Responses to GLIPR1 4–12 and EpCAM 140–148 in cases and controls, and HLA restriction studies.

<p>Responses to <b>a</b>) GLIPR1 (4–12) <b>b</b>) and EpCAM (140–148) in T1D, at risk and control subjects. In both panels, PBMCs from the indicated patient groups were stimulated with GLIPR1 or EpCAM peptides (filled bars) vs. control peptide (open bars) overnight. Anti-CD3/CD28 stimulation was used as a positive control and all responses to antibody stimulation were too numerous to count (data not shown). The graphs display the average raw number of spots from triplicate wells +/− the standard error. The (*) indicates for a given patient that the experimental peptide responses were significantly higher (FDR <0.05) than the response to the control peptide. The number of patients who significantly responded to the each experimental peptide is indicated by the fraction in the graph. PBMC from subject ND2 used for discovery of each epitope were not available and are not included in these graphs. <b>c</b>) HLA-A*0201 binding assay for GLIPR1 epitope TLATIAWMV on T2 cells. 1×10⁶ T2 cells were incubated with 20 µg/ml of the indicated peptide in media for 4 hours at 37°C, stained for HLA-A*0201 and evaluated by flow cytomety. <b>d</b>). Presentation of GLIPR1 eptiope by HLA A*0201. T2 cells were loaded with 10 µg/ml GLIPR1 epitope TLATIAWMV or control epitope from pyruvate dehydrogenase for two hours at room temperature, washed and mixed with purified CD8+ T cells from one responding subject (T1D #2) in a direct IFNγ assay and was found to be statistically significant compared to the control peptide (FDR<0.05).