High-avidity peptide/MHC class I tetramers detect a higher frequency of autoreactive T cells from freshly isolated islets. All numbers indicate percentage of CD8⁺ B220^– tetramer⁺ cells. (a) Pancreatic islets derived from 8-week-old female NOD mice were stained with tetramers associated with the indicated peptides. Data are representative of six independent experiments from mice 6–15 weeks of age. Similar results (but approximately tenfold lower) were observed with peripheral blood (data not shown). (b) Representative examples of TUM, NRP-V7, and INS tetramer staining of islets at 4, 9, 12, and 16 weeks of age. (c) Mean percentage (± SEM) of tetramer-positive cells from mice at 4–5 (n = 4), 7–10 (n = 14), 11–14 (n = 17), and 15–18 (n = 14) weeks of age. NRP staining performed only at 4–5 weeks, n = 3; 7–10 weeks, n = 7; 15–18 weeks, n = 10. Data is shown also for nondiabetic mice at 32 weeks of age (n = 5). ND, not determined; PE, phycoerythrin.*P < 0.001 compared with TUM at each age. (d) Islet cells pooled from 4 mice (13–16 weeks of age) were assayed by ELISpot for IFN-γ secretion in response to TUM, NRP-V7, or INS (n = 4). Naive spleen cells from an 8.3-TCR NOD mouse were used as negative control. *P < 0.0001 compared with TUM, ^#P < 0.05 compared with INS. Inset: representative ELISpot assay: duplicate wells containing 20,000 islet cells from 14-week-old NOD mice.