these autoantigens, those epitopes that are recognized by T cells along the disease process.

The islet autoantigen chosen in the present study is proinsulin. This choice is justified by the fact that protection from disease has been observed in the NOD mouse by injecting insulin (3) or the insulin B chain (3). Furthermore, proinsulin is a predominant {beta}-cell protein and the only candidate autoantigen for which expression is relatively restricted to {beta}-cells. In the mouse, two isoforms of proinsulin encoded by distinct genes coexist. Proinsulin I and proinsulin II are both expressed by {beta}-cells but are differentially expressed by the brain and the thymus: only proinsulin II is expressed in both tissues. Proinsulin I expression is apparently restricted to {beta}-cells (17, 18, 19). Therefore, we could assume that proinsulin I epitopes are preferentially recognized by peripheral T cells in murine diabetes. A striking case of protection from diabetes has been observed, however, in the NOD mouse after administration of insulin B chain peptide 9–23, which is specific of murine proinsulin II B-chain (20, 21). Whether peptide B9–23 is the only determinant recognized on proinsulin II or only one of several epitopes that are recognized by T cells and whether the autoimmune response to insulin extends to proinsulin I remain unknown in this model.

The present study was undertaken to characterize immunogenic epitopes recognized by T cells isolated from islets of diabetes-prone mice. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four epitopes within the islet cell infiltrate of prediabetic female NOD mice. These were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice against individual peptides in CFA. Immunogenic epitopes were found on both isoforms of proinsulin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

NOD mice and ABH/Biozzi mice were bred in our own facilities under specific pathogen-free conditions. The prevalence of spontaneous diabetes in our NOD colony reaches 30% in males and 75% in females by 6 mo of age.

Culture medium

All cultures were maintained in DMEM complemented by 10% FCS, 20 mM HEPES, 1 mM sodium pyruvate, 100 IU/ml penicillin and streptomycin, 50 µM 2-ME, and 2 mM L-glutamine. All these reagents were purchased from Life Technologies (Cergy Pontoise, France).

Islet and {beta}-cell isolation

Langerhans islets were isolated from prediabetic NOD mice as described previously, with slight modifications (22, 23). Briefly, islets were digested for 15 min in 1.5 mg/ml collagenase P (Sigma, Strasbourg, France) in DMEM (see above) by incubation at 37°C and washed in PBS supplemented with 5% FCS. They were isolated on a discontinuous Ficoll gradient (Sigma). After centrifugation for 17 min at 2300 rpm, islets were collected, washed in PBS supplemented with 5% FCS, and handpicked under a microscope. Islets were maintained for 24 h under 5% CO2 at 37°C in MEM (Life Technologies) supplemented with 10% FCS, penicillin, and streptomycin. Islet cell suspensions were prepared by treatment with 10-2 M EDTA (Sigma) for 5 min at 37°C and digestion for 30 min in 0.7 mg/ml dispase (Boehringer-Mannheim, Meylan, France) at 37°C and then washed twice in MEM. All islet cell suspensions used as antigenic source were checked for viability (>=97%) using trypan blue assay.

Generation of T cell hybridomas

Isolated pancreatic islets were pooled and distributed in 24-well culture plates to allow the spontaneous extrusion of infiltrating lymphocytes from the islets. Islets were decanted for 3 h, and then infiltrating lymphocytes were recovered by gentle pipeting and fused. All fusions were performed with CD4-transfected BW5147 thymoma cells. To ensure that all infiltrating T cells were recovered, two fusions were performed in each experiment. The first fusion was performed between infiltrating T cells from prediabetic NOD mice recovered in suspension and BW5147 thymoma cells in polyethylene glycol (Boehringer-Mannheim), as previously described (24). The second fusion was performed between intraislet T cells recovered after dispase digestion and BW5147 thymoma cells. Hybridomas were selected in hypoxanthine-aminopterin-thymidine medium (Sigma). Eight days after fusion, hybridomas were tested for recognition of islet cells and preproinsulin peptides. Hybridomas reactive with peptides were cloned by limiting dilution. Percentages of hybridomas obtained with the different antigenic specificities tested were compared with the {chi}2 test.

Synthesis of preproinsulin peptides

The peptides were synthesized using F-moc chemistry by stepwise solid phase methodology on a multichannel peptide synthesizer, as described previously (25). Protected amino acids were coupled by activation in situ with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate and N-{alpha}-F-moc deprotection was performed as previously described (25). After amino acid assembly, side chain deprotection and cleavage of peptides from the solid support was performed by treatment with reagent K (82.5% trifluoroacetic acid (TFA), 5% phenol, 5% water, 5% thioanisole, 2.5% 1,2-ethanedithiol) for 2.5 h at 20°C (26). Peptides were purified by reversed phase HPLC using a PerkinElmer preparative HPLC system (PerkinElmer, Wellesley, MA) on an Aquapore ODS 15-µm column (PerkinElmer; 100 x 10 mm). Elution was achieved with a linear gradient of aqueous 0.1% TFA (A) and 0.08% TFA in 80% acetonitrile, 20% water (B) at a flow rate of 6 ml/min with UV detection at 220 nm. The purity of each peptide was assessed by analytical reversed phase HPLC on a Beckman instrument (Gagny, France) with a Nucleosil C18 5-µm column (150 x 4.6 mm) using a linear gradient of 0.1% TFA in water and acetonitrile containing 0.08% TFA at a flow rate of 1.2 ml/min. The integrity of each peptide was controlled by matrix-assisted laser desorption and ionization time-of-flight on a Protein TOF mass spectrometer (Bruker, Wissembourg, France).

The peptide set was composed of thirty 15- to 18-mer peptides that overlapped by 10 residues: 14 peptides spanned the preproinsulin I sequence; 14 peptides spanned the preproinsulin II sequence; and 2 C-terminal peptides were common to both isoforms (Table IGo). Peptide II33–47 in isoform II corresponded to peptide B9–23 that was previously reported as eliciting strong protection from the development of diabetes in the NOD mouse (4). Peptide 323–339 of OVA was purchased from Neosystem (Strasbourg, France) with 99% purity.


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  		Table I. Amino acid sequence of preproinsulin I and preproinsulin II peptides

 
Epitope screening

Initial screening of T cell hybridomas for recognition of preproinsulin peptides was performed by testing individual peptides I33–47 and II33–47/B9–23 and four pools of seven peptides (not including peptides I33–47 and II33–47). Altogether, all peptides were included in the initial screening. Each pool was tested as follows. First, each peptide was individually incubated with irradiated (3000 rad) syngeneic spleen cells as APCs for at least 2 h to avoid competition and interaction between peptides for binding to I-Ag7 MHC molecule. Second, APCs incubated with seven distinct peptides were pooled without washing. Pools were immediately distributed into 96-well plates and T cell hybridomas were added. Peptide II33–47 and its homologue on preproinsulin I (peptide I33–47) were tested individually to avoid competition with other peptides that bind to I-Ag7. Mapping of hybridomas that recognized a peptide pool was performed by using individual peptides from the positive pool. The response of hybridomas to islet cells or preproinsulin peptides was assessed by IL-2 production as detected by proliferation of the IL-2-dependent cell line CTLL-2. Proliferation was evaluated by [3H]thymidine incorporation. Results were expressed as cpm per culture. Values >=3 times the IL-2 level measured in wells containing medium alone were considered positive.

Peptide immunization

Immunogenicity of preproinsulin peptides was tested by s.c. immunization of individual NOD mice at the base of the tail with 50 µg of each peptide emulsified in CFA (Sigma). Ten days after immunization, spleens and draining lymph nodes were collected. Cell suspensions were prepared and tested for their capacity to respond to the immunizing peptide. T cell responses were evaluated by IL-2 production in the supernatants, as described above.

Spontaneous responses

Spontaneous T cell responses to preproinsulin peptides were tested in NOD and ABH/Biozzi control mice that also carry the I-Ag7 MHC class II molecule. Spleen cells and lymph node cells were incubated with 10 µg/ml of each preproinsulin peptide for 24 h. Supernatants were analyzed for IL-2 production as described above.

ELISPOT assay

Female NOD mice (8 wk old) were individually immunized against 100 µg of peptide I20–35 or peptide II33–47 in CFA. Eleven days later, splenocytes were dispensed in triplicates at 5 x 105 cells/well in 96-well nitrocellulose-backed plates (Millipore, Bedford, MA) which had been precoated overnight at 37°C in 5% CO2 with 10 µg/ml anti-IFN-{gamma} mAb (R4-6A2; BD PharMingen, San Diego, CA) or with 10 µg/ml anti-IL-4 mAb (11B11; BD PharMingen). Proinsulin peptides were added at a final concentration of 20 µg/ml. Negative control wells contained cells and medium only, and positive control wells contained cells and 5 µg/ml Con A (Sigma). The plates were incubated for 48 h at 37°C in 5% CO2, after which the cells were discarded and a biotinylated anti-IFN-{gamma} mAb (XMG1.2; BD PharMingen) or a biotinylated anti-IL-4 mAb (BVD6-24G2; BD PharMingen) was added at 1 µg/ml for 2 h at room temperature followed by streptavidin-conjugated alkaline phosphatase (Sigma) for an additional hour. Cytokine-producing cells were detected as blue spots after a 30-min reaction with 5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium using an alkaline phosphatase-conjugate substrate kit (Sigma). The spot-forming cells were counted using an ELISPOT reader (Axioplan 2; Zeiss, Oberkochen, Germany). The number of spot-forming cells was reported by millions of cells. The number of spots in positive control was always 250 spots.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Frequency of anti-islet cell-specific and proinsulin-specific hybridomas

Because autoreactive T cells are likely to be located within the islets of Langerhans of the pancreas, we studied islet-cell-specific and preproinsulin-specific T cells within the islet cell infiltrate that precedes the development of diabetes in the NOD mouse. Due to high concentrations of insulin and possibly of other {beta}-cell autoantigens within lymphocyte suspensions recovered from the islets, we did not directly evaluate the response of infiltrating T cells to islet cells and to preproinsulin. Instead, we fused T cells recovered from infiltrated islets with the BW5147 thymoma cell line to generate T cell hybridomas. Then, we evaluated the capacity of these hybridomas to recognize islet cells and preproinsulin peptides. Several fusions were performed with infiltrating T cells recovered from female prediabetic NOD mice of different ages. We found no significant difference between 8- and 14-wk-old female prediabetic NOD mice with regard to the percentage of hybridomas that were responsive to islet cells (Table IIGo); 8 of 42 (19.0%) and 22 of 146 hybridomas (15.1%), respectively, responded to islet cells with IL-2 production >=3 times the IL-2 level measured in wells containing medium alone (cf. Materials and Methods). These percentages reflected the T cell population able to respond to islet Ags presented by the I-Ag7 class II molecule (Fig. 1Go). FACS confirmed that all hybridomas generated were CD3+CD4+CD8- (data not shown).


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  		Table II. Hybridomas specific for islet cells

 


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  		FIGURE 1. Distribution of anti-islet cell hybridomas. The stimulation index is indicated for each T cell hybridoma screened as the ratio between IL-2 production in the presence of islet cells vs that in the absence of islet cells. Hybridomas responding with a stimulation index of 3 were considered islet cell specific.

 
Following the same experimental approach, we characterized preproinsulin I and preproinsulin II peptide-responsive T cells within the islet cell infiltrate. We fused infiltrating T cells from 14-wk-old female NOD mice and tested each hybridoma for IL-2 production in response to peptide I33–47, peptide II33–47, and the four preproinsulin peptide pools described in Table IGo. Peptide II33–47 on isoform II corresponds to peptide B9–23. The latter has been shown previously to contain a dominant epitope recognized by T cell clones generated by in vitro expansion of T cells from diabetic NOD mice in the presence of islet cells (27). This peptide has been shown to elicit a strong protection against the development of diabetes in the NOD mouse. The set of peptides tested was considered random with regard to I-Ag7 anchor residues and exhaustive as including peptides from the hormone leader sequence.

One hundred ninety-two CD4+ T cell hybridomas were analyzed. Seven hybridomas (3.6%) responded to at least one preproinsulin peptide (IL-2 secretion level >=3 times the background). A fraction of hybridomas was also tested onto islet cells as positive controls; 4 of 17 (23.5%) responded to islet cells. This percentage is the same as in the experiments previously mentioned. The percentage of hybridomas specific for preproinsulin peptides was lower than the percentage of hybridomas specific for islet cells (p < 0.05, {chi}2 test). Presumably, this illustrates the diversity of islet autoantigens to which T cells respond in type 1 diabetes. It is also likely that only a minor percentage of autoreactive T cells respond to preproinsulin. Only 2 of the 192 T hybridomas described above (RMS8 (Fig. 2GoD) and LTI100; data not shown) responded to peptide II33–47, and 3 others (LTI97-15 (Fig. 2GoC), IC30, and IC6; data not shown) responded to peptide I33–47. This low percentage exemplifies the minor fraction of infiltrating CD4+ T cells directed against peptide II33–47.



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  		FIGURE 2. Mapping of preproinsulin epitopes recognized by T cell hybridomas. A and B, T cell hybridomas that gave a positive response in the presence of peptide pools 1, 2, 3, or 4 tested in the first screening were reevaluated against each individual peptide contained in the pool. Each hybridoma (2 x 104 cells/well) was tested in the presence of 5 x 105 irradiated (3000 rad) NOD spleen cells per well and 3 µg/ml peptide. A, Assay of LTI152-38 against each peptide of pool 2. B, Assay of LTI27-41 against each peptide of pool 3. C and D, Hybridoma positive against peptide I33–47 (C) or peptide II33–47 (D) in initial screening were tested for cross-reactivity against the corresponding peptide of the alternative preproinsulin isoform. Columns correspond to mean IL-2 production of triplicates (expressed in cpm) as described in Materials and Methods.

 
Preproinsulin epitope mapping

Thus, we confirmed that T cells that recognize peptide II33–47 were spontaneously present in the islet infiltrate recovered from prediabetic mice. However, we detected concomitantly T cells recognizing other preproinsulin peptides, especially peptide I33–47 that differs from peptide II33–47 by 1 aa residue at the N-terminal position (proline in preproinsulin I and serine in preproinsulin II). To identify the preproinsulin epitopes recognized by the two T cell hybridomas that recognized neither peptide II33–47 nor peptide I33–47 (LTI27-41 and LTI152-38), we tested their reactivity against each individual peptide included in the peptide pools (i.e., pools 2 and 3, respectively). Hybridomas LTI27-41 and LTI152-38 were shown to recognize peptide II26–41 and peptide I71–86, respectively (Fig. 2Go, A and B).

Noticeably, hybridomas generated from the islet cell infiltrate were specific for a single peptide and showed no cross-reactivity with any other peptides included in the peptide set tested or with the corresponding peptide on the other insulin isoform. This is particularly striking in the case of hybridomas RMS8 and LTI97-15, which are specific for peptide I33–47 and peptide II33–47, respectively (Fig. 2Go, C and D). We verified that the reactivity of the preproinsulin-specific hybridomas was not attributable to molecular mimicry with glutamate decarboxylase (GAD) 65. We tested proinsulin-specific hybridomas for reactivity to peptide GAD217–236 and peptide GAD524–543, as reported in a workshop on the spontaneous T cell proliferation test in the NOD mouse (28). None of the proinsulin-specific hybridomas that we obtained was responsive to GAD peptides (data not shown). These data further show that islet-infiltrating T cells recognize both preproinsulin I and preproinsulin II peptides, namely I33–47, I71–86, II26–41, and II33–47. This is remarkable if one considers that proinsulin II is expressed in the thymus while proinsulin I expression is restricted to pancreatic {beta}-cells (18, 19). As expected, all hybridomas generated with infiltrating T cells were restricted to the NOD haplotype I-Ag7 (Fig. 3Go).



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  		FIGURE 3. Absence of cross-reactivity and I-Ag7 restriction. Each hybridoma (2 x 104 cells/well) was tested in the presence of 5 x 105 irradiated NOD spleen cells, CBA spleen cells, or C57BL/6 spleen cells per well and 10 µg/ml of each individual peptide. Columns correspond to the mean of IL-2 production of triplicates as in Fig. 2Go.

 
Response of T hybridomas LTI27-41, LTI97-15, LTI152-38, and RMS8 to islet cells

Because hybridomas generated from islet cell infiltrate responded to four preproinsulin peptides, it was important to determine whether corresponding peptides were naturally processed and efficiently presented within the islets. For this purpose, we tested the capacity of peptide-specific hybridomas to respond to islet cells in vitro in the absence of synthetic peptides. Hybridoma specific for peptide I71–86 was mildly responsive, and hybridomas specific for peptides II26–41, II33–47, and I33–47 were highly responsive to islet cells in the presence of irradiated NOD spleen cells (Fig. 4Go). Hybridomas responded equally well to islet cells whether or not APCs were added. This indicates that peptides II26–41, II33–47, I71–86 and I33–47 were efficiently processed by intraislet APCs (data not shown).



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  		FIGURE 4. Recognition of islet cell (IC) suspension by T cell hybridomas specific for preproinsulin peptides. Hybridomas specific for peptides I71–86 (A), II 26–41 (B), I33–47 (C), and II33–47 (D) (2 x 104 cells/well) were tested for their response to the homologous peptide (3 µg/ml) or islet cells (2 x 104 cells/well) in the presence of 5 x 105 irradiated (3000 rad) of NOD spleen cells. Columns correspond to the mean of IL-2 production of triplicates as in Fig. 2Go.

 
Immunogenicity of preproinsulin peptides and spontaneous response of peripheral T cells and perpancreatic T cells

Because the frequency of preproinsulin-specific hybridomas generated from the islet cell infiltrate was low, hybridomas that recognized other preproinsulin peptides were possibly missed during the fusion process. We evaluated T cell responses to preproinsulin peptides directly by immunizing individual NOD mice against each peptide emulsified in CFA. A T cell response was detected against five preproinsulin I and three preproinsulin II peptides (Table IIIGo), including peptides recognized along the insulitis process (I33–47, I71–86, and II33–47). Immunization against II26–41 (recognized by LTI27-41), however, elicited no T cell response in the same experimental conditions at any of the peptide concentration range tested (0.03–100 µg/ml) and with mice of any age (data not shown). Then, we evaluated the spontaneous response of NOD mice of different ages against preproinsulin peptides by using two techniques. The IL-2 response of spleen cells collected from unimmunized 4-, 8-, and 12-wk-old diabetic female NOD mice was evaluated in vitro in supernatants of 5 x 105 cells incubated with 20 µg/ml of each individual preproinsulin peptide for 24 h, as indicated in Materials and Methods. The spleen cell response was further tested by enumerating IL-2, IL-4, and IFN-{gamma} spots in culture of 5 x 105 spleen cells incubated with 20 µg/ml peptide during 48 h. No spontaneous response was observed, whereas a significant IL-2 and IFN-{gamma} response of spleen cells was observed in female NOD mice immunized against proinsulin peptide I86–101 used as control (data not shown).


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  		Table III. Alignment of possible anchoring positions of preproinsulin peptides recognized on the NOD (I-Ag7) genetic background

 
Because it was possible that proinsulin-specific T cells were undetectable peripherally, we tested the spontaneous response of prepancreatic T cells from diabetic female NOD mice against immunogenic proinsulin peptides plus peptide II26–41. Any spontaneous response was detectable (Fig. 5Go).



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  		FIGURE 5. Spontaneous response of prepancreatic cells of diabetic NOD mice to preproinsulin-immunogenic peptides. T cells prepared from prepancreatic lymph nodes (2 x 105 cells/well) were obtained from unimmunized diabetic female NOD mice and tested in triplicate in the presence of 5 x 105 irradiated (3000 rad) NOD spleen cells per well added with 30, 10, or 3 µg/ml of each individual peptide. Columns correspond to the mean of IL-2 production of triplicates as in Fig. 2Go.

 
Finally, because protection from diabetes has been observed in the NOD mouse by injecting insulin B chain peptide B9–23 (20), we evaluated IL-4 and IFN-{gamma} production by NOD T cells after immunization against peptide II33–47 in CFA. Proinsulin peptide I20–35, which has not been reported to induce protection from diabetes, was used as a control. As shown in Fig. 6Go, no IFN-{gamma} response was observed to peptide II33–47 as opposed to a significant response to peptide I20–35. In the case of IL-4 response, a significant response was observed against peptide I20–35. No response was observed against peptide II33–47.



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  		FIGURE 6. IFN-{gamma} and IL-4 responses to proinsulin peptides II33–47 and I20–35 after immunization in CFA. Mice were immunized against peptide II33–47 or, as a control, peptide I20–35 in CFA. Spleen cells responses of immunized-mice were evaluated using an ELISPOT assay as described in Materials and Methods. Each column represents the mean number of spots ± SD of triplicate wells in the absence ({blacksquare}) and presence ({square}) of each individual peptide tested. A, IFN-{gamma} ELISPOT; B, IL-4 ELISPOT. SFC, Spot-forming cells.

 
Crystallographic data have been recently described (29, 30) and constitute an important advance in the definition of anchoring residues within the I-Ag7 binding cleft. On the basis of these data, we analyzed possible anchoring residues shared by immunogenic preproinsulin peptides defined by our experiments. Peptides I7–23, I33–47,I71–86, II33–47, and II71–88 could be fitted using the optimal alignment for the P4, P6, P7, and P9 positions (Table IIIGo). Peptide II14–30 shared optimal residues for P4, P6, and P9 pockets with the aforementioned peptides. Peptide I20–35 and peptide I77–92 could be fitted only to P7 and P9 pockets.

TCR sequences of peptide II33–47-specific hybridomas

It has previously been reported that a majority of T cell clones specific for peptide II33–47 in the NOD mouse use a V{alpha}13 TCR {alpha}-chain (20), identifying a novel V{alpha} subfamily referred to as V{alpha}13.3 (31). Thus, we sequenced the 3' end of the TCR V{alpha}-chain used by RMS8 hybridoma, which turned out to use a V{alpha}13.3 gene associated with a J{alpha}34 gene: MYFCAARGSNAKLTFGKGT. Because the T cell clones in previous experiments were obtained by limiting dilution cloning (27), in vitro culture could possibly introduce a bias by selecting a particular V{alpha} subfamily. Therefore, we decided to analyze the frequency of T cell hybridomas generated after immunization against peptide II33–47. The spleen and lymph nodes were fused and the 3' ends of the V{alpha}-chains used by hybridomas specific for peptide II33–47 were sequenced. Twenty-five percent of hybridomas specific for peptide II33–47 were observed to use V{alpha}13 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mapping the epitopes that are recognized by T cells during the development of the autoimmune reaction to {beta}-cells is required to help clarifying the molecular basis of the selection of T cells specific for {beta}-cell Ags within the thymus and their presentation in the periphery. An extensive characterization of immunogenic T cell epitopes of GAD in the NOD mouse has been previously reported (32, 33). In the present study, the islet cell autoantigen chosen was preproinsulin.

To identify epitopes of preproinsulin, we studied NOD T cell responses to a set of 15- to 18-mer peptides that overlap by 10 aa and span the entire sequence of preproinsulin I and preproinsulin II. First, we evaluated T cell responses to preproinsulin peptides by fusing T cells recovered from the islet cell infiltrate of 14-wk-old female NOD mice. Fusions were performed in these experiments for two reasons: 1) the presence of endogenous insulin within the islet environment precludes direct testing of the response of infiltrating T cells to exogenous insulin; 2) ex vivo fusions of infiltrating T cells avoid biases related to in vitro expansion of T cells and are likely to provide a close image in terms of epitope recognition of the repertoire of T cells involved in the autoimmune reaction to {beta}-cells. T cell hybridomas were screened for recognition of preproinsulin peptides. Four distinct proinsulin peptides were identified as recognized by at least one hybridoma generated from the islet infiltrate.

To identify further proinsulin epitopes that could have been missed in the fusion process, complementary experiments were performed to determine the full spectrum of proinsulin peptides that could potentially be recognized on the NOD genetic background. We evaluated the T cell response against each individual peptide contained in the set of 30 proinsulin peptides used in the previous experiments. We observed no spontaneous response of spleen cells from 4- to 12-wk-old NOD mice or diabetic NOD mice. This is probably related to the fact that it is difficult to detect spontaneous response to GAD peptides or the B9–23 insulin peptide in the NOD mouse, as seen in a previously reported workshop (28). We further immunized individual NOD mice against each peptide in CFA. We identified eight immunogenic preproinsulin epitopes. They were localized on both preproinsulin I and preproinsulin II. They were distributed in the leader (L), C-peptide, and insulin B chain sequences. Two peptides overlapped the L and B chain sequences and one the C-peptide and A chain sequences. Three immunogenic peptides were among those defined as being recognized by T cell hybridomas generated by fusion of islet-infiltrating T cells.

The frequency of hybridomas specific for proinsulin peptides was low compared with that of hybridomas specific for islet cell suspensions within the islet cell infiltrate. This finding fits with the likelihood that preproinsulin is only one among several islet autoantigens recognized along the autoimmune process directed against {beta}-cells (1, 34, 35). This low frequency is similar to that previously reported in GAD-specific hybridomas (32). Besides proinsulin and GAD, T cell responses have been identified in the NOD mouse against a variety of autoantigens, including carboxypeptidase H, peripherin (36), heat shock protein 65 kDa (10), the tyrosine phosphatase-like IA2 Ag (37, 38), islet Ag p69 (39), and other, as yet unidentified autoantigens (40). Considering the diversity of the autoimmune reaction directed against {beta}-cells, it is still unclear whether a unique autoantigen has top priority in the initiation of the autoimmune reaction to {beta}-cells in type 1 diabetes. A unique peptide of the insulin B chain has previously been reported as recognized by CD4+ T cells. This peptide, peptide II33–47, is equivalent to the peptide B9–23 described by Wegmann’s group (4) and it was shown later to exert striking preventive properties under different routes of administration in the NOD mouse (20). A second peptide encompassing residues 48–57 at the B-C junction of proinsulin has been proposed as another early autoantigen epitope in the pathogenesis of type 1 diabetes (41). However, our data bring evidence that a larger panel of proinsulin epitopes are potentially recognized in the NOD mouse and that at least four peptides, i.e., peptides I33–47, I71–86, II26–41, and II33–47, are recognized by T cells that contribute to insulitis. The number of proinsulin epitopes that we identified is probably related to the intramolecular epitope spreading of the autoimmune reaction to proinsulin (42). Our experimental approach restricted our study to mice carrying high grade insulitis to allow recovery of a sufficient number of T cells and efficient generation of T cell hybridomas. This may explain why we did not identify T cells specific for peptide II48–57 on proinsulin II (i.e., peptide 24–33 in Ref. 42).

The set of preproinsulin peptides recognized by T cells in the NOD mouse is remarkable in several respects. 1) Peptides recognized by T cell hybridomas span the preproinsulin II sequence as well as the preproinsulin I sequence. This indicates that preproinsulin II expression within the thymus (17, 18, 19) does not preclude positive selection of preproinsulin II-specific T cells that participate in the autoimmune reaction directed against {beta}-cells in the periphery. An alternative hypothesis is that precursor T cells with high affinity for preproinsulin II peptides are negatively selected within the thymus while allowing positive selection of T cells with lower affinity for peptide II33–47, as reported in transgenic mice expressing a lymphocytic choriomeningitis virus nucleoprotein (43, 44). We are currently performing experiments with NOD mice lacking expression of the preproinsulin II gene to discriminate between these two hypotheses. 2) The frequency of hybridomas specific for peptide II33–47 within the islet cell infiltrate is not higher than that of hybridomas specific for the other preproinsulin epitopes. This is particularly puzzling considering the evidence that the insulin B chain and the corresponding B chain peptide II33–47 induce a strong protection in NOD mice after s.c. immunization in IFA (20, 21) or intranasal administration (20). T cell clones that are specific for peptide II33–47 have been shown to be predominant when obtained from the spleen or the islet infiltrate in the NOD mouse after in vitro expansion in the presence of islet cells (4). Expansion of T cells in vitro under successive steps of stimulation does not preclude, however, artificial selection of a restricted T cell set. 3) It is remarkable that peptide II26–41, which was recognized by infiltrating T cells, was not identified as being immunogenic after immunization of NOD mice. This discrepancy may be related to the stability of peptide II26–41 in CFA. Alternatively, immunogenicity of peptide II26–41 may differ from that of the whole proinsulin molecule due to the presence of a disulfide bond involving cysteine in position 31. In this line, the tertiary conformation of the insulin molecule has been shown previously to be an important factor in the generation of antigenic sequences recognized by T cell hybridomas (45). Influence of protein quaternary structure on Ag processing has seemingly been evidenced in the case of recognition of human chorionic gonadotrophin by T cells (46). 4) Hybridomas specific for peptides I33–47, I71–86, II26–41, and II33–47 were able to recognize islet cell suspensions. This indicates that corresponding epitopes were naturally processed from native proinsulin. Corresponding T cells are thus likely to contribute to the autoimmune reaction to {beta}-cells in the NOD model.

Recent crystallographic data provide important advances in the knowledge of anchoring residues of peptides interacting with I-Ag7 (29, 30). By analyzing primary sequences of proinsulin epitopes, we were able to identify residues possibly involved in I-Ag7 binding. According to the crystallographic data, P4, P6, and P9 positions are key anchoring positions. P7 is considered an alternative position. Seven of nine preproinsulin peptides that elicit a T cell response on the NOD genetic background carry expected residues at the P4, P6, and P9 pockets. Three of these peptides (peptides I7–23, I71–86, and II26–41) carry the expected residues at all four P4, P6, P7, and P9 positions. Peptides I20–35 and I77–92 carry the expected residues only at the P7 and P9 positions.

Our study is the first systematic study of preproinsulin epitope recognition in the NOD mouse. Such a study is a prerequisite to the understanding of prior reports, indicating that proinsulin peptides exert a strong protective action against the development of diabetes in this model. It should help designing therapeutic strategies using peptides in type 1 diabetes. Our data indicate that a multiplicity of proinsulin epitopes are seen by CD4+ T cells on the NOD genetic background on both proinsulin 1 and proinsulin 2 isoforms. This should help understanding the imprint exerted by thymic expression of proinsulin on the peripheral repertoire of T cells specific for the hormone produced by {beta}-cells.


   Acknowledgments
 
We thank Pierre Bougnères, Jean-Claude Carel, Patricia Krief, Sarah Boudaly, and Sylvie Lagaye for comments and suggestions; Jean-Gérard Guillet for reviewing the manuscript and interesting discussion; Joëlle Morin and Nicolas Martin for excellent technical assistance; Muriel Delacroix for secretarial assistance; and Karine Vallon-Geoffroy for breeding mice.


   Footnotes
 
1 This work was supported by an Institut National de la Santé et de la Recherche Médicale grant, a Biomed grant from the European Community (BMH4-CT98-3448), an Action Concertée Incitative grant Biologie du Développement et Physiologie Integrative 2000 No. 42, and Juvenile Diabetes Foundation Grant 1-2001-751. Back

2 Address correspondence and reprint requests to Dr. Christian Boitard, Institut National de la Santé et de la Recherche Médicale Unité 561, Hôpital St. Vincent de Paul, 82 avenue Denfert Rochereau, 75014 Paris, France. E-mail address: boitard{at}cochin.inserm.fr Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; TFA, trifluoroacetic acid; GAD, glutamate decarboxylase. Back

Received for publication November 14, 2001. Accepted for publication June 28, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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