ANALYSIS OF THE ALPHA BETA T-CELL RECEPTOR REPERTOIRE BY COMPETITIVE AND QUANTITATIVE FAMILY-SPECIFIC PCR WITH EXOGENOUS STANDARDS AND HIGH-RESOLUTION FLUORESCENCE-BASED CDR3 SIZE IMAGING/

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an import ant tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR reperto ire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results . We have developed a strategy utilizing exogenous standards with homo logous primer binding sites for the quantitative analysis of the alpha /beta T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PC R (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstr ate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire, In addition, the proposed method reveals dir ect information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions, We di scuss variables such as cell number and experimental conditions influe ncing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of t he fine specificity of the T-cell repertoire in peripheral and organ-i nfiltrating T-lymphocytes. The analyses revealed information about pol yclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets a s well as antigen-driven shaping of the alpha/beta T-cell repertoire i n autoimmune and infectious diseases. (C) 1997 Elsevier Science B.V.