a simple method for the measurement of sjtrec levels in blood

Mechanisms of Ageing and Development

121 (2000) 59–67

A simple method for the measurement ofsjTREC levels in blood

Richard Aspinall *, Jeff Pido, Deborah AndrewDepartment of Immunology, Imperial College School of Medicine, Chelsea and Westminister Hospital,

369 Fulham Road, London SW 10 9NH, UK

Received 2 July 2000; received in revised form 21 July 2000; accepted 30 July 2000

Abstract

We have developed a relatively rapid, safe and simple method for the quantification ofsjTREC levels in samples of peripheral blood. The assay uses an image analysis package tomeasure the brightness of PCR product bands on an image of the standard agarose gel.Comparison of the brightness of the band with that obtained from a standard curve providesa read-out of the amount of sjTRECs in the sample. We have compared the sjTREC levelswe obtained with this method with those obtained from real time analysis PCR using aLightcycler and found that they are comparable. © 2000 Elsevier Science Ireland Ltd. Allrights reserved.

Keywords: sjTREC; Thymic output; PCR

www.elsevier.com/locate/mechagedev

1. Introduction

Extrapolation of the rate of loss of thymic tissue in humans suggests that itwould be completely absent by the time the individual reaches the age of 120 years(Steinmann, 1986). Loss of thymic tissue over this period is not uniform but morelikely biphasic with a period of rapid loss preceding middle age followed by aperiod of relatively slow tissue loss calculated by some to be less than 1% per year(Steinmann, 1986; Kendall et al., 1980; Bertho et al., 1997). In addition to the lossof thymopoetically active tissue there is the gradual deposition of fat within the

* Corresponding author. Tel.: +44-20-87465993; fax: +44-20-87465997.E-mail address: [email protected] (R. Aspinall).

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R. Aspinall et al. / Mechanisms of Ageing and De6elopment 121 (2000) 59–6760

organ. At 20 years of age approximately 20% of the wet weight of the thymus iscontributed by fat, but by 60 years of age this figure is closer to 70% (Kendall etal., 1980). The major function of the thymus is to produce T cells for the peripheralT cell pool, and a result of this loss of active thymic tissue is that the contributionof naı̈ve T lymphocytes by the thymus to the peripheral T cell pool declines withincreasing age.

Measurement of thymic output in humans is indirect and dependant uponfollowing the changes in the number of naı̈ve T cell with age, a naı̈ve T cell beingdefined as a T cell which has not yet encountered its cognate antigen presentedcorrectly to the T cell receptor. Such naı̈ve T cells have in the past been identifiedon the basis of phenotypic markers displayed on the cell’s surface. Using thistechnique several groups have shown that there is a decline in the number naı̈ve Tcells in the blood and an increase in the number of memory T cells with increasingage (Cossarizza et al., 1996; Hulstaert et al., 1994).

More recently measurement of change in thymic output has been achieved by theanalysis of the amount of specific DNA excision products known as T cell receptorrearrangement excision circles (TRECs) within the T cell population. These excisioncircles are a by-product of the process of TCR gene rearrangement and recombina-tion and are present within the T cell but do not have the capacity to replicateduring cellular proliferation. Thus only one of the daughter cells produced duringdivision carried the TREC which consequently becomes diluted within the popula-tion during subsequent divisions. TREC levels are therefore highest in populationsof T cell recently produced by the thymus and lower in T cells populations whichhave undergone several rounds of division (Douek et al., 1998; Kong et al., 1998;Al Harthi et al., 2000; McFarland et al., 2000; Poulin et al., 1999; Zhang et al.,1999).

With the age-related decline seen in thymic output and the proposed expansionof memory T cells to compensate for this decline and maintain T cell numberswithin defined limits one would anticipate that the number of TREC moleculeswithin the total T cell pool in the blood should decline with age. Several studieshave shown this to be the case. TREC levels do show a progressive decline with age(Douek et al., 1998).

In the analysis of TREC levels one can choose whether to analyse the TRECgenerated from the TCRa (Douek et al., 1998) or TCRb (Poulin et al., 1999) chainsince excision circles of DNA would be generated in their production. Here we havechosen to analyse signal joint TCR rearrangement excision circles (sjTRECs)generated during the process of aTCR gene rearrangement and recombination(Douek et al., 1998; Kong et al., 1998; Livak and Schatz, 1996). There are severalreasons for this choice. The first is that TCRa chain gene rearrangement occursafter TCRb chain rearrangement. Any excision circles generated from TCRb chainrearrangement could be more diluted within the recent thymic migrant populationthan the TCRa chain excision circles because of the number of cell divisionsundergone by the cells between TCRb chain production and expression and TCRachain production and expression. Secondly these sjTRECs all possess an identicalDNA sequence, spanning from the dREC gene segment to the cJa gene segment

R. Aspinall et al. / Mechanisms of Ageing and De6elopment 121 (2000) 59–67 61

which allows their use as specific markers for abTCR expressing recent thymicmigrants (Douek et al., 1998). Finally, sjTREC’s have been shown to be stable anddetectable in phenotypically naı̈ve abTCR T cells (CD45RA+) but are undetectablein gd TCR T cells, and B cells (Douek et al., 1998). The aim of this work was todetermine whether we could discover a rapid and sensitive method for the quantita-tion of sjTREC’s.

2. Materials and methods

2.1. Blood collection and DNA purification

Samples of peripheral blood were taken from healthy female volunteers chosenon the criteria that they were healthy at the time of sample extraction. 30 ml ofvolunteers’ peripheral blood was collected into EDTA-K3 containers (BectonDickinson, Dorset, UK) with their informed consent and in accordance with theguidelines set by the Riverside Ethics Committee. Peripheral blood mononuclearcells (PBMCs) were isolated by density gradient centrifugation using Histopaque(Sigma, Dorset, UK). DNA was extracted from 5 × 107 PBMCs using the Pure-gene DNA purification kit (Gibco, UK). b actin polymerase chain reaction (PCR)amplification and agarose gel analysis was performed for each sample in order todetermine the quality of the DNA. The concentration of the DNA was determinedby spectroscopy.

2.2. sjTREC PCR amplification and quantification

sjTREC DNA were detected according to the technique of Douek et al. (1998).The sjTREC bands were visualised on 1.2% agarose gels containing 0.005%ethidium bromide (Sigma) and analysed using the Scion Imager (Meyer Instru-ments). The light intensity values of the bands were used to calculate the number ofsjTREC molecules from the standard curve constructed (details below). Resultswere used if the light intensity value of the positive controls’ DNA band differed tothe actual values on the standard curve by no more than 10%.

To determine the sjTREC levels in volunteers’ DNA samples, a standard curvewas constructed via the PCR amplification of known starting numbers of standardsjTREC molecules (i.e. 103, 104, 105, 106, 107, 108). PCR products were thenseparated on 1.2% agarose gels containing 0.005% ethidium bromide and analysedusing the Scion imager software. An area was defined around each band and thebrightness was read. The size and shape of the area was kept constant throughoutall of the measurement. For the final analysis a background reading was taken fromthe negative control lane at an equivalent position of the sjTREC band in a positivelane of the gel. This background reading was subtracted from the readings obtainedfrom each PCR product band. A best fit straight-line graph (R2 = 0.97) composedof the starting amount of sjTREC molecules used for the PCR against DNA bandintensity was constructed.


2.3. sjTPEC analysis using the Lightcycler

DNA samples and known starting copy numbers of standard sjTREC DNAmolecules (from 103 to 108) were amplified using the Lightcycler (Roche, UK) andthe Sybr Green Light Cycler kit (Roche, UK) in accordance with the manufacturersinstructions. Values for the volunteers’ sjTREC levels were considered valid whenthe sjTREC standard curve had an R2 value of 0.99–1.0.

2.4. Separation of T cell subsets

5 × 106 purified PBMCs were treated with 1 mg of mouse IgG monoclonalantibody (mAb) to CD45RA or CD45RO and incubated for 30 min at 4°C. The cellswere then washed with PBS/0.1% BSA. Dynabeads pan mouse IgG were added tothe amount of approximately 4 beads/cell (beads were first washed with PBS/0.1%BSA 3 times prior addition to the cells) to the samples and incubated for 20 min withgentle mixing. The cells were placed on a Dynal MPC magnetic column and left for2 min. The fluid in the tubes was removed by pippetting and the cells resuspendedin PBS/0.1% BSA, then left for 2 min. The fluid was again removed by pippettingand the beads resuspended in PBS/0.1% BSA and left on the Dynal MPC for 2 min(this step was repeated two times). The purified CD45RA or CD45RO cells were thenresuspended in PBS/0.1% BSA. The purified CD45RA and CD45RO cells were thentreated with 1 mg of mouse IgM mAb to either CD4 or CD8 and incubated for 30min at 4°C and then washed with PBS/0.1% BSA. Dynabeads pan mouse IgM wereadded to the cells and the subsequent procedure used for the CD45RA and CD45ROcell purification was undertaken to obtain CD4 cells expressing CD45RA or CD45ROand CD8 cells expressing CD45RA or CD45RO.

2.5. FACS analysis of CD3 and CD45RA, CD62L T lymphocyte populations

100 ml of peripheral blood obtained from subjects were treated with the followingmonoclonal antibodies; mouse anti-human CD3 conjugated to FITC (Sigma), mouseanti-human CD45RA conjugated to Quantum red (Pharmingen, Dorset, UK) andmouse anti-human CD62L conjugated to PE (Pharmingen) or to the appropriateisotype matched negative controls and incubated on ice for 30 min. The red cells werethen lysed by the addition of 2 ml of Ortholyse (Ortho Diagnostic Systems,Buckinghamshire, UK) and the samples were analysed using the BD FACSCalibur.

3. Results

3.1. Detection of sjTREC in naı̈6e but not memory T cells

We separated T cell subsets to determine firstly whether the technique we usedcould discriminate in the detection of sjTREC in phenotypically defined naı̈ve and


memory cells and secondly whether there was a difference in the CD4 and CD8subset expression of these sjTREC. Our results, shown in Fig. 1 reveal that sjTRECwere detectable in naı̈ve (CD45RA+) cells in both CD4 and CD8 subpopulations.However we failed to detect sjTREC in the memory (CD45RO+) populations ofeither CD4 or CD8 subset using this same PCR conditions which provided positiveresults for the naı̈ve T cell subpopulations.

3.2. Quantitation by scanning the image

The results of the PCR analysis using differing initial amounts of the sjTRECpositive control molecule for the construction of a standard curve is shown in Fig.2. The results seen here are the means and standard deviations from four separateexperiments. The line of best fit drawn on the points obtained had an R2 value of0.97 indicating the close correlation between the brightness of the band and theinitial sjTREC copy number. Inset in the graph is an example of the results fromone of the gels containing the PCR products arising from different starting sjTRECnumbers.

3.3. Comparison with Lightcycler results

The results of the sjTREC levels in blood from female donors of three differentages is shown in Fig. 3(a) and (b). Fig. 3(a) shows the results obtained using theLightcycler and Fig. 3(b) shows the results from the Scion Image scanning method.Comparison of the two results indicates that both clearly show an age-related

Fig. 1. Detection of sjTREC in T cell subsets. The gel shows the presence of the sjTREC band in DNAderived from CD4+CD45RA+ (Lane 2) and CD8+CD45RA+ (Lane 7), but not in CD4+CD45RO+

(Lane 4) and CD8+CD45RO+ (Lane 6) cells. The molecular weight markers are in Lane 1 and theshorter sjTREC molecule used as a positive control is in Lanes 3 and 5.


Fig. 2. Standard curve generated from the sjTREC positive control. Brightness analysis of the bandsproduced from PCR amplification of different starting copy numbers, ranging from 103 to 108, of theshortened sjTREC gene. Each point shown is the mean 9 1 S.D. from 4 readings. The result from onegel is shown above the points.

decrease in sjTREC numbers with both detecting comparable numbers of sjTRECwithin the samples. The results showing a decline in sjTREC levels with age are inagreement with those observed in previous studies as well as reflecting the resultsobtained from phenotypic analysis. Furthermore as expected these samples showthe number of true naı̈ve T cells (CD3+CD45+CD62L+) to decline with age.

4. Discussion

The measurement of episomal DNA circles in the nucleus of T cells is a sensitivemethod for the determination of thymic output with age. The use of sjTRECmeasurement either in relation to the DNA content of the sample (Douek et al.,1998 ) or the total CD3+ T cell content of the sample as here provides a means ofassessing the naı̈ve T cell subpopulation of the sample.

This paper describes the detection of sjTREC in CD45RA+ cells in both the CD4and CD8 T cell subsets, but the failure to detect these products in CD45RO+ Tcells in either the CD4 or CD8 subsets. This result would be anticipated fromprevious work, which shows that the sjTREC episomal circles are not replicatedwith the cell and so are subsequently diluted out by cell division. It is not that thesjTREC molecules are absent from the CD45RO+ populations, but that they arepresent at such a low frequency per cell that the PCR conditions used failed todetect them. The CD45RA+ T cells population contains the naı̈ve T cell emigrantsfrom the thymus which are a non-dividing population with the highest frequency ofTRECS per T cell. Naı̈ve T cells only enter division once activated by antigen andenters the memory/activated T cell pool (CD45RO+).


The identification of recent thymic migrants on the basis of phenotype has beenpossible in some rodent studies because of the use of intrathymic labeling withfluorescein (Scollay et al., 1980) or the identification that the recent thymic migranthas distinctive phenotype (Hosseinzadeh and Goldschneider, 1993). However inhuman systems this has proved to be somewhat more complex. The problem hasarisen from studies showing an ability of cells with some of the phenotypiccharacteristics of naı̈ve T cell to arise from the memory T cell pool (Nociari et al.,1999). These populations have been shown to contain virus specific clones after theresolution of the specific infection (Wills et al., 1999) and in addition it has beenclaimed that this change in phenotype has been linked to the loss of CD28 (Nociariet al., 1999). The use of TREC’s as an additional marker to identify recent thymic

Fig. 3. Levels of sjTREC per 5 × 107 of each of volunteers peripheral blood mononuclear cells asderived by the Lightcycler (a) or by the scanning method (b). The age and number of naı̈ve(CD3+CD45RA+CD62L+) T cells in the sample of 5 × 107 PBMC was (i) 21 years old and 11 373 000naı̈ve T cells, (ii) 33 years old and 5 397 600 naı̈ve T cells and (iii) 55 years old and 3 250 000 naı̈ve Tcells.


migrants has provided a clearer means of identification of recent thymic migrantsand assessing thymic output (Al Harthi et al., 2000; McFarland et al., 2000; Poulinet al., 1999; Zhang et al., 1999).

One of the major problems with the use of TREC measurements is that previousstudies have used either radioisotopes (Douek et al., 1998), ELISA based methods(Al Harthi et al., 2000) or expensive equipment to measure TREC levels with realtime PCR (Zhang et al., 1999). The use of radioisotopes was the first method to beused to quantitate TREC levels and although sensitive and effective has some costand safety implications and in some cases problems with availability. The ELISAmethod of detection uses PCR in the presence of Dig-UTP, and the product iscaptured on a 96 well plate coated with streptavidin by a biotin conjugated proberecognizing an internal sequence and an ELISA is performed using an anti-Digperoxidase antibody. The resulting analysis is sensitive and quantitative, but as withall multi-step processes is dependant on all stages and reagents working correctly.The measurement of product in a real time PCR is a fast direct method ofassessment, but the initial outlay to purchase the equipment is prohibitive in forsome laboratories.

This paper describes a rapid, relatively safe and simple method of measuring theamount of sjTREC’s in a sample of T cells which does not depend upon usingradioisotopes. The method uses the brightness of the DNA band in the gelmeasured on a captured image using an analysis programme freely available on theInternet. As with all methods there are some critical stages to the assay. Theexponential shape of the graph for different amounts of positive control sampleused clearly shows that the correct number of PCR cycles are being used. For asample to be assessed correctly it is important that it falls within the limits of thegraph used to produce the standard curve. Clearly it is important that theconditions used for the analysis of the DNA under test be as close to those used todetermine the standard curve. So considerations such as the thickness and runningconditions of the gel, amount of ethidium bromide and settings on the imagecapture equipment must be kept constant. Repeated running of the positive controlstandards and the construction of a standard curve as we have done here must bea first priority and is a good measure of the repeatability of the system.

We have compared our method with results we have obtained using the Lightcy-cler which measures real time PCR products from the reaction. The results withinthis small sample reveal a remarkable similarity from the two methods, indicatingthat that the gel image scanning method is a valid and reliable method of sjTRECanalysis.

Acknowledgements

We would like to thank D. King for help with the cell separation studies, and DrD. Douek for providing help with the TREC analysis. This work was supported bythe Luard family (J.P. is the Luard scholar) and the Welcome Trust (grant number051541).


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a simple method for the measurement of sjtrec levels in blood

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