RESUMO
Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.
Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Isoenzimas/imunologia , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Células Cultivadas , Diabetes Mellitus Tipo 1/etiologia , Feminino , Glutamato Descarboxilase/isolamento & purificação , Glutamato Descarboxilase/metabolismo , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Biblioteca de Peptídeos , Estado Pré-Diabético/imunologia , Homologia de Sequência de Aminoácidos , Linfócitos T/transplanteRESUMO
Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.