Abstract
Gluten-specific T cells in the small intestinal mucosa are thought to play a central role in the pathogenesis of celiac disease (CD). The vast majority of these T cells recognize gluten peptides when presented by HLA-DQ2 (DQA1*05/DQB1*02), a molecule which immunogenetic studies have identified as conferring susceptibility to CD. We have previously identified and characterized three DQ2-restricted gluten epitopes that are recognized by intestinal T cells isolated from CD patients, two of which are immunodominant. Because almost all of the gluten epitopes are restricted by DQ2, and because we have detailed knowledge of several of these epitopes, we chose to develop peptide-DQ2 tetramers as a reagent to further investigate the role of these T cells in CD. In the present study, stable soluble DQ2 was produced such that it contained leucine zipper dimerization motif and a covalently coupled peptide. We have made four different peptide-DQ2 staining reagents, three containing the gluten epitopes and one containing a DQ2-binding self-peptide that provides a negative control for staining. We show in this study that peptide-DQ2 when adhered to plastic specifically stimulates T cell clones and that multimers comprising these molecules specifically stain peptide-specific T cell clones and lines. Interestingly, T cell activation caused severe reduction in staining intensities obtained with the multimers and an Ab to the TCR. The problem of TCR down-modulation must be taken into consideration when using class II multimers to stain T cells that may have been recently activated in vivo.
Highlights
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We show in this study that peptide-DQ2 when adhered to plastic stimulates T cell clones and that multimers comprising these molecules stain peptidespecific T cell clones and lines
Using a panel of peptide-specific T cell clones (TCCs) and lines derived from celiac disease (CD) patients, we demonstrated that the peptide-DQ2 multimers stained T cells in a peptide-specific manner
Summary
CD, celiac disease; TCC, T cell clone; TCL, T cell line; SA-R-PE, streptavidin-R-PE; TCL CTRL, negative control TCL. GLIADIN-DQ2 MULTIMERS complexes that, when coated onto plastic, efficiently stimulated T cell clones (TCCs). By incubating these enzymatically biotinylated peptide-DQ2s with streptavidin-R-PE (SA-R-PE) we were able to generate tetrameric and multimeric complexes that we refer to in this study as peptide-DQ2 multimers. Using a panel of peptide-specific TCCs and lines derived from CD patients, we demonstrated that the peptide-DQ2 multimers stained T cells in a peptide-specific manner. This phenomenon must be taken into consideration when using peptide-DQ2 multimers to stain T cells of active celiac lesion, and has serious implications for the use of class II multimer reagents in general
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