Human type 1 diabetes is associated with T cell autoimmunity to zinc transporter 8

Abstract

Recently we demonstrated that zinc transporter 8 (ZnT8) is a major target of autoantibodies in human type 1 diabetes (T1D). Because the molecules recognized by T1D autoantibodies are typically also targets of autoreactive T cells, we reasoned that this would likely be the case for ZnT8. To test this hypothesis, IFN-γ-producing T cells specific for ZnT8 in the peripheral blood of 35 patients with T1D (<6 mo after onset at blood draw) and 41 age-matched controls were assayed by ELISPOT using a library of 23 overlapping dipeptide pools covering the entire 369 aa primary sequence. Consistent with our hypothesis, patients showed significantly higher T cell reactivity than the matched controls, manifest in terms of the breadth of the overall response and the magnitude of responses to individual pools. Therefore, the median number of pools giving positive responses (stimulation index ≥ 3) in the control group was 1.0 (range, 0-7) compared with 6.0 (range, 1-20; p < 0.0001) for the patients. Similarly, the median stimulation index of positive responses in controls was 3.1 versus 5.0 in the patients (p < 0.0001). Individually, 7 of 23 pools showed significant disease association (p < 0.001), with several of the component peptides binding the disease associated HLA-DR3 (0301) and -DR4 (0401) molecules in vitro. We conclude that ZnT8 is also a major target of disease-associated autoreactive T cells in human T1D, and we suggest that reagents that target ZnT8-specific T cells could have therapeutic potential in preventing or arresting the progression of this disease.

FIGURE 1

T cell responses to ZnT8 in newly diabetic individuals. A, Schematic representation of the ZnT8 protein and peptide library. The transmembrane helices (solid bars), peptide pools (alternating pairs of faint and bold lines), and polymorphic residue 325 are indicated. B–G, PBMCs from 35 newly diabetic subjects and 41 age-matched controls were analyzed by IFN-γ ELISPOT as described in methods using 23 ZnT8 di-peptide pools. B, Total (spots - background) values of summed ZnT8 incubations from each subject are shown. C, The number of ZnT8 peptide pools giving positive responses (SI ≥ 3) in each subject is shown. D, The number of ZnT8 peptide pools giving positive responses (SI ≥ 2.1) in each subject is shown. E, The number of ZnT8 peptide pools giving positive responses (SI ≥ 5) in each subject is shown. F, The number of positive ZnT8 peptide pools (SI ≥ 3) in each subject is shown after correction for potentially non-redundant responses. G, The median SI of positive responses to ZnT8 (SI ≥ 3) in each subject is shown. In B – E the median and interquartile ranges of each group is indicated.

FIGURE 2

Effect of the HLA genotype on proinflammatory T cell reactivity to ZnT8. PBMCs from 35 newly diabetic subjects and 41 age-matched controls were analyzed by IFN-γ ELISPOT as described in methods using 23 ZnT8 di-peptide pools. Individuals were stratified on the basis of their expression of selected HLA molecules, and the total responses (spots - background) in each included subject is shown. A, Subjects expressing HLA-DR3/DQ2 and/HLADR4/DQ8. B, Controls were stratified on the basis of HLA-DQB1*0602 or HLA-DRB1*0403 expression. C, The control group was restricted to subjects who did not express either HLA-DQB1*0602 or HLA-DRB1*0403. D, The control group was restricted to subjects who expressed either HLA-DQB1*0602 and/or HLA-DRB1*0403. The median and interquartile ranges of each group is indicated.

FIGURE 3

Binding of ZnT8 peptides to recombinant HLA-DR3 and -DR4 in vitro. A library of 52 15mers encompassing the entire 369aa sequence of ZnT8 was tested for binding to recombinant HLA-DR3(0301) (A) and HLA-DR4(0401) (B) using the Class II REVEAL^™ binding assay (Proimmune). The boxes above each profile indicate the peptide pools used in the ex vivo assays, and are filled to indicate statistical association with T1D in relevant groups (*p < 0.05; **p < 0.01, supplementary table III). The solid lines show the cut-off for positivity of the assay.

FIGURE 4

T1D association of proinflammatory T cell reactivity to ZnT8. PBMCs from 35 newly diabetic subjects and 41 age-matched controls were analyzed by IFN-γ ELISPOT as described in methods using 23 ZnT8 di-peptide pools, and stratified on the basis of the 6 non-redundant pools giving the most significant disease association (Table III). A, The number of selected peptide pools giving positive responses (SI ≥ 3) in each subject is shown. B, The number of selected peptide pools giving positive responses in subjects who expressed at least 1 HLA-DR3/DQ2 or HLA-DR4/DQ8 haplotype is shown. The median and interquartile ranges of each group is indicated. The dotted line shows a cut-off for positivity based on the mean + 3SD of the respective control group.