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T cell reconstitution is a critical feature of the recovery of the adaptive immune response and has 2 main components: thymic output of new T cells and peripheral homeostatic expansion of preexisting T cells. It has been shown that though thymic function declines with age, a reasonable output is still maintained into late adult life.(1) In many clinical situations, thymic output is crucial to the maintenance and competence of the T cell effector immune response.
Thymic function can be determined by T-cell receptor excision circle (TREC) analysis. TRECs are extrachromosomal DNA byproducts of T-cell receptor (TCR) rearrangement, which are nonreplicative. TRECs are expressed only in T cells of thymic origin and each cell is thought to contain a single copy of TREC. Hence, TREC analysis provides a very specific assessment of T-cell recovery (eg, after hematopoietic cell transplantation) or numerical T-cell competence. There are several TRECs generated during the process of TCR rearrangement and the TCR delta deletion TREC (deltaREC psi-J-alpha signal joint TREC) has been shown to be the most accurate TREC for measuring thymic output.(2) This assay measures this specific TREC using quantitative, real-time PCR.
Clinical use of TRECs in HIV and Antiretroviral Therapy:
HIV infection leads to a decrease in thymic function. Adult patients treated with highly active antiretroviral therapy (HAART) show a rapid and sustained increase in thymic output.(1)
Clinical use of TRECs in Hematopoietic Cell Transplantation (HCT) and Primary Immunodeficiencies (PID):
Following HCT, there is a period of prolonged immunodeficiency that varies depending on the nature and type of stem cell graft used and the conditioning regimen, among other factors. This secondary immunodeficiency also includes defects in thymopoiesis.(3-5) It has been shown that numerical T cell recovery is usually achieved by day 100 posttransplant, though there is an inversion of the CD4:CD8 ratio that can persist for up to a year.(4) Also, recovery of T-cell function and diversity can take up to 12 months, although this can be more rapid in pediatric patients. However, recovery of T-cell function is only possible when there is numerical reconstitution of T cells. T cells, along with the other components of adaptive immunity, are key players in the successful response to vaccination post-HCT.(6)
Recently, it has been shown in patients who received HCT for severe combined immunodeficiency (SCID) that T cell recovery early after stem cell transplant is crucial to long-term T cell reconstitution.(7) Patients who demonstrated impaired reconstitution were shown to have poor early grafting, as opposed to immune failure caused by accelerated loss of thymic output or long-term graft failure. In this study, the numbers of TRECs early after HCT were most predictive for long-term reconstitution. This data suggests that frequent monitoring of T-cell immunity and TREC numbers after HCT can help identify patients who will fail to reconstitute properly, which would allow additional therapies to be instituted in a timely manner.(7) It would be reasonable to extrapolate such a conclusion to other diseases that are also treated by HCT.
TREC Copies and Thymic Output in Adults:
Since the adult thymus involutes after puberty and is progressively replaced by fat with age, thymus-dependent T cell recovery has been assumed to be severely limited in adults. However, with TREC analysis it has been shown that the change in thymic function in adults is a quantitative phenomenon rather than a qualitative one and thymic output is not totally eliminated.(1,8,9) Thus, after HCT or HAART, the remaining thymic tissue can be mobilized in adults to replenish depleted immune systems with a potentially broader repertoire of naive T cells. Douek et al have shown that there is a significant contribution by the thymus to immune reconstitution after myeloablative chemotherapy and HCT in adults.(8) In fact, this data shows that there is both a marked increase in the TREC numbers and a significant negative correlation of TREC copies with age posttransplant.
In addition to the specific clinical situations elucidated above, TREC analysis can be helpful in identifying patients with primary immunodeficiencies and assessing their numerical T-cell immune competence. It can also be used as a measure of immune competence in patients receiving immunotherapy or cancer vaccines, where maintenance of, T-cell outputis integral to the immune response against cancer.
The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 am and noon, with no change between noon and afternoon. Natural killer (NK) cell counts, on the other hand, are constant throughout the day.(10) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(11-13) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(11) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening,(14) and during summer compared to winter.(15) These data, therefore, indicate that timing and consistency in timing of blood collection are critical when serially monitoring patients for lymphocyte subsets.
Measuring T-cell output or reconstitution (thymopoiesis) following hematopoietic cell transplantation or highly active antiretroviral therapy
Evaluating thymic function in patients with cellular or combined primary immunodeficiencies, or receiving immunotherapy or cancer vaccines
Assessing T-cell recovery following thymus transplants for DiGeorge syndrome
T-cell receptor excision circles (TRECs) generally show an inverse correlation with age, though there can be substantial variations in TREC copies relative to T-cell count within a given age group.
Following hematopoietic cell transplantation (HCT), highly active antiretroviral therapy (HAART), thymic transplants, etc, TREC typically increases from absent or very low levels (below age-matched reference range) to baseline levels or exceeds baseline levels, showing evidence of thymic rebound, which is consistent with recovery of thymic output and T-cell reconstitution.
When a patient is being monitored for thymic recovery posttransplant treatment, this assay recommends that a pretransplant (prior to myeloablative or nonmyeloablative conditioning) or a pretreatment baseline specimen be provided so that appropriate comparisons can be made between the pre- and posttransplant treatment specimens. Since there is substantial variability between individuals in TREC copies, the best comparison is made to the patient's own baseline specimen rather than the reference range (which provides a guideline for TREC copies for age-matched healthy controls).
A consultative report will be generated for each patient.
While indicative of thymic function and T-cell recovery, T-cell receptor excision circle (TREC) results cannot be taken as a direct measure of thymic output because TREC are diluted by peripheral T cell division and intracellular degradation. In addition, the longevity of naive T cells in the periphery precludes TREC from being regarded as recent thymic emigrants. The assay provides a quantitative measure of TREC, ie, TREC copies per million CD3 T cells; however, this number should be regarded as a relative, rather than absolute, number because of the caveats explained above.
The TREC assay should not be ordered on adults over age 60 due to physiological decline in thymic function in the sixth and seventh decades of life.
Assay results are dependent on the patient's T-cell counts and in patients with profound lymphopenia it may be impossible to perform the assay if there are insufficient numbers of cells.
Temperature and time are critical to the performance of the assay. Temperatures that exceed or drop below 20 to 25 degrees C can dramatically affect the assay. High temperatures can cause substantial hemolysis that will interfere with the methodology used to perform the assay. Transportation delays may result in significant TREC degradation.
Timing and consistency in timing of blood collection are critical when serially monitoring patients for lymphocyte subsets. See Clinical Information.
The appropriate age-related reference values will be provided on the report.
1. Douek DC, McFarland RD, Keiser PH, et al: Changes in thymic function with age and during the treatment of HIV infection. Nature 1998;396:690-694
2. Hazenberg MD, Verschuren MC, Hamann D, et al: T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation. J Mol Med 2001;79:631-640
3. Parkman R, Weinberg K: Immunological reconstitution following hematopoietic stem cell transplantation. In Hematopoietic Cell Transplantation. Second edition. Edited by ED Thomas, KG Blume, SJ Forman. Blackwell Scientific, Oxford, UK, 1999, pp 704-711
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5. Weinberg K, Annett G, Kashyap A, et al: The effect of thymic function on immunocompetence following bone marrow transplantation. Biol Blood Marrow Transplant 1995;1:18-23
6. Auletta JJ, Lazarus HM: Immune restoration following hematopoietic stem cell transplantation: an evolving target. Bone Marrow Transplant 2005;35:835-857
7. Borghans JA, Bredius RG, Hazenberg MD, et al: Early determinants of long-term T cell reconstitution after hematopoietic stem cell transplantation for severe combined immunodeficiency. Blood 2006;108:763-769
8. Douek DC, Vescio RA, Betts MR, et al: Assessment of thymic output in adults after hematopoietic stem cell transplantation and prediction of T cell reconstitution. Lancet 2000;355:1875-1881
9. Jamieson BD, Douek DC, Killian S, et al: Generation of functional thymocytes in the human adult. Immunity 1999;10:569-575
10. Carmichael KF, Abayomi A: Analysis of diurnal variation of lymphocyte subsets in healthy subjects and its implication in HIV monitoring and treatment. 15th Intl Conference on AIDS, Bangkok, Thailand, 2004, Abstract B11052
11. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. Blood 2009 May 21;113(21):5134-5143
12. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411
13. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Psychosom Med 1997;59:42-50
14. Malone JL, Simms TE, Gray GC, et al: Sources of variability in repeated T-helper lymphocyte counts from HIV 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J AIDS 1990;3:144-151
15. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516