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.
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.
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 been largely used over the last 15 years to evaluate the extent of thymic output,1 the KREC assay, which was initially developed to measure B-cell proliferation and its contribution to B-cell homeostasis in health and disease,2 has been only recently proposed as a marker of bone marrow output.3,4 Here, we describe the method we developed for the simultaneous quantification of both TRECs and KRECs.4
With this combined method, the variability associated to DNA quantification by real-time PCR is eliminated by the use of a unique standard curve obtained by diluting a triple-insert plasmid containing fragments of TRECs, KRECs and T-cell receptor alpha constant (TCRAC) gene in a 1:1:1 ratio. This allows a more accurate evaluation of the TREC and KREC copy number. Furthermore, the simultaneous quantification of the two targets in the same reaction allows reagent cost reduction.
The proposed TREC/KREC assay can be useful to measure the extent of T- and B-cell neo-production in children or adults with Severe Combined Immunodeficiency (SCID),4 Common Variable Immunodeficiency,5 autoimmune diseases,6-8 and HIV infection.9 Furthermore, it can be used to monitor the immune recovery after hematopoietic stem cell transplantation,10 enzyme replacement,11 and antiviral9 or immunomodulating therapies.6-8 Finally, because SCID patients are recognized using TREC assay despite the underlying genetic defects, and agammaglobulinemia patients can be identified using KREC quantification, the TREC/KREC assay can be also used to detect immunodeficiencies in newborn screening programs.12 In this case, the test must be performed on DNA extracted from small spots of blood blotted and dried on filter paper, must be highly sensitive and specific for the target diseases, as well as highly reproducible and cost-effective.
The introduction of KREC quantification in the test should improve performances of newborn screening for immunodeficiencies, which has routinely been performed in the some parts of the USA (WI, MA, CA) since 2008 when Wisconsin became the first to introduce the analysis of TRECs in its postnatal screening program.13
NOTE: Ethics statement: This protocol follows the guidelines of our institution, the Spedali Civili di Brescia
1. Preparation of a “Triple-Insert” Plasmid
TRECs | forward | 5’-AAA GAG GGC AGC CCT CTC CAA GGC-3’ |
reverse | 5’-GGC TGA TCT TGT CTG ACA TTT GC-3’ | |
KRECs | forward | 5’-CCC aag ctt TCA GCG CCC ATT ACG TTT CT-3’ |
reverse | 5’-CCC aag ctt GTG AGG GAC ACG CAG CC-3’ | |
TCRAC | forward | 5’-Gac tag tAT GAG ACC GTG ACT TGC CAG-3’ |
reverse | 5’-Gac tag tGC TGT TGT TGA AGG CGT TTG C-3’ |
Table 1. Primers used for the cloning procedures. At the 5'-end, in lower case are shown nucleotides corresponding to restriction enzyme sites, whereas in italics are shown added flanking nucleotides.
Figure 1. Triple-insert plasmid map. Triple-insert plasmid map showing the position of TREC, KREC, TCRAC sequences and restriction enzyme sites. Please click here to view a larger version of this figure.
2. Standard Curve Preparation
NOTE: Prepare all dilutions in 0.1x TE buffer in a place specially designed for containment of DNA carry-over.
Copy # | x 5.311 x 10-18 g | Mass of plasmid DNA (g) |
1 x 106 | 5.311 x 10-12 | |
1 x 105 | 5.311 x 10-13 | |
1 x 104 | 5.311 x 10-14 | |
1 x 103 | 5.311 x 10-15 | |
1 x 102 | 5.311 x 10-16 |
Table 2. Mass of plasmid needed for each standard curve dilution point.
Mass of plasmid DNA (g) | ÷ 5 μl | Final concentration of plasmid DNA (g/μl) |
5.311 x 10-12 | 1.062 x 10-12 | |
5.311 x 10-13 | 1.062 x 10-13 | |
5.311 x 10-14 | 1.062 x 10-14 | |
5.311 x 10-15 | 1.062 x 10-15 | |
5.311 x 10-16 | 1.062 x 10-16 |
Table 3. Calculation of plasmid concentrations needed for each dilution point.
Initial conc. (g/μl) | Plasmid DNA vol (μl) | Diluent vol (μl) | Final Vol. (μl) | Final conc. (g/μl) | Final copy number of plasmid DNA/5μl |
C1 | V1 | V2-V1 | V2 | C2 | |
1 x 10-7 | 5 μl | 45 μl | 50 μl | 1 x 10-8 | N/A |
1 x 10-8 | 5 μl | 495 μl | 500 μl | 1 x 10-10 | N/A |
1 x 10-10 | 5 μl | 465 μl | 470 μl | 1.062 x 10-12 | 1 x 106 |
1.062 x 10-12 | 50 μl | 450 μl | 500 μl | 1.062 x 10-13 | 1 x 105 |
1.062 x 10-13 | 50 μl | 450 μl | 500 μl | 1.062 x 10-14 | 1 x 104 |
1.062 x 10-14 | 50 μl | 450 μl | 500 μl | 1.062 x 10-15 | 1 x 103 |
1.062 x 10-15 | 50 μl | 450 μl | 500 μl | 1.062 x 10-16 | 1 x 102 |
Table 4. Dilution calculations.
3. DNA Extraction from Target Samples
4. Real-time PCR for Quantification of TRECs and KRECs
TRECs+KRECsa | TCRACb | TRECs+KRECs | TCRAC | TRECs+KRECs | TCRAC | |||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | 106 copies | 106 copies | 106 copies | 106 copies | 1 | 1 | 1 | 1 | 9 | 9 | 9 | 9 |
B | 105 copies | 105 copies | 105 copies | 105 copies | 2 | 2 | 2 | 2 | 10 | 10 | 10 | 10 |
C | 104 copies | 104 copies | 104 copies | 104 copies | 3 | 3 | 3 | 3 | 11 | 11 | 11 | 11 |
D | 103 copies | 103 copies | 103 copies | 103 copies | 4 | 4 | 4 | 4 | 12 | 12 | 12 | 12 |
E | 102 copies | 102 copies | 102 copies | 102 copies | 5 | 5 | 5 | 5 | 13 | 13 | 13 | 13 |
F | 10 copies | 10 copies | 10 copies | 10 copies | 6 | 6 | 6 | 6 | 14 | 14 | 14 | 14 |
G | CTRL+ | CTRL+ | CTRL+ | CTRL+ | 7 | 7 | 7 | 7 | 15 | 15 | 15 | 15 |
H | NTC | NTC | NTC | NTC | 8 | 8 | 8 | 8 | 16 | 16 | 16 | 16 |
Table 5. Sample real-time PCR plate.
TRECs/KRECs | TCRAC | ||
H2O | 2 μl | H2O | 4.75 μl |
KRECs for 20 pmol/μl | 1.125 μl | TCRAC for 20 pmol/μl | 1.125 μl |
KRECs rev 20 pmol/μl | 1.125 μl | TCRAC rev 20 pmol/μl | 1.125 μl |
KRECs probe 10 pmol/μl | 0.5 μl | TCRAC probe 10 pmol/μl | 0.5 μl |
TRECs for 20 pmol/μl | 1.125 μl | 2x TaqMan Universal PCR Master Mix | 12.5 μl |
TRECs rev 20 pmol/μl | 1.125 μl | ||
TRECs probe 10 pmol/μl | 0.5 μl | ||
2x TaqMan Universal PCR Master Mix | 12.5 μl |
Table 6. Volume of reagents needed for the indicated wells.
TRECs | forward | 5’-CAC ATC CCT TTC AAC CAT GCT-3’ |
reverse | 5’-TGC AGG TGC CTA TGC ATC A-3’ | |
probe | 5’-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT-TAMRA-3’ | |
KRECs | forward | 5’-TCC CTT AGT GGC ATT ATT TGT ATC ACT-3 |
reverse | 5’-AGG AGC CAG CTC TTA CCC TAG AGT-3’ | |
probe | 5’-HEX-TCT GCA CGG GCA GCA GGT TGG-TAMRA-3 | |
TCRAC | forward | 5’-TGG CCT AAC CCT GAT CCT CTT-3’ |
reverse | 5’-GGA TTT AGA GTC TCT CAG CTG GTA CAC-3 | |
probe | 5’-FAM-TCC CAC AGA TAT CCA GAA CCC TGA CCC-TAMRA-3’ |
Table 7. Sequence of primer and probes for the real-time PCR assay.
Figure 2. Standard curves for TRECs, KRECs, and TCRAC. Plots of the standard curve points and log-regression line estimates for TRECs (A), KRECs (B), and TCRAC (C) are built to verify compliance with the ideal exponential amplification rate (slope = 3.32) that would correspond to an efficiency of 100%. Ct: threshold cycle; R2: regression coefficient of determination. Please click here to view a larger version of this figure.
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 decreases more rapidly in men than in women.5 This may create problems in setting appropriate reference ranges, which should be differentiated on the basis of the age and possibly gender, if a very precise estimation of normality is needed.
Figure 3. TREC quantification in healthy donors. The amount of TRECs was determined as TRECs/106 cells in healthy children (A) and adults (B) and then calculated as TRECs/ml in the same subjects (C, D). Clear circles: females; filled circles: males. Lines were obtained by linear regression using log-transformed data. In children, two regression lines were fitted to better represent the variable rate of TREC decrease with age. In adults, dashed lines represent the trend of decrease with age of TRECs seen in females, which is slower than in males (continuous line). Please click here to view a larger version of this figure.
The number of KRECs decreases with the age with a pattern similar to TRECs only in children, whereas in the adults the bone marrow output is fairly stable throughout life and does not depend on gender.
Figure 4: KREC quantification in healthy donors. The amount of KRECs was determined as KRECs/106 cells in healthy children (A) and adults (B) and then calculated as KRECs/ml in the same subjects (C, D). Clear circles: females; filled circles: males. Lines were obtained by linear regression using log-transformed data. In children, two regression lines were fitted to better represent the variable rate of KREC decrease with age. Please click here to view a larger version of this figure.
For representative purposes, simple log-linear regression models were fitted to depict the pattern of TREC/KREC decrease with age, but more refined non-linear models may be fitted in an attempt to better predict the seemingly exponential KREC/TREC decrease with age.
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 of targets. From the methodological point of view, a critical point is that the frozen plasmid dilutions corresponding to the standard curve points may degrade sooner than expected. This requires that new dilutions from the originally stored plasmid need to be prepared before each experimental procedure, in particular the most dilute ones. In the final calculations, a non-perfect equivalence between lymphocyte plus monocyte count as an estimate of the PBMC quantity may add some bias in the conversion between values expressed per 106 and values expressed per ml of blood. An improvement can be made to determine the amount of two targets (TRECs and KRECs) from a known amount of DNA extracted directly from whole blood, thus eliminating the need of the monocyte plus lymphocyte counts.14 In this specific case, a reference gene is not strictly necessary to perform the quantification, but may be used as an additional DNA quality and quantity check.
The result interpretation can be affected by some issues. First of all, it should be taken into account that reference ranges change with gender and age, determining marked differences between children and adults. Furthermore, in case of certain immune deficiencies or autoimmune syndromes, a homeostatic peripheral replication of existing memory cells can be present, which dilutes TREC/KREC content and lowers the result of the assay when expressed only per 106 lymphocytes. This may lead to the erroneous conclusion that the thymic/bone marrow output is lower than it actually is. Therefore, it is advisable to always estimate lymphocyte neoproduction by also calculating TRECs/KRECs per ml of blood, which makes results independent of the proliferation rate of memory cells. Third, whatever is the unit of measure, the test result may lead to biased overestimates, due to persistence of some long-lived naïve lymphocytes that still bear TRECs or KRECs. Therefore, those who are interested in the best possible estimate of “true” lymphocyte neoproduction can take advantage of some more complicated mathematical models that also takes into account the lymphocyte apoptosis rate.15
An improvement of assay sensitivity to enhance its use is to perform TRECs and KRECs quantification starting from a limited amount of capillary whole blood spotted on Guthrie card filter paper. This modified version of the assay can be applied with success to the newborn screening of congenital immune deficiencies affecting lymphocyte production,12 and was proved beneficial in terms of cost-efficacy. This simplified version of assay also paves the way to new potential applications, especially considering the high number of potent immune modulating drugs that have been developed in the last few years. However, the “classical” TREC/KREC assay herein described has been already applied with success as a routine test in the follow up of children with severe congenital immune deficiencies following hematopoietic stem cell transplantation,4 or for the monitoring of the therapy outcome of autoimmune diseases and acquired immune deficiencies.6-9,11
The authors have nothing to disclose.
The authors have no acknowledgements.
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 |