Detection of T cell receptor circles (TRECs) as biomarkers for de novo T cell synthesis using a quantitative polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA)

Currently, phenotypic markers that distinguish between recent thymic emigra nts/de novo T cells and the rest of the peripheral T cell pool are lacking. This distinction is critical in studies aimed at evaluating immune reconst itution following intensive chemotherapy, in immunodeficiency-related thera pies, or in the elucidation of the kinetics of thymic function. During V(D) J T cell receptor rearrangement, DNA extrachromosomal excision products are generated. These products, known as T cell receptor excision circles (TREC s), are not replicated during mitosis and are thus diluted with each round of cell division. Therefore, TRECs can be used as an indicator of recent th ymic emigrants. Thus far, quantitative competitive-polymerase chain reactio n (QC-PCR) and real time PCR were used to measure TREC levels. However, QC- PCR relies on radioactivity, is cumbersome when processing many samples at once and the cost of real time PCR does not make it a viable option for man y laboratories. We describe here the development of a quantitative PCR-ELIS A method for the measurement of coding joint TRECs generated from V alpha J alpha recombination. Our assay is ultra sensitive, relies on biotin labeli ng rather than radioactivity, is based on a 96-well format making multiple process sampling relatively easy, and is cost effective. Using this PCR-ELI SA method, we evaluated thymic output among 22 normal subjects, ranging in age from 22-53 years, and among HIV-infected individuals following highly a ctive antiretroviral therapy (HAART). We demonstrate that an inverse relati onship exists between TREC levels and aging in normal individuals and that, among some HIV patients, HAART treatment leads to enhanced thymic output. Our assay has direct relevance in projects examining normal and abnormal th ymic function and in immune reconstitution studies. (C) 2000 Elsevier Scien ce B.V. All rights reserved.