Identification of overrepresented T cell receptor genes in blood and tissue biopsies by PCR-ELISA

Abstract

The analysis of T cell receptor variable (TCR V) gene repertoires in blood or tissues may provide important information when studying immunopathological mechanisms. The overexpression of a TCR gene may indicate the expansion of the corresponding T cell subset. In autoimmune diseases, clonally expanded T cell subsets in the affected organs may represent pathogenic lymphocytes. We describe a simple, rapid and sensitive method to determine the TCR AV and BV gene repertoire using a PCR-ELISA method. RNA is extracted from lymphocytes, transcribed to cDNA, which is then used as a template for PCR with 19 different TCR AV gene and 20 BV gene specific primers as the forward primer, and a digoxigenin (DIG) labeled AC/BC primer as the reverse primer. The DIG labeled PCR amplicons are hybridized with a fluorescein isothiocyanate (FITC) labeled TCR C region specific probe. Finally, the amplicons are quantified by ELISA using anti-FITC coated microtiter plates, and an anti-DIG conjugated peroxidase. Although PCR-ELISA cannot accurately quantify the expression level of a given TCR gene, overrepresented TCR V genes are easily identified by comparing the relative expression levels of each individual V gene in the total V gene repertoire. We demonstrate that this technique can be used to determine TCR profiles in blood and tissue samples containing as few as 50,000 T cells. In combination with CDR3 fragment size analysis, this method is an efficient tool to identify clonally expanded T cell subsets in the synovial biopsies of rheumatoid arthritis patients.