Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65)

Abstract

T cell recognition of autoantigens is critical to progressive immunemediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulindependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4 ؉ T cell response in IDDM.T ype I diabetes (insulin-dependent diabetes mellitus, IDDM), like many autoimmune diseases, exhibits exquisite target organ specificity. IDDM is characterized by immune-mediated destruction of beta cells in the pancreatic islet, coincident with the sparing of neighboring alpha and delta cells. The precise target-cell specificity in this disease implies the existence of antigenic self proteins derived from beta cells that are recognized by autoimmune T lymphocytes. Extensive analysis of serum antibodies in patients with IDDM has documented several self proteins that are candidates for this role (1, 2). The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is expressed in pancreatic beta cells at high levels, and antibodies to hGAD65 are present in up to 70% of newly diagnosed diabetics. These antibodies are also often present for several years before the development of clinical diabetes, providing a useful serum marker for prediction of disease onset (1, 3, 4). Recently, it has been shown that the suppression of GAD expression in nonobese diabetic (NOD) mice prevents autoimmune diabetes (5), directly implicating GAD as a likely participant in IDDM progression.Studies of T cell reactivity to autoantigens in diabetics have confirmed the immunogenicity of hGAD65 with reports of both CD4 ϩ and CD8 ϩ T cell responses (6)(7)(8)(9)(10)(11)(12)(13)(14). Approximately 70% of Caucasoid diabetics express the DRB1*0401, *0404, and *0405 MHC class II alleles. These DR4 ϩ alleles are in linkage disequilibrium with the DQB1*0302 gene, the HLA-DQ marker most highly associated with IDDM (15). T cell responses to the hGAD65 proteins that are restricted by HLA-DR molecules predominate in patients with these HLA disease-susceptibility haplotypes, and previous studies using overlapping synthetic peptides identified approximately 10 peptides that were capable of efficient binding to DR4 molecules. Indeed, three of these candidate autoantigen epitopes, corresponding to residues 115-127, 274-286, and 554-566 of hGAD65, were immunogenic when used to immunize mice transgenic for HLA-DR4 (16). A separate study, also using DR4-transgenic mice, found that these same three epitopes ...

MS and clinically isolated syndromes: Shared specificity but diverging clonal patterns of virus‐specific IgG antibodies produced in vivo and by CSF B cells in vitro

“…Interestingly, GAD65-specific T cell epitopes from one of the SPS patients (SPS 2) were localised within the C-terminal region (residues 474-484 and 555-565) of GAD65. This is in line with previous findings in T1D showing that T cell epitopes to GAD65 localise to residues 481-495, 511-525 and 551-585 (Patel 1997;Nepom 2001). Furthermore, the C-terminal region is also targeted by CD8 + T cells and of interest, tolerance-inducing DNA coding for the Cterminal region, GAD 500-585 , has been shown to protect NOD mice from diabetes (Quinn 2001;Han 2005).…”

T-cell vaccination leads to suppression of intrapancreatic Th17 cells through Stat3-mediated RORγt inhibition in autoimmune diabetes

“…Auto-reactive T cells directed against one or more β-cell autoantigens are believed to be involved in the autoimmune process of T1D [1,31,32]. Sequential spreading seems to orchestrate T1D, with insulin being required for the initiation of the disease [33], whereas GAD-reactive T lymphocytes are more involved at later stages of T1D [34,35]. Thus, for an antigen-specific therapy to be effective and practical against T1D, it would have to target late-stage epitopes that could counter diverse aggressive T-cell specificities.…”

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

“…While the decreasing proinsulin-specific responses and the increasing of IGRP [265][266][267][268][269][270][271][272][273] reactivity are reminiscent of NOD mouse data (6), the finding of a similar phenomenon for GAD and IA-2 is unparalleled. This is not surprising, since GAD and IA-2 are thought to be important target Ags in human type 1 diabetes (15,17,26,(37)(38)(39) but not in the NOD mouse (40 -43). Consideration of additional epitopes poorly recognized at type 1 diabetes onset (17,18) may reveal further specificities becoming immunodominant at later time points.…”