T-cell epitope mapping. (a) The peptide library covering the entire human glucose-6-phosphate-isomerase (G6PI) sequence contained 137 peptides of 15 amino acids length, overlapping by 11 amino acids (aa). (b) Epitope mapping with two-dimensional peptide pools. Pools are represented by horizontal lines or vertical columns (white numbers on black background). For example, Pool 14 contains peptides 13 through 24 (numbers on white background). Each peptide is represented in two pools. For example, peptide 22 is represented in Pools 10 and 14. For epitope mapping, DBA/1 mice were immunized with 100 μg G6PI in complete Freund's adjuvant (n = 3 per experiment). Single-cell suspensions were prepared from draining lymph nodes and the spleen 12 days after immunization, and proliferation assays were performed as described in Materials and methods. Three independent experiments were performed. The one peptide (peptide 22) that scored positive in all three experiments is highlighted in bold italic; peptides identified in two experiments are highlighted by bold underlined numerals (peptides 17, 118, 125 and 30); peptides identified in only one experiment are highlighted in bold and the other peptides are given in grey. (c) Results from one exemplary experiment are shown. Results are displayed as the stimulation index. A stimulation index >2 was considered positive.