Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time 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-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are small circularized DNA elements that are excised in a proportion of T- and B-cells, respectively, during a genomic DNA recombination process, leading to the formation of a highly diversified repertoire of T- and B-cell receptors. They have no function, but because they are stable and cannot be replicated, they are diluted after each cell division, thus persisting only in one of the two daughter cells. Therefore, their levels in the peripheral blood can be assumed as an estimate of the thymic and bone marrow output.

While the TREC assay has bee....

Protocol

NOTE: Ethics statement: This protocol follows the guidelines of our institution, the Spedali Civili di Brescia

1. Preparation of a “Triple-Insert” Plasmid

1. Selection and preparation of appropriate starting material:
   1. Obtain a sample containing cells very likely to have TRECs and KRECs detectable by PCR, such as peripheral blood of a young healthy subject collected into EDTA tubes.
      NOTE: Originally, we had developed the method using thymocytes for TRECs and mononuclear cells from tonsil fragments for KRECs, but healthy subjects usually have an amount of TRECs and KRECs sufficient to allow their detection by PCR. T....

Results

The assay was performed in a representative sample of 87 healthy controls: 42 children aged 0 - 17 (male/females: 25/17) and 45 adults aged 24 - 60 (males/females: 29/16). Results were obtained as TRECs and KRECs per 10⁶ PBMC, and then the TRECs and KRECs per ml of blood were calculated.

The number of TRECs decreases with age due to thymic involution,⁴ in particular in a very sharp fashion from 0 to 3 - 4 years. In adults, the TREC number also depends on gender because it.......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 pmol/µl
AmpliTaq DNA Polymerase: including 10x Buffer II and 25 mM 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 pmol/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