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Recently we demonstrated that zinc transporter 8 (ZnT8) is a major target of autoantibodies in human type 1 diabetes (T1D). Since 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 (< 6mo post-onset at blood draw) and 41 age-matched controls were assayed by ELISPOT using a library of 23 overlapping di-peptide pools covering the entire 369aa primary sequence. Consistent with our hypothesis, patients showed significantly higher T cell reactivity than the matched controls, manifest both in terms of the breadth of the overall response, and the magnitude of responses to individual pools. Thus, the median number of pools giving positive responses (stimulation index ≥ 3) in the control group was 1.0 (range 0 – 7) compared to 6.0 (range 1 – 20; p < 0.0001) for the patients. Similarly, the median SI of positive responses in controls was 3.1 versus 5.0 in the patients (p < 0.0001). Individually 7/23 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 suggest that reagents that target ZnT8-specific T cells may have therapeutic potential in preventing or arresting the progression of this disease.

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n=27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n=31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n=11) and control subjects (n=11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.

This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course.

Risk of type 1 diabetes at 3 years is high for initially multiple and single Ab+ IT and multiple Ab+ NT. Genetic predisposition, age, and male sex are significant risk factors for development of Ab+ in twins.

Using a multicentre blind-coded setup and three different T-cell assays, we have validated PPI and GAD epitopes as commonly recognized CD8+ T-cell targets in recently diagnosed T1D patients. Both TMrs and PMrs showed higher detection sensitivity than the CD8+ T-cell interferon-γ enzyme-linked immunospot assay. However, there are some important methodological issues that need to be addressed in using these sensitive techniques for detecting low frequency responses.

SUMMARYIn a prospective study. CD45 isoform expression on T lymphocytes was analysed in seven patienis with haemophagocytic lymphohistiocytosis (HLH) and their family members. Six patients, their parents and seven healthy age-matched controls showed the normal pattern of three subpopulations of CD45R expression. In one patient, his mother and one of his healthy brothers only two subpopulations could be identified, which might indicate that the maturation from a CD45RA/RO double-positive to a CD45RO single-positive phenotype does not occur in these individuals. It is not clear if this finding had any impact on the development of HLH in this patient and if the pattern of CD45RA and RO expression observed in this family might represenl a predisposition to HLH. In another patient a marked increase of the CD45RO single-positive T cell population was observed during a f»eriod of increased disease activity. The clinical relevance of ihis observation and whether CD45RO expression could serve as a marker for disease activity, or in selected individuals as a susceptibility marker for HLH. remain unknown.

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