Zinc transporter (ZnT)8186–194 is an immunodominant CD8+ T cell epitope in HLA-A2+ type 1 diabetic patients
- First Online:
- Cite this article as:
- Scotto, M., Afonso, G., Larger, E. et al. Diabetologia (2012) 55: 2026. doi:10.1007/s00125-012-2543-z
- 681 Downloads
Anti-zinc transporter (ZnT)8 autoantibodies are commonly detected in type 1 diabetic patients. We hypothesised that ZnT8 is also recognised by CD8+ T cells and aimed to identify HLA-A2 (A*02:01)-restricted epitope targets.
Candidate epitopes were selected by ZnT8 plasmid DNA immunisation of HLA-A2/DQ8 transgenic mice and tested for T cell recognition in peripheral blood mononuclear cells of type 1 diabetic, type 2 diabetic and healthy participants by IFN-γ enzyme-linked immunospot.
White HLA-A2+ adults (83%) and children (60%) with type 1 diabetes displayed ZnT8-reactive CD8+ T cells that recognised a single ZnT8186–194 (VAANIVLTV) epitope. This ZnT8186–194-reactive fraction accounted for 50% to 53% of total ZnT8-specific CD8+ T cells. Another sequence, ZnT8153–161 (VVTGVLVYL), was recognised in 20% and 25% of type 1 diabetic adults and children, respectively. Both epitopes were type 1 diabetes-specific, being marginally recognised by type 2 diabetic and healthy participants (7-12% for ZnT8186–194, 0% for ZnT8153–161).
ZnT8-reactive CD8+ T cells are predominantly directed against the ZnT8186–194 epitope and are detected in a majority of type 1 diabetic patients. The exceptional immunodominance of ZnT8186–194 may point to common environmental triggers precipitating beta cell autoimmunity.
KeywordsAutoimmunity Beta cell Environment Islet SCL30A8 Type 1 diabetes
Intracellular part of protein tyrosine phosphatase 2
Mycobacterium avium paratuberculosis
Peripheral blood mononuclear cell
The autoimmune cascade leading to beta cell destruction in type 1 diabetes is propagated by T cell recognition of specific epitopes. Epitope mapping is therefore critical for staging of autoimmunity during the disease, and for developing and monitoring immunomodulatory therapies. Indeed, a combination of multiple epitope markers is likely to track beta cell autoimmunity more efficiently than any single epitope. Likewise, restoring immune tolerance to a broad antigen spectrum may be clinically more effective .
Zinc transporter (ZnT)8 was recently identified as a major type 1 diabetes antigen, with 60–80% of recent-onset type 1 diabetic participants harbouring antibodies against its C-terminal domain . As for other beta cell antigens, such autoantibodies are likely to be accompanied by autoreactive T cells targeting ZnT8 . Indeed, ZnT8-reactive CD4+ and CD8+ T cells were recently described in white and Chinese type 1 diabetic patients, respectively [3, 4]. Given their central role in beta cell autoimmunity [5, 6], further characterisation of these CD8+ T cells is needed. Here, we identify a highly immunodominant ZnT8186–194 epitope recognised by such cells.
ZnT8 peptide library
We synthesised 70 nonamer peptides (>75% purity; ProImmune, Oxford, UK) that had been selected as potential binders to HLA-A1, -A2, -A3, -A24, -B7 and -B8 (electronic supplementary material [ESM] Table 1). Binding to recombinant HLA-A2 molecules was assessed using Class I REVEAL assays (ProImmune). Candidate epitopes were re-synthesised at >90% purity.
HLA-A2/DQ8 transgenic (A*02:01/DQB1*03:02) H-2Db−/−β2m−/−IAbβ−/−IAbα−/−IEbβ−/− mice were obtained by crossing HLA-A2 and HLA-DQ8 transgenic mice [7, 8]. These mice were pretreated with cardiotoxin and vaccinated twice with non-coding plasmid or plasmid encoding full-length human ZnT8 (1:1 mixture of Arg/Trp325 isoforms) cloned into pcDNA3.1, as described previously . The study was approved by the local ethics committee. Splenocytes were plated (5 × 105/well) in IFN-γ enzyme-linked immunospot (ELISpot) plates (Millipore, Molsheim, France) along with peptides (10 μmol/l). After culturing for 20 to 24 h, plates were treated with a biotinylated anti-IFN-γ antibody (U-CyTech, Utrecht, the Netherlands), alkaline phosphatase-conjugated streptavidin and NBT-BCIP (Sigma, Lyon, France). Triplicate wells were counted and averaged using a Bioreader 5000 Pro-SF equipment (BioSys, Karben, Germany).
We recruited the following participant groups in Paris and Turin: (1) new-onset autoantibody-positive type 1 diabetic adults (n = 12; 8 men, 4 women; median age 31 years [range 24–60]; diabetes duration 39 days [0–140]) and children (n = 10; 6 boys, 4 girls; median age 12 years [1–16]; diabetes duration 5 days [0–80]); (2) type 2 diabetic patients (n = 24; 15 men, 9 women; median age 60 years [12–77]; diabetes duration 12 years [0–42]); and (3) healthy controls (n = 27; 12 men, 15 women; median age 36 years [22–60]). Serum antibodies were measured by RIA, using 35S-labelled GAD65, the intracellular part of protein tyrosine phosphatase-2 (IA-2ic) and ZnT8 (construct C-term CRCW JH6.2). All participants were HLA-A2+ (HLA-A*02:01) by genotyping and gave written informed consent. Local ethics committees approved the study.
Peripheral blood mononuclear cells (PBMCs) prepared as described previously  were used fresh or frozen-and-thawed with similar results [5, 9]. PBMCs (3 × 105/well) were seeded in anti-IFN-γ-coated ELISpot plates in AIM-V medium (Life Technologies, Saint Aubin, France) containing 0.5 ng/ml IL-7 and 10 μmol/l peptide. A viral peptide mix (Flu MP58–66, Epstein–Barr virus BMLF280–288, cytomegalovirus pp65495–503) and phytohaemagglutinin (1 μg/ml) were used as positive controls. Pyruvate dehydrogenase (PD)5–13 and DMSO diluent were negative controls. After 20 to 24 h, plates were developed and counted as above. For blocking experiments, anti-CD8 (OKT8, 25 μg/ml) or anti-HLA-A2 (BB7.2, 50 μg/ml, both in-house) were added to wells. Readouts are expressed as spot-forming cells (SFC)/106 PBMCs after subtracting background responses against PD5–13 and no peptide (which were identical in all cases). The positive cut-off was set at 3 SD above the average background, as determined by receiver-operator characteristics analysis . Intra- and inter-assay CVs are 14% and 9%, respectively .
Values are expressed as mean ± SD or median (range), according to distribution. Comparisons between proportions were made with Fisher's exact test.
Binding assays to recombinant HLA-A2 molecules were performed in parallel (Fig. 1c). While most (8/10; 80%) candidate epitopes identified by DNA immunisation were strong HLA-A2 binders, two epitopes (number 50, ZnT8253–261 and number 57, ZnT8280–288) were weak binders. In addition, several epitopes (22/30; 73%) that were strong HLA-A2 binders did not elicit significant responses in DNA-immunised mice. Thus, compared with epitope selection based on HLA-binding affinity, DNA immunisation allowed us to focus on fewer candidates (10/70 vs 30/70; 14% vs 43%), including two weakly binding peptides. Moreover, DNA immunisation selects candidates on the additional criterion of natural processing and presentation .
The median frequency of ZnT8 epitope-reactive CD8+ T cells in new-onset type 1 diabetic patients (34.5 SFC/106 PBMCs, i.e. 0.003%, range 0.0007–0.08%) was similar to that previously observed for other beta cell epitopes [5, 7]. Moreover, one type 1 diabetic adult patient (identified as A03) was studied at four different time points (Fig. 2c). While IFN-γ responses to a viral mix remained fairly stable over time, frequencies of ZnT8186–194-reactive T cells secreting IFN-γ gradually increased over time, while the corresponding ZnT8153–161-reactive T cells rapidly disappeared.
The immunodominance of ZnT8186–194 was also evident when considering its relative representation among all epitopes recognised (Fig. 2d). Indeed, ZnT8186–194 was the most frequently targeted peptide in type 1 diabetic adults and children (53% and 50% of total ZnT8-reactive CD8+ T cell responses, respectively) compared with other epitopes.
ZnT8 is clearly a major target of autoreactive CD8+ T cells in white new-onset type 1 diabetic patients. Strikingly, a single HLA-A2-restricted ZnT8186–194 epitope was recognised by the overwhelming majority of ZnT8-reactive T cells (50–53%), and in both type 1 diabetic adults (83%) and children (60%). Another epitope, ZnT8153–161, ranked second, being recognised in 20% to 25% of type 1 diabetic patients. Such a high degree of epitope focusing is surprising, as autoreactivity is usually more widely distributed against different sequences . Indeed, the two most immunodominant epitopes described to date are GAD114–123  and preproinsulin (PPI)15–24 , which are recognised in ~50% of type 1 diabetic adults compared with the 83% scored by ZnT8186–194. An increase in ZnT8186–194-reactive IFN-γ responses during the disease was also observed in one patient, contrary to observations for most epitope reactivities, including ZnT8153–161, which rapidly wane after diagnosis . ZnT8253–261 was recognised in 25% of type 2 diabetic patients, a population seldom included in previous T cell studies. The phenotype of these patients was not distinctive; they were all antibody-negative and all but one not being treated with insulin. However, it is possible that larger ad hoc studies focusing on patients of uncertain classification may reveal a discrete subset.
The epitopes here identified do not overlap with those targeted by CD4+ T cells . The subdominant ZnT8153–161 epitope was also recognised by PBMCs of Chinese HLA-A2+ recent-onset type 1 diabetic patients . In contrast, the novel ZnT8186–194 emerges here as the major epitope recognised. Intriguingly, we recently described the same ZnT8186–194 epitope and its contiguous ZnT8178–186 sequence as targets of autoantibody responses in ~60% of Sardinian type 1 diabetic patients . These autoantibodies were cross-reactive with homologous sequences of the Mycobacterium avium paratuberculosis (MAP) 3865c protein, prompting the hypothesis that MAP could be an environmental trigger of type 1 diabetes via a molecular mimicry mechanism . It would therefore be relevant to investigate whether ZnT8186–194-reactive CD8+ T cells also cross-recognise MAP3865c133–141, which, if confirmed, could explain their high prevalence.
We wish to thank all the patients who contributed to this research with their blood donations, as well as A. Falaschi-Jones (Inserm U986, Paris, France), S. Marchand and C. Dumange (Hôtel Dieu, Paris, France) for their help with patient databases.
This study was supported by grants from JDRF (grant 1-2008-106), the European Foundation for the Study of Diabetes (EFSD/JDRF/Novo Nordisk European Programme in Type 1 Diabetes Research 2007), the Inserm Programme National de Recherche sur le Diabète 2007, the Fondation Recherche Médicale (grant Installation Nouvelle Equipe), the Ile-de-France CODDIM (grant Soutien aux Jeunes Equipes) and the Societé Francophone du Diabète to R. Mallone. R. Mallone is an Inserm Avenir Investigator and recipient of an APHP-Inserm Contrat Hospitalier de Recherche Translationelle. J. C. Hutton and H. W. Davidson acknowledge the JDRF Grant 4-2007-1056, and University of Colorado Diabetes Research Center NIH grants P30-DK-57516 and R01-DK-052068.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
MS and GA designed and performed experiments, participated in data analysis and interpretation, and edited the manuscript. EL and CB participated in study design and data interpretation, provided blood samples and clinical data, and edited the manuscript. CR, JCC, DDL, BB, DL, JFG, OL and GB participated in research discussions, provided blood samples and clinical data, and reviewed the manuscript. FAL and LAS participated in the study design and data interpretation, provided reagents and reviewed the manuscript. JCH and HWD participated in the study design, data analysis and interpretation, performed experiments, provided reagents and edited the manuscript. RM coordinated the study, designed experiments, participated in data analysis and interpretation, and wrote the manuscript. All authors gave final approval for the article.