(A) Five clones isolated from a patient with type 1 diabetes stained with anti-CD8 and HLA-A2 tetramers loaded with PPI_15–24. Tetramer-stained cells were detected in the upper-right quadrant. A CD8⁺ T cell clone obtained in the same expansion that was negative for PPI_15–24–Tmr (3C9) and a clone (2D9) raised against the CMV pp65 epitope NLVPMVATV are shown. (B) Staining of the same clones with HLA-A2 tetramers loaded with the CMV pp65 epitope NLVPMVATV. (C) PPI_15–24–specific T cell clones produce TNF-α in a dose-dependent fashion, shown here as the percentage of 1E6 clone cells staining for intracellular cytokine in response to HLA-A2⁺ PBMCs pulsed with varying concentrations of PPI_15–24. (D) 1E6 clone cells proliferate in response to PPI_15–24 peptide-pulsed monocyte-derived DCs, as measured by [³H]thymidine incorporation. No response was observed using antigen-presenting cells lacking HLA-A2 (data not shown). (E) 1E6 clone cells also recognize the PPI_15–24 epitope when naturally processed by K562-PPI-A2 cells, since no TNF-α was produced after coculture with K562-A2 cells, but in the presence of PPI_15–24 peptide-pulsed K562-A2 (1 μg/ml) or K562-PPI-A2 cells, copious amounts of cytokine were produced. Isotype control staining was similar to control-activated clone cells. Proliferation assays were performed in triplicate; bars represent means, error bars are SEMs. Values inside flow cytometry quadrants indicate percentage of positive cells. Data are representative of more than 5 individual experiments.