HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blancou, P. Articles by Bach, J.-M. Search for Related Content PubMed PubMed Citation Articles by Blancou, P. Articles by Bach, J.-M.

Immunization of HLA Class I Transgenic Mice Identifies Autoantigenic Epitopes Eliciting Dominant Responses in Type 1 Diabetes Patients¹

Philippe Blancou^2,*, Roberto Mallone^,, Emanuela Martinuzzi^,, Sabine Sévère^*, Sylvie Pogu^*, Giulia Novelli^¶, Graziella Bruno^¶, Bernard Charbonnel^*,, Manuel Dolz, Lucy Chaillous^*,, Peter van Endert^, and Jean-Marie Bach^2,*

^* Immuno-Endocrinology Unité Mixte de Recherche 707, Institut National de la Recherche Agronomique/Ecole Nationale Vétérinaire de Nantes/Université, Nantes, France; Institut National de la Santé et de la Recherche Médicale, Unité 580, Paris, France; Université Paris Descartes, Paris, France; Clinique d’Endocrinologie, Hôpital Hôtel-Dieu, Nantes, France; and ^¶ Department of Internal Medicine, University of Turin, Turin, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 
Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic cells. CD8⁺ T cells have recently been assigned a major role in cell injury. Consequently, the identification of autoreactive CD8⁺ T cells in humans remains essential for development of therapeutic strategies and of assays to identify aggressive cells. However, this identification is laborious and limited by quantities of human blood samples available. We propose a rapid and reliable method to identify autoantigen-derived epitopes recognized by human CD8⁺ T lymphocytes in T1D patients. Human histocompatibility leukocyte Ags-A*0201 (HLA-A*0201) transgenic mice were immunized with plasmids encoding the T1D-associated autoantigens: 65 kDa glutamic acid decarboxylase (GAD) or insulinoma-associated protein 2 (IA-2). Candidate epitopes for T1D were selected from peptide libraries by testing the CD8⁺ reactivity of vaccinated mice. All of the nine-candidate epitopes (five for GAD and four for IA-2) identified by our experimental approach were specifically recognized by CD8⁺ T cells from newly diagnosed T1D patients (n = 19) but not from CD8⁺ T cells of healthy controls (n = 20). Among these, GAD_114–123, GAD_536–545 and IA-2_805–813 were recognized by 53%, 25%, and 42% of T1D patients, respectively.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 
Type 1 diabetes (T1D)³ is an autoimmune disease diagnosed when most pancreatic cells are destroyed by autoreactive T lymphocytes. Clinical diagnosis is thus preceded by a prediabetic asymptomatic phase characterized by an active cell destruction process. However, there is limited information on the dynamics of the cellular immune response to cell autoantigens during the course of preclinical disease, and no autoimmune marker closely revealing the pathogenic process leading to T1D exists for humans. Even if T1D risk assessment and diagnosis essentially relies on autoantibody detection, autoantibodies are not accountable for cell injury (1). Similarly, although the association of MHC class II polymorphism with genetic T1D susceptibility (2) has stimulated strong interest in the role of CD4⁺ T cells in the disease, observations made in the NOD mouse model demonstrate the essential role of CD8⁺ T cells.

The pathogenic role of autoreactive CD8⁺ T cells in cell aggression has been documented in the NOD mouse model in every step of autoimmune cell injury. The early initiators of islet cell destruction may be MHC class I-restricted T lymphocytes. Mice lacking MHC class I molecules do not develop insulitis (3, 4), and some islet-specific CD8⁺ T cell clones transfer diabetes in the absence of CD4⁺ T lymphocytes (5, 6). As the pathogenic process progresses, CD8⁺ T cells eventually develop to a stage that can cause disease (7). Independent of autoreactive CD4⁺ T cells, CD8⁺ T cells could also be implicated in the progression of destructive insulitis and overt diabetes when they express high-affinity TCR (8). Finally, direct detection of autoreactive CD8⁺ T cells in peripheral blood closely reflects cell aggression (9).

In humans, CD8⁺ T cells are major components of insulitis as seen in few unfortunate cases of death upon disease presentation or in pancreas biopsies obtained under laparoscopy from adult T1D patients upon disease onset (10, 11, 12, 13, 14, 15). The characterization of naturally processed cell epitopes recognized by CTLs in humans may be crucial for a more accurate prediction of T1D than autoantibody or CD4⁺ T cell reponses. Identification of cell epitopes would also provide new tools for T1D risk assessment and follow-up, as well as for the elaboration of new immunotherapeutic strategies targeting islet-specific CD8⁺ T lymphocytes. High priority should therefore be given to the identification of cell autoantigen-derived peptides recognized by CD8⁺ T cells in humans. However, identification requires labor-intensive methodologies, including either in vitro proteasome (16, 17) or the screening of algorithm selected peptides (18, 19, 20). In vitro digestion of autoantigens using proteasome complexes has been described as successfully identifying epitopes derived from proinsulin, presented by HLA-A*0201 molecules (16, 17) and recognized by human CD8⁺ T cells from diabetic patients (16). However, this approach is limited by the length of the autoantigenic protein tested and cannot be used for high m.w. cell autoantigens, such as glutamic acid decarboxylase (GAD), insulinoma-associated protein (IA-2), or islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Exhaustive screening of peptide libraries with human PBMCs is also strongly limited by the quantity of human blood that can be obtained from T1D patients (in particular young T1D subjects) and the low frequency of autoreactive CD8⁺ T cells in human peripheral blood.

Among cell autoantigens, proinsulin, IA-2, and GAD 65kDa (GAD65) are considered essential self-proteins associated with T1D. Two epitopes derived from proinsulin (16, 21), two from IA-2 (20), and one from GAD65 (22) have been identified as being recognized by HLA-A2-restricted CD8⁺ T cells in humans. GAD65 (23, 24) and IA-2 (23, 24), especially the catalytic unit of the intracellular domain (25), are major islet Ags targeted by humoral autoimmunity in human T1D. Several studies indicate that GAD is implicated in the aggravation (26, 27) or regulation (28) of cell aggression in NOD mice. However, even if the role of GAD-specific CD4⁺ T cells have been disputed (29), the importance of GAD-specific CD8⁺ T cells is still unknown in the NOD mouse model. More than a decade ago, the human GAD_114–123 CD8⁺ epitope was reported as being associated with two T1D patients and with one preclinical T1D individual (22). However, no extensive study was performed on this particular epitope. GAD65 is expressed at very low levels in NOD mouse cells compared with human cells, which synthesize large amounts of GAD65. Mouse cells express the GAD67 isoform (30, 31). Besides, although the expression of both GAD65 and GAD67 isoforms is well established in human thymus (32), no GAD expression is detected in mouse thymus (31). Thus, the role of GAD65 in humans may be very different from that recorded in mice (30).

HLA-A*0201 is the most commonly expressed HLA class I allele in Europeans (40%). Moreover, it may contribute to susceptibility to disease in humans (33, 34, 35), and aggravate it when introduced in NOD mice (36). HHDII mice are transgenic for the HLA-A*0201.1 1 and 2 extracellular domains on an H-2 class I knockout background. These mice exhibit a qualitatively normal diversified CD8⁺ T cell repertoire, HLA-A*0201-restricted education and cytolytic activity of CD8⁺ T lymphocytes (37). DNA vaccination allows direct intracellular synthesis of entire antigenic protein leading to the presentation of naturally processed epitopes and robust and long-lasting cytolytic CD8⁺ T cell responses (reviewed in Ref. 38). Genetic immunization has already been used with success in the identification of subdominant viral CTL epitopes in infectious disease models (38).

We took advantage of DNA vaccination in HHDII mice to identify new epitopes associated with T1D. Nine new epitopes derived from GAD65 and IA-2 targeted in T1D patients were identified. Recognition of GAD_114–123, GAD_536–545, and IA-2_805–813 by a substantial proportion of T1D patients renews interest in GAD- and IA-2-specific CD8⁺ T cells in human T1D.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 
Peptides

Peptides of nine and ten amino acids containing putative binding motifs for HLA-A*0201 molecules were selected using the SYFPEITHI algorithm (39) and the human GAD and IA-2 sequences retrieved from GenBank (accession number M74826 and L18983, respectively). All peptides were synthesized by ProImmune.

Animals

HLA-A*0201-transgenic ₂m^–/– HHD mice were provided by Dr. F. Lemonnier (Pasteur Institute, Paris, France) and were previously described (37).

Plasmid construction and mouse immunizations

Plasmids used to vaccinate HHDII mice encoding GAD65 (pc-GAD) or the catalytic unit of the intracellular domain of IA-2 (pc-IA-2). pc-GAD were previously described (40). The catalytic domain of IA-2 was amplified by PCR (TaqDNA polymerase; Promega) from ICA512.bdc pCRII (provided by Dr. C. Levy-Marchal, Hospital Robert Debré, Paris, France) using a sense oligonucleotide containing an EcoRI site (5'-GGAATTCTAACATGGACATCTCCACGGG-3'; MWG) and an antisense primer containing an XbaI site (5'-CGGGCCCTCTAGAGCCTGGGGCAGGGCCTT-3'). Pc-GAD and pc-IA-2 were obtained by cloning the respective EcoRI-XbaI-digested amplicons into pcDNA3.1/B/V5 (Invitrogen). Constructs were verified by sequencing (Millegen). Recombinant proteins produced by transiently transfected mammalian cells (293T cells) were detected by immunoblotting. HHDII mice were randomly allocated and DNA vaccinated twice at 6-wk intervals as previously described (40).

Mouse immunization experiments

HHDII mice were immunized s.c. with 100 µg IA-2_828–837, IA-2_830–837, and IA-2_830–839, or DMSO alone in CFA along with 140 µg of HBcAg_128–140 helper peptide (TPPAYRPPNAPIL) at the base of the tail. After 12–14 days, an i.p. boost was given at half-dose in IFA without helper peptide. After another 12–14 days, mice were sacrificed and splenocytes challenged at 5 x 10⁵/well in an ELISPOT recall assay (see below).

HLA-A*0201 binding assay

HLA-A*0201 binding assays were performed by ProImmune (Reveal module 2).

Cytotoxicity assay

HHDII mice were immunized by i.p. injection of 100 µg of HLA-A*0201-restricted peptides together with 140 µg of HBcAg_128–140 helper peptide emulsified in IFA (Sigma-Aldrich). Spleens were removed 12 days after injection, and spleen cells were restimulated using irradiated (2000 rad) HHDII lymphoblasts previously pulsed for 2 h with 10 µg/ml HLA-A*0201-restricted peptide at a concentration of 5 x 10⁶ cells/ml. Restimulated cells were tested 6 days later for their cytotoxicity using a nonradioactive CARE-LASS as previously described (54). Effector cells were incubated 2 h in PBS-5% FCS with 8 x 10³ HLA-A*0201 T2 target cells/well previously stained with calcein (Molecular Probes). Percentage of lysis was defined as (experimental release – spontaneous release from effectors alone – spontaneous release from targets alone) x 100/(maximum – minimum releases from targets). Assays were performed in quadruplicates.

Patients

New-onset adult (>16 yr old) T1D patients with acute onset of symptoms requiring permanent insulin treatment from the time of diagnosis were recruited from the Registry of the Province of Turin (Italy) and from the GOFEDI Network (France) (see Appendix). Recruitment of 20 healthy control subjects took place at the same institutions. T2D patients were defined as Ab^– subjects with normal fasting C-peptide levels, and they had no secondary complications. All participating subjects gave informed consent and the relevant ethics committees in Italy and France approved the study.

Blood processing

Upon arrival, blood samples were rapidly HLA-A2 screened with the BB7.2 mAb, followed by genotyping using the Olerup SSP HLA*02 kit (GenoVision/Qiagen). PBMCs were isolated and immediately used or stored frozen (10% DMSO in pooled human male AB serum) as appropriate. Serum samples were also stored for subsequent Ab determinations following protocols previously evaluated within the Diabetes Ab Standardization Program (lab no. 137) (41). Sensitivities in the 2005 Diabetes Ab Standardization Program were 84% for anti-GAD, 76% for anti-IA-2, and 28% for insulin autoantibodies where the specificity was set at 95%. Islet cell Abs were assayed by indirect immunofluorescence on frozen sections of human blood group O pancreas.

ELISPOT assay

Mouse IFN- ELISPOT were performed with 10⁵ immunomagnetically selected (anti-CD8 microbeads; Miltenyi Biotec) CD8⁺ T lymphocytes/well from two DNA-immunized HLA-A*0201 transgenic mice in the presence of 4 x 10⁵ mitomycin-treated spleen cells/well and 5 µM peptide following manufacturer’s instructions (ELISPOT set; BD Biosciences). Preliminary experiments performed on individual mice showed an important interindividual variability in IFN- ELISPOT responses against the peptides tested. These results and the low number of CD8⁺ T cells in the HHD mice (8% of spleen cells) prompted us to perform ELISPOT assays on CD8⁺ T cells pooled from two animals. To test the complete synthesized peptide libraries, IFN- ELISPOT assays were performed in single wells, and three independent experiments were performed for each autoantigen. Epitope-specific responses were considered positive when the number of spots was greater than three times the number detected with the control peptide.

For human IFN- assays, 96-well plates (Millipore) were coated overnight with anti-human IFN- Ab (U-CyTech). Plates were subsequently blocked with RPMI 1640 plus 10% human serum. PBMCs (3 x 10⁵) were added in triplicate with the peptide (10 µM) and IL-2 (0.5 IU/ml; R&D Systems) and cultured for 20–24 h. Following PBMC removal, IFN- secretion was visualized with a biotin-conjugated anti-human IFN- Ab (U-CyTech), alkaline phosphatase-conjugated streptavidin and NBT-5-bromo-4-chloro-3-indolyl phosphate reagents (both obtained from Sigma-Aldrich). All ELISPOT results are expressed as spot-forming cells per 10⁶ human PBMCs or mouse CD8⁺ cells.

Statistical analysis

Spots were counted using an AID reader (Auttoimmune Diagnostika GmbH) and means of triplicate wells calculated. For human IFN- ELISPOT, the mean +3SD of the two negative control triplicates (DMSO and HIV-1 gag77–85) was selected as cutoff for positive response base on receiver-operator characteristic analysis. The Mann-Whitney U test was used to determine statistical differences between the age of T1D patients and controls. Correlations between T cell responses and autoantibodies were analyzed using the Spearman rank correlation test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 
CD8⁺ T cell responses in HLA-A*0201 transgenic mice following DNA vaccination

HHDII mice were immunized with plasmid-encoding human GAD65 or the IA-2 intracellular catalytic domain. CD8⁺ T cell responses were detected after two genetic immunizations using IFN- ELISPOT assays on positively selected CD8⁺ spleen cells from vaccinated mice. This method allowed us to screen large libraries of peptides binding HLA-A*0201 molecules based on their SYFPEITHI algorithm scores (39).

Three independent experiments were performed for each autoantigen (Table I). To check for specificity, HLA-A*0201 mice were vaccinated with plasmid-encoding rat preproglucagon and tested for GAD and IA-2 reactivity. Responses against GAD and IA-2 peptides in these mice did not exceed basal responses to diluent alone, indicating that peptide responses in GAD and IA-2 vaccinated mice were Ag-specific (data not shown). As indicated in Table I, five GAD peptides and two IA-2 peptides tested positive in each experiment performed, whereas eight peptides for GAD and three for IA-2 were positive in one or two of the three assays. Of note is that all 10-mers tested gave the same result when their overlapping 9-mers were tested (positive peptide pairs: GAD65_110–118/GAD65_110–119; GAD65_114–122/GAD65_114–123; GAD65_140–149/GAD65_141–149; negative peptide pairs: GAD65_39–47/GAD65_39–48; GAD65_291–299/GAD65_290–299; GAD65_293–301/GAD65_293–302).

View this table: [in this window] [in a new window]   Table I. IFN- ELISPOT assay performed in DNA vaccinated HLA-A*0201 micea

Some viral or tumoral epitopes relevant in human were recently identified as strongly recognized by HHDII murine CD8⁺ T cells following peptide immunization of these mice (37, 42, 43, 44, 45, 46). To select candidate epitopes for testing with human PBMC, we applied the following criteria. All peptides testing positive at least twice in the three ELISPOT experiments performed were included for human T cell assays. Moreover, we also selected two peptides, GAD_536–545 and IA-2_962–970, that elicit high responses in a single assay (Table I).

Some overlapping 9- and 10-mer peptides were both found to be positive in the IFN- ELISPOT assay (see above). In these cases, due to limited material available from T1D patients, we decided to use the peptide with the highest SYFPEITHI algorithm score, except for the GAD65_114–122/GAD65_114–123 couple for which peptide GAD65_114–123 described previously was selected (22). Concerning IA-2_828–837 and IA-2_830–839 peptides that overlap by 8 aa, immunization with peptide IA-2_828–837 elicited a CD8⁺ T cell reactivity to both peptides whereas immunization with peptide IA-2_830–839 induced exclusively a response against the immunizing peptide (Fig. 1). Considering that this phenomenon might suggest antigenicity of the 8-mer overlapping epitope core, we tested recognition of the 8-mer overlap by 10-mer immunized mice (Fig. 1). This experiment showed that the 8-mer peptide can indeed be recognized by T cells from mice immunized with the 10-mer 828–837 but not with 830–839 suggesting that IA-2_828–837 cross-reactivity for IA-2_830–839 occurs via the 8-mer recognition. Based on these results, we chose to test the more broadly recognized peptide IA-2_830–839 in humans.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Cross-reactivity of CD8+ T cells from HLA-A*0201 transgenic mice immunized with IA-2828–837, IA-2830–839, or IA-2830–837 peptides. HHDII mice were immunized s.c. either with the IA-2828–827, IA-2830–837 peptide or with the IA-2830–839 peptide. Splenocytes were subsequently challenged in an IFN- recall assay using medium alone (white bars), IA-2828–837, IA-2830–839, or IA-2830–837 peptides (black bars, light grey, and dark grey bars, respectively) or ConA (1 µg/ml, hatched bars). Results are expressed as the average ± SD of ELISPOT triplicate. *, Not tested.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Cytotoxicity of HHDII CD8+ T cells for GAD and IA-2 epitopes. Two mice were injected with each GAD and IA-2 epitope studied. Spleen cells from each mouse were then tested for their lysis activity against the immunizing peptide and the Muc-1 control peptide. Results are expressed as mean percentage of lysis ± SD for each quadruplicate performed at various effector/target ratios.

CD8⁺ T cell reactivities to candidate epitopes in T1D patients

The ten selected peptides were evaluated for their reactivity in new-onset T1D patients. Patients (n = 19) selected were HLA-A*0201⁺, and recently diagnosed as having T1D with a median duration of 11 days (range 3–180). All patients were tested for GAD, insulin, and IA-2 Abs (Table II). A parallel healthy control cohort (n = 20) was aged-matched with the T1D population (29 ± 11.1 vs 32 ± 8.8 years, respectively; p = 0.52).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 
CD8⁺ T cells have been assigned recently a major role in cell injury leading to T1D. Defining cell autoantigenic epitopes is essential for the development of T cell assays and the design of peptide-based immunotherapy in T1D. Moreover, definition of relevant CD8⁺ T cell epitopes might open avenues for immunointervention with native or altered peptide ligands (47). Our study provides evidence for a strong and disease-specific CD8⁺ T cell autoreactivity to new GAD and IA-2 epitopes in T1D patients using an original method based on the plasmid-DNA immunization of HLA-A*0201 transgenic mice.

Different approaches have been tested recently to identify epitopes derived from cell autoantigens and targeted by CD8⁺ T cells in T1D patients. Among them, exhaustive screening of large algorithm-selected peptide libraries with human PBMCs is severely limited by the quantity of human blood that can be obtained from T1D patients and the low frequency of autoreactive CD8⁺ T cells in human peripheral blood. Reverse immunology approaches including proteasome digestion may afford the most efficient epitope selection; however, for practical reasons, such approaches are likely to remain limited to small autoantigens like insulin. Ten epitopes presented by HLA-A2 class I alleles to CD8⁺ cells have been defined so far with theses techniques: two from insulin (insB_10–18 and insB_18–27; (16, 21)), two from IAPP (IAPP_5–13 and IAPP_9–17; (19, 20, 48)), two from IA-2 (IA-2_172–180 and IA-2_482–490; (20)), two from IGRP (IGRP_152–160 and IGRP_215–223; (19, 20)), one from GFAP (GFAP_143–152 and GFAP_214–222; (19)) and one from GAD (22). DNA vaccination of HLA-A*0201 transgenic mice with human autoantigens should speed-up the process, allowing to simultaneously evaluate both natural processing by APCs and recognition by CD8⁺ T cells before testing on human samples. All the epitopes identified by this method and tested by ELISPOT scored positive in T1D patients and negative in control subjects. In the recent past, HHDII mice have been used to facilitate the generation of HLA-A*0201-restricted CTLs and the study of immunization protocols for effective treatment of human infectious diseases or cancers (42, 43, 44, 45, 46, 49). In this study, we present the first report of plasmid-DNA immunization aimed at identifying CD8⁺ T cells in human autoimmune disease. Our study demonstrates that genetic immunization of HHDII mice is an attractive approach for the rapid identification of autoantigenic epitopes presented by HLA-A*0201 molecules. This approach is particularly useful in autoimmune diseases for which limited pools of human autoreactive CD8⁺ precursors are present. This method is also faster and cheaper compared with extensive screening of potentially binding epitopes and other reverse immunology approaches.

Although all epitopes were recognized by patient PBMC, the magnitude of responses differed greatly. T cells specific for some epitopes were observed in a small percentage of the patient population (10%), questioning the relevance of these T cells in the pathogenesis of T1D, and for developing T cell monitoring assays. However, it is possible that T cells recognizing such epitopes are implicated in the initial stages of cell injury and are more difficult to detect at the final stage of insulitis, when the disease is diagnosed.

Of interest was the high frequency of GAD65_114–123 and GAD65_536–545 peptide responses by CD8⁺ T cells from T1D patients. These results confirm the differential relevance of this autoantigen in diabetes pathogenesis between humans and mice. Indeed, GAD is a major islet Ag targeted by humoral autoimmunity in human T1D (23, 24), whereas GAD autoantibodies are not diabetes-specific in the NOD mouse model (50). IA-2 is also a major islet Ag targeted by humoral autoimmunity (23, 24) but its relevance in autoreactive CD8⁺ T cell responses has remained unclear. Outside the catalytic unit of the IA-2 intracellular domain considered by us, two epitopes (IA-2_172–180 and IA-2_482–490), have recently been identified by a peptide affinity algorithm method and are reactive to 18% and 27% of recent-onset T1D patients (20). The high incidence (> 40% of T1D patients) of cellular immunity to IA-2_805–813 at diabetes onset we observed here provides the second report of T1D-associated human CD8⁺ T cell reactivity to IA-2, as opposed to previously reported responses against IA- 2_797–805 that were equally found in healthy individuals (51). Interestingly, IA-2_805–813 overlaps with an HLA class II-restricted dominant CD4⁺ T cell epitope (IA-2_805–820) detected in islet Ab-positive at-risk individuals (52).

The selected panel of peptides used in human IFN- ELISPOT assays discriminates efficiently between T1D patients and healthy individuals, as > 70% of T1D patients were positive for at least one epitope and 42% of T1D patients showed reactivity to more than two epitopes. Interestingly, a comparison of HHDII mouse (Table I) and T1D patient (Table III) responses in IFN- ELISPOT assays revealed that peptides harboring the highest reactivity in patients (GAD65_114–123 and GAD65_536–545 and IA-2_805–813) induced intermediate CD8⁺ responses (mean spot numbers 473, 264 and 216, respectively) or no response at all in some mice (1/3, 2/3 and 1/3, respectively). Although, we did not find histological evidence of insulitis in mice vaccinated with plasmid encoding GAD or IA-2 (data not shown), we cannot rule out that some CD8⁺ T lymphocytes activated in mice by DNA vaccination migrated to the pancreas or pancreatic lymph-nodes, thereby escaping detection among spleen cells. Conversely, some peptides were strongly immunogenic for CD8⁺ T cells in DNA-vaccinated mice (Table I), but elicited CD8⁺ reactivity only in few T1D patients (Table III). This observation may be linked to the fact that CD8⁺ clones with the highest TCR avidity for self-Ag are deleted during thymic T cell education, while clones of intermediate avidity circulate in peripheral blood (53). Consequently, other peptides inducing weak responses in mice (Table II), may be relevant candidate epitopes in humans and need to be investigated in diabetic patients (GAD65_293–302, GAD65_319–328, GAD65_406–414, GAD65_443–452, IA-2_917–925).

In conclusion, DNA vaccination of HLA class I transgenic mice emerges as an efficient strategy for the identification of self epitopes relevant to autoimmunity. This may apply specifically to autoantigens with higher molecular weight which are difficult to analyze in proteasome digests or by screening of exhaustive peptide libraries. Moreover, our study identifies GAD65 and IA-2 epitopes recognized by PBMC in >25% of patients, underlining the interest of these autoantigens in diagnostic tests for T1D.

	Note added in proof.

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 
A second paper by our groups was recently published (Diabetes 56: 613-21, 2007), where these study cohorts were further analyzed to develop an ELISPOT-based T cell assay for type 1 diabetes.

	Acknowledgments

 
We thank François Lemonnier for the gift of HLA-A*0201 transgenic mice and useful discussion and Claude Chevalier for excellent technical assistance. We are grateful to patients and control subjects for blood donation. The following colleagues and Institutions contributed to the study with patient recruitment: Piedmont Study Group for Diabetes Epidemiology, Italy (coordinators: G. Bruno, G. Pagano): S. Cianciosi, Avigliana; A. Perrino, Carmagnola; C. Giorda, E. Imperiale, Chieri; A. Chiambretti, R. Fornengo, Chivasso; V. Trinelli, D. Gallo, Ciriè-Lanzo; A. Caccavale, Collegno; F. Ottenga, Cuneo; R. Autino, P. Modina, Cuognè; L. Gurioli, L. Costa- Laia, Ivrea; C. Marengo, M. Comoglio, Moncalieri; T. Mahagna, Nichelino; M. Trovati, F. Cavalot, San Luigi Hospital, Orbassano; A. Ozzello, P. Gennari, Pinerolo-Pomaretto-Torre Pellice; S. Bologna, D. D’Avanzo, Rivoli; S. Davì, M. Dore, Susa; S. Martelli, E. Megale, Giovanni Bosco Hospital, Turin; S. Gamba, A. Blatto, Maria Vittoria Hospital, Turin; P. Griseri, C. Matteoda, Martini Hospital, Turin; A. Grassi, A. Mormile, Mauriziano Hospital, Turin; P. Cavallo-Perin, E. Pisu, G. Grassi, V. Martina, V. Inglese, R. Quadri, Molinette Hospital, Turin; G. Petraroli, L. Corgiat-Mansin, Ophtalmologic Hospital, Turin; F. Cerutti, C. Sacchetti, Regina Margherita Pediatric Hospital, Turin; A. Clerico and L. Richiardi, Valdese Hospital, Turin; G. Bendinelli, A. Bogazzi, Venaria.GOFEDI (Groupe Ouest-France pour l’Etude du Diabète Insulino-dépendant), France (coordinator: L. Chaillous): C. Briet, Amiens; P.H. Ducluzeau and V. Rohmer, Angers; V. Kerlan and E. Sonnet, Brest; M. Dolz and B. Charbonnel, Nantes; R. Marechaud, Poitiers; P. Lecomte, Tours.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. 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 study was supported by the Association de Langue Française pour l’Etude du Diabéte et des maladies Métaboliques and the Juvenile Diabetes Research Foundation Grant 1-2005-39.

² Address correspondence and reprint requests to Drs. Philippe Blancou and Jean-Marie Bach, Unité Mixte de Recherche 707, Immuno-Endocrinology Unit, Ecole Nationale Vétérinaire de Nantes, BP 40706, Nantes, France. E-mail addresses: blancou{at}vet-nantes.fr and bach{at}vet-nantes.fr

³ Abbreviations used in this paper: T1D, type 1 diabetes; GAD, glutamic acid decarboxylase; IA-2, insulinoma-associated protein 2; IGRP, islet-specific glucose-6-phosphatase.

Received for publication November 22, 2006. Accepted for publication March 2, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Note added in proof. Disclosures References

 

1. Martin, S., D. Wolf-Eichbaum, G. Duinkerken, W. A. Scherbaum, H. Kolb, J. G. Noordzij, B. O. Roep. 2001. Development of type 1 diabetes despite severe hereditary B-lymphocyte deficiency. N. Engl. J. Med. 345: 1036-1040. [Free Full Text]
2. Nepom, G. T., W. W. Kwok. 1998. Molecular basis for HLA-DQ associations with IDDM. Diabetes 47: 1177-1184. [Abstract]
3. Serreze, D. V., E. H. Leiter, G. J. Christianson, D. Greiner, D. C. Roopenian. 1994. Major histocompatibility complex class I-deficient NOD-B₂m null mice are diabetes and insulitis resistant. Diabetes 43: 505-509. [Abstract]
4. Wicker, L. S., E. H. Leiter, J. A. Todd, R. J. Renjilian, E. Peterson, P. A. Fischer, P. L. Podolin, M. Zijlstra, R. Jaenisch, L. B. Peterson. 1994. ₂-microglobulin-deficient NOD mice do not develop insulitis or diabetes. Diabetes 43: 500-504. [Abstract]
5. Graser, R. T., T. P. DiLorenzo, F. Wang, G. J. Christianson, H. D. Chapman, D. C. Roopenian, S. G. Nathenson, D. V. Serreze. 2000. Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions. J. Immunol. 164: 3913-3918. [Abstract/Free Full Text]
6. Wong, F. S., I. Visintin, L. Wen, R. A. Flavell, C. A. Janeway, Jr. 1996. CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells. J. Exp. Med. 183: 67-76. [Abstract/Free Full Text]
7. Serreze, D. V., H. D. Chapman, D. S. Varnum, I. Gerling, E. H. Leiter, L. D. Shultz. 1997. Initiation of autoimmune diabetes in NOD/Lt mice is MHC class I-dependent. J. Immunol. 158: 3978-3986. [Abstract]
8. Amrani, A., J. Verdaguer, P. Serra, S. Tafuro, R. Tan, P. Santamaria. 2000. Progression of autoimmune diabetes driven by avidity maturation of a T cell population. Nature 406: 739-742. [Medline]
9. Trudeau, J. D., C. Kelly-Smith, C. B. Verchere, J. F. Elliott, J. P. Dutz, D. T. Finegood, P. Santamaria, R. Tan. 2003. Prediction of spontaneous autoimmune diabetes in NOD mice by quantification of autoreactive T cells in peripheral blood. J. Clin. Invest. 111: 217-223. [Medline]
10. Bottazzo, G. F., B. M. Dean, J. M. McNally, E. H. MacKay, P. G. Swift, D. R. Gamble. 1985. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N. Engl. J. Med. 313: 353-360. [Abstract]
11. Hanninen, A., S. Jalkanen, M. Salmi, S. Toikkanen, G. Nikolakaros, O. Simell. 1992. Macrophages, T cell receptor usage, and endothelial cell activation in the pancreas at the onset of insulin-dependent diabetes mellitus. J. Clin. Invest. 90: 1901-1910. [Medline]
12. Itoh, N., T. Hanafusa, A. Miyazaki, J. Miyagawa, K. Yamagata, K. Yamamoto, M. Waguri, A. Imagawa, S. Tamura, M. Inada, et al 1993. Mononuclear cell infiltration and its relation to the expression of major histocompatibility complex antigens and adhesion molecules in pancreas biopsy specimens from newly diagnosed insulin-dependent diabetes mellitus patients. J. Clin. Invest. 92: 2313-2322. [Medline]
13. Somoza, N., F. Vargas, C. Roura-Mir, M. Vives-Pi, M. T. Fernandez-Figueras, A. Ariza, R. Gomis, R. Bragado, M. Marti, D. Jaraquemada, et al 1994. Pancreas in recent onset insulin-dependent diabetes mellitus: changes in HLA, adhesion molecules and autoantigens, restricted T cell receptor V usage, and cytokine profile. J. Immunol. 153: 1360-1377. [Abstract]
14. Conrad, B., E. Weidmann, G. Trucco, W. A. Rudert, R. Behboo, C. Ricordi, H. Rodriquez-Rilo, D. Finegold, M. Trucco. 1994. Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology. Nature 371: 351-355. [Medline]
15. Foulis, A. K., M. McGill, M. A. Farquharson. 1991. Insulitis in type 1 (insulin-dependent) diabetes mellitus in man—macrophages, lymphocytes, and interferon- containing cells. J. Pathol. 165: 97-103. [Medline]
16. Toma, A., S. Haddouk, J. P. Briand, L. Camoin, H. Gahery, F. Connan, D. Dubois-Laforgue, S. Caillat-Zucman, J. G. Guillet, J. C. Carel, et al 2005. Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients. Proc. Natl. Acad. Sci. USA 102: 10581-10586. [Abstract/Free Full Text]
17. Hassainya, Y., F. Garcia-Pons, R. Kratzer, V. Lindo, F. Greer, F. A. Lemonnier, G. Niedermann, P. M. van Endert. 2005. Identification of naturally processed HLA-A2–restricted proinsulin epitopes by reverse immunology. Diabetes 54: 2053-2059. [Abstract/Free Full Text]
18. Takaki, T., M. P. Marron, C. E. Mathews, S. T. Guttmann, R. Bottino, M. Trucco, T. P. DiLorenzo, D. V. Serreze. 2006. HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes. J. Immunol. 176: 3257-3265. [Abstract/Free Full Text]
19. Standifer, N. E., Q. Ouyang, C. Panagiotopoulos, C. B. Verchere, R. Tan, C. J. Greenbaum, C. Pihoker, G. T. Nepom. 2006. Identification of novel HLA-A*0201-restricted epitopes in recent-onset type 1 diabetic subjects and antibody-positive relatives. Diabetes 55: 3061-3067. [Abstract/Free Full Text]
20. Ouyang, Q., N. E. Standifer, H. Qin, P. Gottlieb, C. B. Verchere, G. T. Nepom, R. Tan, C. Panagiotopoulos. 2006. Recognition of HLA class I-restricted cell epitopes in type 1 diabetes. Diabetes 55: 3068-3074. [Abstract/Free Full Text]
21. Pinkse, G. G., O. H. Tysma, C. A. Bergen, M. G. Kester, F. Ossendorp, P. A. van Veelen, B. Keymeulen, D. Pipeleers, J. W. Drijfhout, B. O. Roep. 2005. Autoreactive CD8 T cells associated with cell destruction in type 1 diabetes. Proc. Natl. Acad. Sci. USA 102: 18425-18430. [Abstract/Free Full Text]
22. Panina-Bordignon, P., R. Lang, P. M. van Endert, E. Benazzi, A. M. Felix, R. M. Pastore, G. A. Spinas, F. Sinigaglia. 1995. Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes. J. Exp. Med. 181: 1923-1927. [Abstract/Free Full Text]
23. Bingley, P. J., E. Bonifacio, A. J. Williams, S. Genovese, G. F. Bottazzo, E. A. Gale. 1997. Prediction of IDDM in the general population: strategies based on combinations of autoantibody markers. Diabetes 46: 1701-1710. [Abstract]
24. Kulmala, P., K. Savola, J. S. Petersen, P. Vahasalo, J. Karjalainen, T. Lopponen, T. Dyrberg, H. K. Akerblom, M. Knip. 1998. Prediction of insulin-dependent diabetes mellitus in siblings of children with diabetes, a population-based study: the Childhood Diabetes in Finland study group. J. Clin. Invest. 101: 327-336. [Medline]
25. Kawasaki, E., L. Yu, M. J. Rewers, J. C. Hutton, G. S. Eisenbarth. 1998. Definition of multiple ICA512/phogrin autoantibody epitopes and detection of intramolecular epitope spreading in relatives of patients with type 1 diabetes. Diabetes 47: 733-742. [Abstract]
26. Tisch, R., X. D. Yang, S. M. Singer, R. S. Liblau, L. Fugger, H. O. McDevitt. 1993. Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366: 72-75. [Medline]
27. Zekzer, D., F. S. Wong, O. Ayalon, I. Millet, M. Altieri, S. Shintani, M. Solimena, R. S. Sherwin. 1998. GAD-reactive CD4⁺ Th1 cells induce diabetes in NOD/SCID mice. J. Clin. Invest. 101: 68-73. [Medline]
28. Tarbell, K. V., M. Lee, E. Ranheim, C. C. Chao, M. Sanna, S. K. Kim, P. Dickie, L. Teyton, M. Davis, H. McDevitt. 2002. CD4⁺ T cells from glutamic acid decarboxylase (GAD)65-specific T cell receptor transgenic mice are not diabetogenic and can delay diabetes transfer. J. Exp. Med. 196: 481-492. [Abstract/Free Full Text]
29. Jaeckel, E., L. Klein, N. Martin-Orozco, H. von Boehmer. 2003. Normal incidence of diabetes in NOD mice tolerant to glutamic acid decarboxylase. J. Exp. Med. 197: 1635-1644. [Abstract/Free Full Text]
30. Kim, J., W. Richter, H. J. Aanstoot, Y. Shi, Q. Fu, R. Rajotte, G. Warnock, S. Baekkeskov. 1993. Differential expression of GAD65 and GAD67 in human, rat, and mouse pancreatic islets. Diabetes 42: 1799-1808. [Abstract]
31. Faulkner-Jones, B. E., D. S. Cram, J. Kun, L. C. Harrison. 1993. Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic cells and other peripheral tissues of mouse and rat. Endocrinology 133: 2962-2972. [Abstract]
32. Pugliese, A., D. Brown, D. Garza, D. Murchison, M. Zeller, M. J. Redondo, J. Diez, G. S. Eisenbarth, D. D. Patel, C. Ricordi. 2001. Self-antigen-presenting cells expressing diabetes-associated autoantigens exist in both thymus and peripheral lymphoid organs. J. Clin. Invest. 107: 555-564. [Medline]
33. Fennessy, M., K. Metcalfe, G. A. Hitman, M. Niven, P. A. Biro, J. Tuomilehto, E. Tuomilehto-Wolf. 1994. A gene in the HLA class I region contributes to susceptibility to IDDM in the Finnish population: Childhood Diabetes in Finland (DiMe) study group. Diabetologia 37: 937-944. [Medline]
34. Demaine, A. G., M. L. Hibberd, D. Mangles, B. A. Millward. 1995. A new marker in the HLA class I region is associated with the age at onset of IDDM. Diabetologia 38: 623-628. [Medline]
35. Robles, D. T., G. S. Eisenbarth, T. Wang, H. A. Erlich, T. L. Bugawan, S. R. Babu, K. Barriga, J. M. Norris, M. Hoffman, G. Klingensmith, et al 2002. Millennium award recipient contribution: identification of children with early onset and high incidence of anti-islet autoantibodies. Clin. Immunol. 102: 217-224. [Medline]
36. Marron, M. P., R. T. Graser, H. D. Chapman, D. V. Serreze. 2002. Functional evidence for the mediation of diabetogenic T cell responses by HLA-A2.1 MHC class I molecules through transgenic expression in NOD mice. Proc. Natl. Acad. Sci. USA 99: 13753-13758. [Abstract/Free Full Text]
37. Pascolo, S., N. Bervas, J. M. Ure, A. G. Smith, F. A. Lemonnier, B. Perarnau. 1997. HLA-A2.1-restricted education and cytolytic activity of CD8⁺ T lymphocytes from ₂ microglobulin (₂m) HLA-A2.1 monochain transgenic H-2Db ₂m double knockout mice. J. Exp. Med. 185: 2043-2051. [Abstract/Free Full Text]
38. Gurunathan, S., D. M. Klinman, R. A. Seder. 2000. DNA vaccines: immunology, application, and optimization. Annu. Rev. Immunol. 18: 927-974. [Medline]
39. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, S. Stevanovic. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219. [Medline]
40. Gauvrit, A., M. Debailleul, A. T. Vu, P. Sai, J. M. Bach. 2004. DNA vaccination encoding glutamic acid decarboxylase can enhance insulitis and diabetes in correlation with a specific Th2/3 CD4 T cell response in non-obese diabetic mice. Clin. Exp. Immunol. 137: 253-262. [Medline]
41. Bingley, P. J., E. Bonifacio, P. W. Mueller. 2003. Diabetes antibody standardization program: first assay proficiency evaluation. Diabetes 52: 1128-1136. [Abstract/Free Full Text]
42. Loirat, D., F. A. Lemonnier, M. L. Michel. 2000. Multiepitopic HLA-A*0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization. J. Immunol. 165: 4748-4755. [Abstract/Free Full Text]
43. Graff-Dubois, S., O. Faure, D. A. Gross, P. Alves, A. Scardino, S. Chouaib, F. A. Lemonnier, K. Kosmatopoulos. 2002. Generation of CTL recognizing an HLA-A*0201-restricted epitope shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 tumor antigens: implication in a broad-spectrum tumor immunotherapy. J. Immunol. 169: 575-580. [Abstract/Free Full Text]
44. Gallez-Hawkins, G., M. C. Villacres, X. Li, M. C. Sanborn, N. A. Lomeli, J. A. Zaia. 2003. Use of transgenic HLA A*0201/Kb and HHD II mice to evaluate frequency of cytomegalovirus IE1-derived peptide usage in eliciting human CD8 cytokine response. J. Virol. 77: 4457-4462. [Abstract/Free Full Text]
45. Faure, O., S. Graff-Dubois, L. Bretaudeau, L. Derre, D. A. Gross, P. M. Alves, S. Cornet, M. T. Duffour, S. Chouaib, I. Miconnet, et al 2004. Inducible Hsp70 as target of anticancer immunotherapy: identification of HLA-A*0201-restricted epitopes. Int. J. Cancer 108: 863-870. [Medline]
46. Komori, H., T. Nakatsura, S. Senju, Y. Yoshitake, Y. Motomura, Y. Ikuta, D. Fukuma, K. Yokomine, M. Harao, T. Beppu, et al 2006. Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma. Clin. Cancer Res. 12: 2689-2697. [Abstract/Free Full Text]
47. Hartemann-Heurtier, A., L. T. Mars, N. Bercovici, S. Desbois, C. Cambouris, E. Piaggio, J. Zappulla, A. Saoudi, R. S. Liblau. 2004. An altered self-peptide with superagonist activity blocks a CD8-mediated mouse model of type 1 diabetes. J. Immunol. 172: 915-922. [Abstract/Free Full Text]
48. Panagiotopoulos, C., H. Qin, R. Tan, C. B. Verchere. 2003. Identification of a cell-specific HLA class I restricted epitope in type 1 diabetes. Diabetes 52: 2647-2651. [Abstract/Free Full Text]
49. Garcia, F., P. Sepulveda, P. Liegeard, J. Gregoire, E. Hermann, F. Lemonnier, P. Langlade-Demoyen, M. Hontebeyrie, Y. C. Lone. 2003. Identification of HLA-A*0201-restricted cytotoxic T cell epitopes of Trypanosoma cruzi TcP2 protein in HLA-transgenic mice and patients. Microbes Infect. 5: 351-359. [Medline]
50. Bonifacio, E., M. Atkinson, G. Eisenbarth, D. Serreze, T. W. Kay, E. Lee-Chan, B. Singh. 2001. International workshop on lessons from animal models for human type 1 diabetes: identification of insulin but not glutamic acid decarboxylase or IA-2 as specific autoantigens of humoral autoimmunity in nonobese diabetic mice. Diabetes 50: 2451-2458. [Abstract/Free Full Text]
51. Takahashi, K., M. C. Honeyman, L. C. Harrison. 2001. Cytotoxic T cells to an epitope in the islet autoantigen IA-2 are not disease-specific. Clin. Immunol. 99: 360-364. [Medline]
52. Honeyman, M. C., N. L. Stone, L. C. Harrison. 1998. T cell epitopes in type 1 diabetes autoantigen tyrosine phosphatase IA-2: potential for mimicry with rotavirus and other environmental agents. Mol. Med. 4: 231-239. [Medline]
53. Mallone, R., S. A. Kochik, H. Reijonen, B. Carson, S. F. Ziegler, W. W. Kwok, G. T. Nepom. 2005. Functional avidity directs T cell fate in autoreactive CD4⁺ T cells. Blood 106: 2798-27805. [Abstract/Free Full Text]
54. Lichtenfils, R., W. E. Biddison, H. Schulz, A. B. Vogt, R. Martin. 1994. CARE-LASS (calcium-release-assay), an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity. J. Immunol. Methods 172: 227-239. [Medline]

This article has been cited by other articles:

				E. Martinuzzi, G. Novelli, M. Scotto, P. Blancou, J.-M. Bach, L. Chaillous, G. Bruno, L. Chatenoud, P. van Endert, and R. Mallone The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment Diabetes, May 1, 2008; 57(5): 1312 - 1320. [Abstract] [Full Text] [PDF]

				E. Enee, E. Martinuzzi, P. Blancou, J.-M. Bach, R. Mallone, and P. van Endert Equivalent Specificity of Peripheral Blood and Islet-Infiltrating CD8+ T Lymphocytes in Spontaneously Diabetic HLA-A2 Transgenic NOD Mice J. Immunol., April 15, 2008; 180(8): 5430 - 5438. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blancou, P. Articles by Bach, J.-M. Search for Related Content PubMed PubMed Citation Articles by Blancou, P. Articles by Bach, J.-M.