Extended Data Fig. 2: Evaluation of T cell reactivity and relative MHCII binding affinity in peptides selected from the MHCII peptidome.

a, Pooled islet and pLN cells from 8-10-week old female NOD mice were challenged with each indicated peptide for two cycles, and ELISPOT assays were conducted to read IFN-γ production in the cells upon recalling with the same peptide (see Methods). The results show positive T cell responses to two immunogenic β-cell-derived peptides, Ins1C:33-61 and InsB:9-23, indicating the presence of the effector T cells to these peptides. No IFN-γ responses were observed to the HEL protein or the I-A^g7-binding HEL:11-25 peptide, demonstrating that the culture assay did not generate de novo T cells. Data (mean ± s.e.m.) are representative of n = 3 experiments using n = 5 mice per experiment. b, Different numbers of pre-activated HEL-reactive T cells were spiked into the normal two-cycle culture, followed by recall with the HEL peptide and ELISPOT assay. The data depict a ~1:1 recovery ratio of the spiked HEL-reactive T cells with the IL-2 spots. The assay detected as few as ~20 pre-activated HEL-reactive T cells, indicating a high sensitivity. Data (mean ± s.e.m.) are representative of n = 2 experiments using n = 8 mice per experiment. c, An example of the competitive binding assay and the presentation of the data. A standard APC line (C3g7) was cultured with 1 µM HEL:11-25 peptide together with serial dilutions of a competitor peptide: the response of a specific T cell hybridoma to HEL:11-25 was probed by standard antigen presentation assay (see Methods). In every experiment, each competitor peptides were compared to a reference peptide, g7-MIME, with known strong binding to I-A^g7. The amounts of the peptide required to compete half-maximal T cell response to HEL:11-25 (IC50) was estimated and compared to the reference peptide. A higher amount indicated a weaker binding affinity. The table (right) depicts the IC50 calculated from several representative peptides. The results are presented as the percent of reference after normalization to the reference g7-MIME peptide. Data are presentative of n = 4 independent experiments with similar results.