Levels of Recent Thymic Emigrant Cells Decrease in Children Undergoing Partial Thymectomy during Cardiac Surgery

The human thymus is required for establishment of a T-cell poolin fetal life, but postnatal thymectomy is not known to causeimmunodeficiency. T-cell emigration from thymus (thymic recentemigrants [TRECs]) is a continuous thymic-dependent process.We studied TREC levels pre- and post-partial thymectomy in childrenundergoing cardiac surgery. TRECs were quantitated by real-timePCR in peripheral blood lymphocytes of 24 children (0 to 12years). TREC values were 47916 ± 9271 pre-partial thymectomyand 33157 ± 8479 post-partial thymectomy in 11 pairedpatients (P = 0.014). Interval between pre- and post-partialthymectomy was 8.8 days ± 5.8 days. Another group of8 children had 30384 ± 9748 TRECs 16 days to 6 yearspost-partial thymectomy. There was a significant drop in TRECvalues post-partial thymectomy in the immediate postoperativeperiod compared to prethymectomy TREC levels. While decreasedthymic output may persist, the long-term implications were notevaluated in this patient population.

INTRODUCTION

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Introduction

The thymus plays a crucial role in the development of T cellsproviding an inductive microenvironment in which bone marrow-derivedprogenitors undergo proliferation, T-cell receptor gene rearrangementand thymocyte differentiation into mature T cells.

Two types of cells are generated in the thymus. Most expressa T-cell receptor composed of ${alpha}$ and ß chains, but asmall population expresses a receptor of ${gamma}$ and ${delta}$ chains. All ofthese chains are encoded by variable (V), diversity (D), andjunctional (J) gene segments, which are rearranged during T-celldevelopment in a process called V (D) J recombination. Thisprocess involves cleavage of DNA at the recombination signalsequences that flank T-cell receptor gene segments in theirgerm line configuration. When the intervening stretches of DNAare excised, then the coding ends are joined to form a functionalT-cell receptor gene in the chromosomal DNA, and the signalends join to form extra chromosomal DNA circles termed T-cellreceptor rearrangement excision circles (TRECs). The T-cellreceptor- ${delta}$ locus is embedded in the T-cell receptor- ${alpha}$ locus; soT-cell receptor- ${delta}$ sequences are specifically deleted in all ${alpha}$ ßT cells. During T-cell receptor rearrangement there are tworearrangement events which happen, first producing a signaljoint TREC and second producing a coding joint TREC (11).

In humans there is no known way to distinguish phenotypicallybetween cells that have recently emigrated the thymus and long-livednaïve cells in the periphery. One proposed marker for recentthymic emigrants is the episomal DNA circle that is generatedduring excision rearrangement of T-cell receptor genes. TRECsare stable and are not duplicated during mitosis (3). Signaland coding joint TRECs are affected similarly in mature T cells.

Children with congenital heart diseases routinely undergo partialthymectomy during cardiac surgery for better visualization ofstructures. There have been few studies done to determine theeffects of neonatal thymectomy in humans (1, 13). In the reportfocused on neonatal thymectomy in humans, Brearley et al. (1)had shown that in infants younger than 3 months of age thymectomycaused impaired immunity in late childhood and concluded thatthymectomy in pediatric cardiac surgery should be avoided. Wellset al. (13) did not concur on this concept of preserving thethymus and showed that total number of T cells and CD4 cellsdropped 12 months after thymectomy.

In chickens (7) levels of chT1⁺ cells (which correlated to TRECs)disappeared after complete thymectomy. In chickens with partialthymectomies the levels of chT1⁺ T cells in the circulation4 weeks postthymectomy correlated directly with the numbersof residual thymic lobes.

There have been no studies done to determine what happens toTREC levels after partial thymectomy in children with congenitalheart disease. This study was done to study the effect of partialthymectomy on TREC values. Partial thymectomy is defined asremoval of 70 to 90% of thymic gland from the anterior mediastinumto facilitate exposure for cannulation for cardiopulmonary bypass.

MATERIALS AND METHODS

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Materials and Methods

The institutional ethical and research review boards approvedthe study and informed consent was obtained from the parents.Twenty-four children (age range, 0 to 12 years, mean = 4 ±3.8 years) with congenital heart disease were prospectivelyentered into the study. They were subdivided into three groups.Group A included patients undergoing first surgery for congenitalheart disease. Both pre- and post-partial thymectomy sampleswere collected after 3 to 15 days from 11 patients.

Group B included patients undergoing reoperation for congenitalheart disease. (Only post-partial thymectomy samples were collectedfrom 8 patients, after an interval of 16 days to 6 years.)

Group C included patients with congenital heart disease undergoingcardiac catheterization. (Only pre-partial thymectomy sampleswere collected from 5 patients.)

No study patient carried the diagnosis of DiGeorge syndrome.No patients received steroids during the hospitalization.

Cell sources. Peripheral blood samples were drawn from patients. Three millilitersof whole blood was collected in sterile heparinized tubes processedwithin 24 h.

Cell isolation and quantification of TRECs by real-time PCR. Peripheral blood mononuclear cells (PBMC) were isolated by Ficolldensity gradient centrifugation. Cells lysates were preparedby proteinase K digestion TRECs were quantified by real-timePCR analysis, using the TaqMan 5 nuclease assay, and the ABIPrism 7700 sequence detection system (PE-Applied Biosystems).In brief, 0.5 µM primers (forward primer: CACATCCCTTTCAACCATGCTand reverse primer: GCCAGCTGCAGGGTTTAGG), 0.1 µM TaqManprobe (FAM-ACACCTCTGGTTTTTGTAAAGGTGCCCACT-TAMRA), 3.5 mM MgCl₂,0.2 mM deoxynucleoside triphosphates, 0.25 U of uracil DNA glycosylase,0.625 U of Amplitaq Gold DNA polymerase in TaqMan buffer (PEBiosystem), and 100,250 ng of genomic DNA from PBMC were mixedtogether in a 25-L PCR mixture. PCR conditions were 50°Cfor 2 min, 95°C for 10 min, 95°C for 30 s, and 60°Cfor 1 min for 45 cycles. A standard curve was established withknown copies of plasmids containing signal joint TREC fragmentand TREC values for test samples were calculated with the softwareprovided with the ABI Prism 7700 system (Sequence DetectionSystem, version 1.6.3). Each sample was run in duplicate andmean TREC values were used for data analysis. Results were expressedas TRECs/10⁶ cells.

Statistical analysis. The significance of differences between means in TREC valueswas determined by use of the Student t test, Wilcoxon signedrank sum test, and Mann-Whitney U test.

RESULTS

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Results

TREC values were recorded per million peripheral blood mononuclear cells. TREC levels, group A: for patients from group A (n = 11), theaverage interval between the samples was 8.8 days ± 5.8days. The pre-partial thymectomy TREC value was 47,916 ±30,748, (mean ± standard error of mean) with the post-partialthymectomy TREC value being 33,157 ± 28,122, (mean ±standard error of mean) (Fig. 1). This decrease in the post-partialthymectomy TREC value was significant (P = 0.014, Wilcoxon signedrank sum test).

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FIG. 1. Mean TREC values/million PBMC pre- and post-partial thymectomy with standard deviation in group A patients (paired data).

TREC levels in patients in group B (n = 8) were 30,384 ±9,748 (mean ± standard error of mean).

TREC levels in patients in group C (n = 5) were 69,774 ±33,601 (mean ± standard error of mean).

Although no statistical correlation was drawn between groupsB and C as they included different patients, the overall pre-partialthymectomy TREC levels were higher than the post-partial thymectomyTREC levels.

DISCUSSION

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Discussion

The thymus is the primary site of T-cell lymphopoiesis duringfetal and early postnatal life. Studies of thymuses of patientswith immune deficiencies have led to an understanding of themolecular and genetic basis of many immune deficiency diseases,pointing to the importance of the thymus in T-cell development(12). Due to thymic involution, it has been suggested that theadult thymus does not contribute to the new T-cell replacement.However recent data indicate that the thymus contributes tonew T cells even in adulthood (4, 6, 10). Thus, it is highlylikely that a decrease in thymopoiesis would lead to a lastingdeficiency in the ability of the host to generate new T cells.There are no phenotypic markers that distinguish between recentthymic emigrants from the rest of peripheral naïve T-cellpool. The evaluation of TREC frequencies in peripheral T cellscan be a useful indicator of the de novo production of T lymphocytes(3).

In children, studies using TRECs have been performed in patientswith primary and secondary immune deficiency diseases includinghuman immunodeficiency virus (HIV)-infected infants and children(2, 8, 9). Patients with absent thymus, such as in severe combinedimmune deficiency disease or in complete DiGeorge syndrome havevery low TRECs. In HIV-infected infants, TRECs in peripheralblood lymphocytes were decreased when compared to HIV-exposed,uninfected infants; after therapy the TREC levels increasedto levels present in HIV-exposed, uninfected infants (2). Ina recent study, TRECs were markedly reduced in patients manyyears after thymectomy in childhood, concurrently with decreasesin total T cells and phenotypic naïve CD4 T cells (5).

In the present study the TREC levels significantly declinedin all study patients post-partial thymectomy in the immediatepostoperative period. The decreased TREC values seen in patientsin the immediate postoperative period are probably due to severalreasons, including decreased thymic output and changes in bloodvolume distributions. Overall the TREC values in patient groupswithout thymectomy were higher than the patients who had a partialthymectomy. The likelihood of a true decrease in TRECs resultingfrom decreased thymopoiesis is strengthened by the observationthat even patients examined for periods up to six years post-partialtheymectomy had lower TRECs than patients who did not have thymectomy.In the published study of Halnon et al. (5), patients with noresidual thymus had significantly lower TRECs than those withpartial thymectomy.

Whether the decrease in TREC in patients with partial or completethymic removal also leads to a deficiency in thymic-derivedregulatory T cells is unknown. The partial thymectomy in infancyand decrease in TRECs did not appear to adversely influenceimmune function, as there was no known autoimmune diseases inthe study population over a short term follow up. Presumably,minimal residual thymic tissue post-partial thymectomy is sufficientfor immune function.

ACKNOWLEDGMENTS

This study was supported by NIH grant AI48857 to S.P.

FOOTNOTES

* Corresponding author. Present address: Batchelor Children's Research Institute, 1580 NW 10th Avenue, University of Miami School of Medicine, Miami, Florida 33136. Phone: 305-243-7732. Fax: (305) 243-7211. E-mail: spahwa{at}med.miami.edu.

REFERENCES

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