T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR
Summary
Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.
Abstract
T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.
Introduction
T细胞受体删除环（TRECs）和K-删去重组切除圆（KRECs）是切下在T细胞和B细胞，分别在基因组DNA重组过程中的比例小环化的DNA分子，从而导致形成T形的一个高度多元化剧目和B细胞受体。他们没有的功能，但因为它们是稳定的，并且不能被复制，它们稀释每次细胞分裂后，从而持续仅在两个子细胞中的一个。因此，它们在周边血液水平可以假定为胸腺和骨髓输出的估计。
而TREC测定已大量使用在过去15年来评价胸腺输出的范围内，1 KREC测定，这是最初开发用于测量B细胞增殖和B细胞稳态在健康和疾病的贡献，2-已经是最近才提出作为骨质米的标记箭头的输出。3,4在这里，我们描述我们开发了既TRECs和KRECs的同时定量的方法。4
与此相结合的方法，通过实时PCR关联到DNA的定量变异是通过使用通过稀释含有TRECs，KRECs和T细胞受体的α常数的片段三重插入片段的质粒而获得的独特标准曲线（TCRAC），去除基因在以1：1：1的比例。这允许的TREC和KREC拷贝数的更准确的评价。此外，在相同的反应中的两个目标的同时定量能够降低试剂成本。
所提出的TREC / KREC测定可以是有用的测量T-的程度和B细胞的新生产的儿童或成人重症联合免疫缺陷（SCID），4-普通变异型免疫缺陷，5自身免疫性疾病，6-8和HIV感染9此外，它可以用于监测造血干细胞移植，10酶替代，11和抗病毒药9或免疫调节疗法后免疫恢复6-8最后，因为SCID病人正在使用的TREC测定尽管潜在的遗传缺陷识别，丙种球蛋白血症患者可使用KREC量化被识别中，TREC / KREC测定也可以用于检测免疫缺陷在新生儿筛查计划。12在这种情况下，该测试必须对DNA从血液涂抹小斑点萃取执行，并且在滤纸上干燥，必须是高度敏感和特异的目标疾病，以及高度重复性和成本效益。
KREC定量测试中的引入应该改善新生儿筛查的免疫缺陷，这经常被美国（WI，MA，CA）自2008年以来的一些地方进行表演时，威斯康星州成为第一个引种TRECs电子在其出生后的筛查方案的分析。13
Protocol
Representative Results
Discussion
TREC and KREC quantification can be considered a good estimate of recent thymic and bone marrow output provided that some caveats are taken into account. Even though an absolute quantification method employing standard curve requires more reagents and more space on the real-time PCR reaction plate, it ensures highly accurate quantitative results because unknown sample quantities are interpolated from standard curves built upon known amounts of starting material. Moreover this method is better fitted to detect low amount …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Histopaque-1077 | Sigma Aldrich SRL | 10771-500 ML | density gradient separation method |
| QIAamp DNA Blood Mini Kit (250) | QIAGEN | 51106 | DNA extraction |
| Unmodified DNA Oligonucleotides HPSF 0.01 mmol | Eurofins MWG Operon/Carlo Erba Reagents S.r.l | Resuspend the lyophilized product to 100 picmol/µl | |
| AmpliTaq DNA Polymerase: including 10x Buffer II and 25mM MgCl2 | Applied Biosystems/Life-Technologies | N8080156 | |
| GeneAmp dNTP Blend (100 mM) | Applied Biosystems/Life-Technologies | N8080261 | |
| TOPO TA Cloning Kit for Subcloning | Invitrogen/Life-Technologies | K4500-01 | |
| XL1-Blue Subcloning Grade Competent Cells | Stratagene | 200130 | |
| PureYield Plasmid Miniprep System | Promega | A1223 | |
| SpeI 500U | New England Biolabs | R0133S | |
| HindIII-HF 10,000 U | New England Biolabs | R3104S | |
| PureYield Plasmid Midiprep System | Promega | A2492 | |
| XhoI 5,000 U | New England Biolabs | R0146S | |
| TRIS Utrapure | Sigma Aldrich SRL | T1503 | |
| EDTA | Sigma Aldrich SRL | E5134 | |
| TE buffer (1 mM TRIS and 0.1 mM EDTA) | |||
| TaqMan Universal PCR Master Mix | Applied Biosystems/Life-Technologies | 4364338 | |
| Dual labeled probes HPLC 0.01 mmol | Eurofins MWG Operon/Carlo Erba Reagents S.r.l | Resuspend the lyophilized product to 100 picmol/µl | |
| NanoDrop 2000c spectrophotometer | ThermoFisher | ||
| Applied Biosystems 2720 Thermal Cycler | Applied Biosystems/Life-Technologies | 4359659 | |
| Fast 7500 Real-Time PCR system | Applied Biosystems/Life-Technologies | ||
| SDS Sequence Detection Software 1.4 | Applied Biosystems/Life-Technologies |
Referenzen
- Douek, D. C., et al. Changes in thymic function with age and during the treatment of HIV infection. Nature. 396 (6712), 690-695 (1998).
- Zelm, M. C., Szczepanski, T., van der Burg, M., van Dongen, J. J. M. Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion. J. Exp. Med. 204 (3), 645-655 (2007).
- Fronkova, E., et al. B-cell reconstitution after allogeneic SCT impairs minimal residual disease monitoring in children with ALL. Bone Marrow Transplant. 42 (3), 187-196 (2008).
- Sottini, A., et al. Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation. Clin. Immunol. 136 (2), 217-227 (2010).
- Serana, F., et al. Thymic and bone marrow output in patients with common variable immunodeficiency. J. Clin. Immunol. 31 (4), 540-549 (2011).
- Zanotti, C., et al. Opposite effects of interferon-β on new B and T cell release from production sites in sclerosis. J. Neuroimmunol. 240-241, 147-150 (2011).
- Zanotti, C., et al. Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients. Clin. Immunol. 145 (1), 19-26 (2012).
- Sottini, A., et al. Pre-existing T- and B-cell defects in one progressive multifocal leukoencephalopathy patient. PLoS One. 7, e34493 (2012).
- Quiros-Roldan, E., et al. Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients. J. Transl. Med. 10, 94 (2012).
- Mensen, A., et al. Utilization of TREC and KREC quantification for the monitoring of early T- and B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation. J. Transl. Med. 11, 188 (2013).
- Serana, F., et al. The different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine deaminase deficiency. J. Immunol. 185 (12), 7713-7722 (2010).
- Borte, S., et al. Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR. Blood. 119 (11), 2552-2555 (2012).
- Baker, M. W., et al. Implementing routine testing for severe combined immunodeficiency within Wisconsin’s newborn screening program. Public Health Rep. 125 (2), 88-95 (2010).
- Lorenzi, A. R., et al. Determination of thymic function directly from peripheral blood: a validated modification to an established method. J. Immunol. Meth. 339 (2), 185-194 (2008).
- De Boer, R. J., Perelson, A. S. Quantifying T lymphocyte turnover. J. Theor. Biol. 327, 45-87 (2013).
