Dissection of the clonal composition of bovine αβ T cell responses using T cell receptor Vβ subfamily-specific PCR and heteroduplex analysis

Although techniques that permit analysis of the clonal composition of T cell populations have been used extensively to provide a better understanding of the mechanisms that influence efficacy of T cell responses in humans and mice, such methods are lacking for other animal species. In this paper we report the establishment and validation of a panel of V beta subfamily-specific semi-nested PCR assays, and a CDR3 beta heteroduplex technique for analysing the clonal diversity of bovine alpha beta T cell responses. Development of these methods was based on available sequence data for 48 functional V beta genes classified within 17 subfamilies. These techniques were used to determine the clonal composition of parasite-reactive CD8(+) T cells obtained from two animals immunised with the protozoan parasite Theileria parva. Analyses of uncloned T cell lines as well as large panels of cloned T cells derived from each of these lines confirmed the specificity and sensitivity of the assays. Specific PCR products were obtained from 96% of the T cell clones examined, indicating that the currently identified V genes represent most of the functional V subfamilies in cattle. Heteroduplex analyses, coupled with sequencing of PCR products, identified over 20 clonal expansions within each of the T cell lines, distributed over a large number of V beta subfamilies, although a limited number of clonotypes numerically dominated the response in both animals. The development and validation of these methods provides for the first time a generic set of molecular tools that can be used to perform detailed analysis of the TCR diversity and clonal composition of bovine T cell responses. (C) 2008 Published by Elsevier B.V.