Cellular calibrators to quantitate T-cell receptor excision circles (TRECs) in clinical samples.
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.
Dept of Pediatrics, University of California San Francisco, San Francisco, CA 91413, USA.
Divya
D
Punwani
Diana
D
Gonzalez-Espinosa
Anne Marie
AM
Comeau
Amalia
A
Dutra
Evgenia
E
Pak
Jennifer
J
Puck
D001402:B-Lymphocytes / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D002461:Cell Line, Transformed; D056915:DNA Copy Number Variations; D004270:DNA, Circular / Q000032:analysis*; D059788:Dried Blood Spot Testing; D015329:Gene Rearrangement, T-Lymphocyte; D017930:Genes, Reporter; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D016086:Lentivirus / Q000235:genetics; D018655:Lymphocyte Count; D008231:Lymphopenia / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D015997:Neonatal Screening; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D016511:Severe Combined Immunodeficiency / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D013601:T-Lymphocytes / Q000378:metabolism / Q000473:pathology*
D015415:Biomarkers; D004270:DNA, Circular; D011948:Receptors, Antigen, T-Cell