The results of this initial study of cellular autoimmunity to ZnT8 clearly suggest that, like the other “gold-standard” humoral targets, ZnT8 is a significant target of autoimmune T cells in human T1D. Based on overall activity, over 68% of patients but less than 8% of controls, showed significantly expanded numbers of ZnT8-specific pro-inflammatory T cells in their peripheral blood. The presence of positive responses in some controls may appear surprising, but has often been reported in studies of human T cell responses to other diabetes autoantigens (reviewed by (1
)), and may be indicative of the inherent immunogenicity of the antigen. In addition, it should be noted that some of the control subjects are autoantibody negative first-degree relatives of diabetic subjects, who might be more prone to islet autoimmunity than those with more protective genotypes. Using an established cut-off, all of the newly diabetic subjects tested responded to at least one peptide from ZnT8, with the majority exhibiting elevated numbers of pro-inflammatory T cells specific to multiple non-contiguous regions of the protein, likely indicative of “epitope-spreading.” Moreover, the magnitude of the signal elicited by individual pools among responders was also considerably greater in the patients than controls. The time course of the ELISPOT assay is too short to allow significant maturation of naive cells to occur (29
). Consequently the responses we observed presumably reflect the presence of elevated pools of ZnT8-specific effector and/or memory T cells in the peripheral blood of the diabetic subjects, with the most likely explanation for this being that it is indicative of a role of ZnT8-specific T cells in diabetogenesis. However, as is also true for all other human T cell responses to diabetes autoantigens, the relationship between peripheral T cells and those in the target organ remains a subject of debate (for example (30
)). Nevertheless, consistent with an antigen-driven process, post-onset the peripheral frequency of ZnT8-specific IFN-γ producing T cells appeared greatest within 6 months of clinical diagnosis, with those individuals re-tested at later ages typically showing a much reduced response (data not shown). Given that cross-presentation of free 20mer peptides is typically inefficient (32
), we assume that the responses we observed were predominantly, if not exclusively, derived from CD4+
T cells. However, as the library used may contain some truncated peptides we cannot entirely exclude the possibility that CD8+
T cells may also have contributed, although our recent studies using highly purified peptides suggest that this is unlikely to have significantly impacted the results we obtained.
When considered individually, 13 of the 23 di-peptide pools showed statistically significant disease association at the 95% confidence level. Although at first glance this may seem unexpectedly high, the human MHC is highly complex, with any individual expressing up to 12 different class II molecules, most if not all of which have the potential to bind multiple peptides from ZnT8. The existence of ZnT8-specific T cells restricted to many different class II molecules, both within an individual, and in the control and patient populations as a whole, likely explains both the breadth of the auto-response we observed, and the fact that all of the pools gave positive responses in at least 11% of the patients tested. Indeed the degree of complexity of the response only increased when the components of the most reactive pools were tested individually in a separate cohort of patients (data not shown). To date we have only conducted direct binding studies with two of the HLA-DR molecules expressed by the subjects in our study. However, the level of T cell reactivity we observed appears consistent with in silico
predictions that suggest that most of the HLA-DR molecules expressed by our subjects, including some of those deemed “protective”, have the capacity to bind peptides from multiple pools of the library. The bioinformatics based predictions suggest that ZnT8 is inherently immunogenic, and the magnitude of the response we observe implies that its potential as a target of cellular immunity is realized in many patients, perhaps because ZnT8-specific T cells are subject to minimal negative selection. For tissue-specific antigens such as proinsulin central tolerance is believed to depend upon thymic and extra-thymic expression driven by transcriptional activators such as AIRE (33
) and Deaf1 (35
). Interestingly, ZnT8 (slc30a8) was not identified among the genes selectively expressed by mouse thymic medullary epithelial cells (36
) and we are unaware of any evidence of thymic expression in humans. Thus it is tempting to speculate that tolerance to ZnT8 is largely the result of immunological ignorance, and that once this is overcome a robust response may ensue.
That said, it is possible that the actual diversity of the auto-response might be slightly exaggerated by our methodology, with the overlap of the constituent peptides in consecutive pools meaning that some of the disease-associated responses we observed might be non-redundant. For example, part or all of the reactivity to pool 18 could be due to epitopes also in pools 17 and/or 19. Nonetheless, our data strongly suggests the presence of a minimum of 9 disease associated T cell epitopes in human ZnT8. Coincidentally, this value is similar to those obtained in studies using overlapping peptide libraries encompassing GAD65 (37
), and the cytoplasmic domain of IA-2 (38
). The fact that the HLA haplotypes exhibited by the 2 groups were not identical means that we cannot discount the possibility that variations in detection limits within the 2 groups might have influenced the magnitude of the differences in overall reactivity that we observed. However, its contribution is clearly insufficient to account for the results we obtained, and a highly statistically significant disease association was also evident when our analysis was limited to non-overlapping peptide pools (, ), when this concern no longer applies.
Our decision to utilize a library of overlapping peptides rather than focusing solely on those predicted to bind to high risk alleles was intended to ensure an unbiased analysis of the overall proinflammatory CD4+ response in patients and controls. However, the fine mapping of epitopes is highly problematic using this experimental approach, and at present we can neither be certain of the total number of immunodominant ZnT8 epitopes, nor of their restriction to any particular class II molecule. Nevertheless, our results clearly indicate that like ZnT8A, ZnT8-directed T cell autoimmunity is associated with, but not restricted to, both the DR4/DQ8 and DR3/DQ2 high-risk haplotypes. In particular the HLA-DR4 (0401) molecule, which was expressed by 19/35 (54.3%) of the diabetic group, appears to be a major element in ZnT8 autoimmunity, with several peptides that bind this molecule including ZnT88–22 and ZnT815–29 (pool 1), ZnT8120–134 and ZnT8134–148 (pool 9), ZnT8260–274 (pool 17), ZnT8267–281 (pools 17 and 18), and ZnT8295–309 (pool 19) possibly containing disease-related epitopes. Similarly, ZnT8155–169 (pool 10) and ZnT8323–337 (pool 21) may contain HLA-DR3 restricted epitopes.
In a previous study of autoreactivity to IA-2 and proinsulin, Arif and colleagues reported a reciprocal polarization towards IL-10 or IFN-γ production for peripheral T cells from patients and controls specific for the same epitope (14
). In the present study we were unable to measure both IFN-γ and IL-10 responses to the entire peptide library in the majority of our study subjects due to the limited amount of blood available, although preliminary results from the sub-set tested for both cytokines revealed essentially equivalent ZnT8-specific IL-10 responses in both patients and controls (data not shown). Our identification of 7 key disease associated ZnT8 peptide pools will now enable us to address this and other important questions relating to the role of ZnT8 in T1D pathogenesis, such as the temporal appearance/disappearance of ZnT8-specific T cells during disease progression, and whether ZnT8 is also a significant target of “natural” regulatory T cells. It also provides the basis for the development of novel reagents that either alone or in combination could be used for antigen-specific therapeutic intervention to arrest the progression of the disease.