No abstract

Antigens of pathogenic microbes that mimic autoantigens are thought to be responsible for the activation of autoreactive T cells. Viral infections have been associated with the development of the neuroendocrine autoimmune diseases type 1 diabetes and stiffman syndrome, but the mechanism is unknown. These diseases share glutamic acid decarboxylase (GAD65) as a major autoantigen. We screened synthetic peptide libraries dedicated to bind to HLA-DR3, which predisposes to both diseases, using clonal CD4 ؉ T cells reactive to GAD65 isolated from a prediabetic stiff-man syndrome patient. Here we show that these GAD65-specific T cells crossreact with a peptide of the human cytomegalovirus (hCMV) major DNA-binding protein. This peptide was identified after database searching with a recognition pattern that had been deduced from the library studies. Furthermore, we showed that hCMV-derived epitope can be naturally processed by dendritic cells and recognized by GAD65 reactive T cells. Thus, hCMV may be involved in the loss of T cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity.

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4 ؉ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4 ؉ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the ␤ cell-reactive CD4 ؉ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4 ؉ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and͞or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.A large number of T cell-mediated diseases, exemplified by the autoimmune disease insulin-dependent diabetes mellitus (IDDM), are thought to be caused by the activation of CD4 ϩ T cells by antigen-derived peptides in the context of HLA class II (1, 2). For many T cell-mediated diseases, it is not known which antigen(s) cause(s) the activation of pathogenic CD4 ϩ T cells. Identification of antigens and epitopes in T cell-mediated diseases might enable immune intervention strategies with respect to treatment or prevention of disease. Even in the absence of an identified antigen source, peptides that stimulate pathogenic CD4 ϩ T cells may be useful in the development of primary immune intervention approaches via the induction of tolerance or the application of altered peptide ligands (3, 4).Various methods for the identification of T cell epitopes have been described. Most of them are based on identification of the source protein of an epitope by using molecular biology approaches (5-7) or peptide elution from HLA molecules followed by fractionation and sequence analysis (8, 9). These methods are time consuming or require large amounts of biological material. Alternative, fast synthetic peptide library approaches have been shown to be useful in the identification of T cell epitopes only for CD8 ϩ T cells (10, 11). Herein we show that CD4ϩ T cell epitopes can be identified by using a newly developed system for construction and screening synthetic peptide libraries. We applie...

A single T cell clone can be activated by many different peptides in the context of a particular HLA molecule. To quantify the number of peptides that can be recognized by a CD4+ T cell clone, we screened a one‐bead‐one‐peptide synthetic peptide library and a protein database for peptides that stimulate an HLA‐DR3‐restricted, human glutamic acid decarboxylase (GAD65)‐reactive CD4+ T cell clone. Both the library screening and the database analysis indicated that this T cell clone is able to recognize approximately 106 11‐mer peptides at low nanomolar concentration. Furthermore, we determined that the frequency of cross‐reactivity increased only 1.5‐3 times when the peptide concentration increased 10 times, in the range of 0.01 – 1 μM. These data imply that there is a considerable potential for T cell cross‐reactivity and are useful for studies on the role of molecular mimicry in the etiology of T cell‐mediated disease.