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It was examined that the BDC 2.5-like T cell response in NOD mice by sorting and analyzing single cells with I-Ag7 tetramers displaying BDC 2.5 peptide mimotopes with either a negative charge (HHPIWARMDA; hereafter referred to as 2.5mi) or no charge at P9 (G or Q, named P9G and P9Q thereafter, HHPIWARMG/QA). The sequencing of individual TCR segments revealed an increase of negative residues in the CDR3β N-terminal region from cells sorted with P9Q or P9G tetramers (16/31, 52%) as compared with cells sorted with P9D tetramers (5/23, 21%). The latter frequency was comparable to the normal frequency of negative charges found in all purified CD4+ CDR3β sequences in NOD mice. The negative charges found in the CDR3β of the P9Q and P9G populations were encoded by N additions or deletions, suggesting an antigen-driven process. This belief was also supported by the fact that P9G or P9Q tetramer–positive cells were found in higher numbers in the periphery than in the thymus and that over time, the population expanded early (3–4 weeks), then contracted (6–12 weeks) to reexpand later, especially in diabetic animals. In addition, the BDC 2.5 9Q/9G reactive TCR populations were found, up until week twelve, to be larger than the corresponding BDC 2.5 9D populations.
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