An alternative approach to the assessment of γδ T-cell clonality in celiac disease intestinal lesions through cDNA heteroduplex analysis of T-cell receptor VJ junctions

Abstract

We have investigated the clonality of the γδ T lymphocytes infiltrating the intestinal mucosa of CD patients and control subjects by means of a simple and powerful method based on the heteroduplex analysis of the TCR VJ junctions. Each V-specific TCR chain, amplified either from fresh biopsy material or intestinal T-cell-line cDNA, is denatured and renatured to allow the random reshuffling of the various strands carrying different junctional sequences, coamplified in the same reaction. The mismatched chains (heteroduplexes) are separated from the matched ones (homoduplexes) through polyacrylamide gel electrophoresis, and whenever one or more T-cell clones are emerging over the polyclonal background, discrete bands are visible by ethidium-bromide staining. Through this method, we have estimated the diversity of the Vδ1–3 chains and a newly described V gene (Vδ8) whose homologue in mice is abundantly expressed in γδ iLs. We demonstrate that the well-documented expansion of Vγ1 + γδ lymphocytes in the jejunum of CD patients is polyclonal. Overall, the heteroduplex analysis on fresh intestinal and peripheral blood lymphocytes from both healthy and affected subjects shows a polyclonal pattern of all the Vδ + subsets. In contrast, most intestinal T-cell lines produce oligoclonal patterns, suggesting a dramatic in vitro selection effect. The cell expansion in culture is generally not required for the TCR heteroduplex analysis, which can therefore be applied to rapidly monitor the T-cell response in a variety of physiologic and autoimmune reactions, substituting the standard approach of TCR cloning and multiple VJ sequencing. Human Immunology 40, 303–311 (1994)