Molecular mimicry in type 1 diabetes mellitus revisited: T-cell clones to GAD65 peptides with sequence homology to Coxsackie or proinsulin peptides do not crossreact with homologous counterpart.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. Molecular mimicry and T-cell crossreactivity to beta-cell autoantigens and environmental agents with sequence similarities have been a proposed mechanism underlying the pathogenesis of type 1 diabetes, but actual crossreactivity has not yet been demonstrated. We isolated and investigated T cells reactive to GAD65 peptides and homologous peptides of the Coxsackie virus protein P2C and proinsulin from recent onset type 1 diabetes patients, and tested their fine specificity and cytokine production profile. Six T-cell lines specific for GAD65 peptides (amino acids 491-530) with homology to proinsulin (B20-C14) were isolated from six newly diagnosed patients with type 1 diabetes, but none of the stable T-cell lines crossreacted to the homologous proinsulin peptides. Similarly, none of four T-cell lines reactive to GAD65 peptides (amino acids 247-280) with sequence homology to Coxsackie P2C (amino acids 30-50) crossreacted to the homologous viral peptide. Two T-cell lines corecognized a GAD65 peptide and a Coxsackie P2C peptide. However, the antigen-specific T-cell clones from these T-cell lines were reacting either with the GAD65 peptide or the Coxsackie P2C peptide using different restriction elements without crossreacting to the homologous peptide. Our data demonstrate that homologous peptides previously proposed to serve as targets for crossreactivity indeed are immunogenic. Yet, T-cell clones did not crossreact with linear sequence homologies, despite strong T-cell responses to individual peptides.

Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase.

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 stiff-man 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.

T-cell lines reactive to an immunodominant epitope of the tyrosine phosphatase-like autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is the result of destruction of the insulin-secreting beta-cells of the pancreas by a process in which T-cells play a central role. A tyrosine phosphatase-like protein, IA-2, is a major target for autoantibodies and T-cells in the disease. In this study, we have further characterized the T-cell response to IA-2 by the generation and characterization of T-cell lines. T-cell lines responsive to IA-2 antigen were generated from 17 of 32 patients and 3 of 10 control subjects. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. Five lines from diabetic patients responded to one of two peptides representing amino acids 831-850 and 841-860 of IA-2. The overlapping portion may therefore represent an immunodominant region of the molecule. The sixth patient-derived line responded to a peptide representing amino acids 751-770 of IA-2 presented by the DR 4 (DRB1*0401) allele that confers susceptibility to type 1 diabetes. Primary T-cell responses to peptides of the immunodominant region were detected in 9 of 19 (47%) type 1 diabetic patients and 16 of 22 (73%) nondiabetic siblings, consistent with this region having immunostimulatory properties. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule.

Quantitative determination of TCR cross-reactivity using peptide libraries and protein databases.

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 10(6) 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 microM. 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.

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.