Cellular calibrators to quantitate T-cell receptor excision circles (TRECs) in clinical samples
ABSTRACT 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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ABSTRACT: The study of T cell biology has been accelerated by substantial progress at the technological level, particularly through the continuing advancement of flow cytometry. The conventional approach of observing T cells as either T helper or T cytotoxic is overly simplistic and does not allow investigators to clearly identify immune mechanisms or alterations in physiological processes that impact on clinical outcomes. The complexity of T cell sub-populations, as we understand them today, combined with the immunological and functional diversity of these subsets represent significant complications for the study of T cell biology. In this article, we review the use of classical markers in delineating T cell sub-populations, from "truly naïve" T cells (recent thymic emigrants with no proliferative history) to "exhausted senescent" T cells (poorly proliferative cells that display severe functional abnormalities) wherein the different phenotypes of these populations reflect their disparate functionalities. In addition, since persistent infections and chronological aging have been shown to be associated with significant alterations in human T cell distribution and function, we also discuss age-associated and cytomegalovirus-driven alterations in the expression of key subset markers. © 2013 International Society for Advancement of Cytometry.Cytometry Part A 03/2014; 85(1). DOI:10.1002/cyto.a.22351 · 2.93 Impact Factor
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ABSTRACT: In general, human pathogen-related small circular deoxyribonucleic acid (DNA) molecules are bacterial plasmids and a group of viral genomes. Plasmids are extra-chromosomal small circular DNAs that are capable of replicating independently of the host, and are present throughout a variety of different microorganisms, most notably bacteria. While plasmids are not essential components of the host, they can impart an assortment of survival enhancing genes such as for fertility, drug resistance, and toxins. Furthermore, plasmids are of particular interest to molecular biology especially in relation to gene-cloning. Among viruses, genomes of anelloviruses, papillomaviruses, and polyomaviruses consist of small circular DNA. The latter two virus families are known for their potential roles in a number of pathogenic processes. Human papillomaviruses (HPV) are now widely recognised to be associated with a greatly increased risk of cervical cancer, especially oncogenic strains 16 and 18. On the other hand, human cells may contain several types of small circular DNA molecules including mitochondrial DNA (mtDNA). The mitochondrial genome consists of 37 genes that encode for proteins of the oxidation phosphorylation system, transfer ribonucleic acids (tRNAs), and ribosomal RNAs (rRNAs). Though mitochondria can replicate independently of the host; nuclear DNA does encode for several mitochondrial proteins. Mutations in mtDNA contribute to some well characterised diseases; mtDNA is also implicated in several diseases and malignancies with poorly elucidated aetiologies. Furthermore, mtDNA can function as a diagnostic tool. Other extra-chromosomal circular DNAs are usually detected in cancer. This review article is intended to provide an overview of four broad categories of small circular DNAs that are present in non-eukaryotic (plasmids and relevant viral genomes) and eukaryotic (mtDNA and other extra-chromosomal DNAs) systems with reference to human diseases, particularly cancer. For this purpose, a literature search has been carried out mainly from PubMed. Improved understanding of the significance of small circular DNA molecules is expected to have far reaching implications in many fields of medicine.Malaysian Journal of Medical Sciences 05/2014; 21(3):4-18.
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ABSTRACT: : Little is known about different phases of T-cell maturation in gut mucosa. Based on current knowledge about the migratory pathways of naive and memory T cells, it is believed that access to peripheral, nonlymphoid tissues is restricted to memory T cells. Surprisingly, there is increasing evidence of high numbers of naive T cells in the chronically inflamed gut tissue of patients with inflammatory bowel disease. This could partially be explained by new formation of ectopic lymphoid organs. Ongoing recruitment of naive T cells at inflammatory sites might play a role in the immunopathogenesis of inflammatory bowel disease.Inflammatory Bowel Diseases 09/2014; 21(1). DOI:10.1097/MIB.0000000000000221 · 4.46 Impact Factor