Cellular calibrators to quantitate T-cell receptor excision circles (TRECs) in clinical samples.
- Author(s): Punwani, D
- Gonzalez-Espinosa, D
- Comeau, AM
- Dutra, A
- Pak, E
- Puck, J
- et al.
Published Web Locationhttp://www.sciencedirect.com/science/article/pii/S1096719212003599
T-cell receptor excision circles (TRECs) are circular DNA molecules formed during rearrangement of the T-cell receptor (TCR) genes during lymphocyte development. Copy number of the junctional portion of the δRec-ψJα TREC, assessed by quantitative PCR (qPCR) using DNA from dried blood spots (DBS), is a biomarker for newly formed T cells and absent or low numbers of TRECs indicate SCID (severe combined immunodeficiency) or T lymphocytopenia. No quantitation standard for TRECs exists. To permit comparison of TREC qPCR results with a reliable method for counting TRECs across different laboratories, we sought to construct a stable cell line containing a normal human chromosomal constitution and a single copy of the TREC junction sequence. A human EBV (Epstein Barr virus)-transformed B-cell line was transduced with a lentivirus encoding mCherry fluorescence, puromycin resistance and the δRec-ψJα TREC sequence. A TREC-EBV cell line, with each cell carrying a single lentiviral insertion was established, expanded and shown to have one TREC copy per diploid genome. Graded numbers of TREC-EBV cells added to aliquots of T lymphocyte depleted blood showed TREC copy number proportional to TREC-EBV cell number. TREC-EBV cells, therefore, constitute a reproducible cellular calibrator for TREC assays, useful for both population-based screening for severe combined immunodeficiency and evaluation of naïve T-cell production in clinical settings.
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