Cellular calibrators to quantitate T-cell receptor excision circles (TRECs) in clinical samples

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• 作者：Divya Punwani^a ; Diana Gonzalez-Espinosa^a ; Anne Marie Comeau^b ; Amalia Dutra^c ; Evgenia Pak^c ; Jennifer Puck^a ; ^{puckj@peds.ucsf.edu}
• 关键词：TREC ; T©\cell receptor excision circle ; TCR ; T©\cell receptor ; DBS ; dried blood spots ; SCID ; Severe combined immunodeficiency ; HCT ; hematopoietic cell transplantation ; EBV ; Epstein Barr virus ; TREC-EBV ; T©\cell receptor excision circle&mdash ; Epstein Ba
• 刊名：Molecular Genetics and Metabolism
• 出版年：2012
• 出版时间：November, 2012
• 年：2012
• 卷：107
• 期：3
• 页码：586-591
• 全文大小：564 K

文摘

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 ¦ÄRec-¦×J¦Á 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 ¦ÄRec-¦×J¦Á 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 na?ve T©\cell production in clinical settings.