Research paper
A reliable and simplified sj/β-TREC ratio quantification method for human thymic output measurement

https://doi.org/10.1016/j.jim.2009.11.007Get rights and content

Abstract

Current techniques to peripherally assess thymic function are: the signal-joint T-cell receptor excision circle (sj-TREC) level measurement and the naive T cell and CD31+ TREC-rich subset determination. However, all of them are indirect approaches and none could be considered a direct recent thymic emigrant (RTE) marker. To overcome their limitations, Dion et al. (2004) described the sj/β-TREC ratio that allows the peripheral quantification of the double negative to double positive intrathymic proliferation step. Nevertheless, the protocol described is expensive, sample and time-consuming, thus, limiting its usefulness. In this study, we describe a simplified protocol that reduces from 33 to 9 the amount of PCR reaction needed but maintaining the sensitivity and reproducibility of the original technique. In addition, we corroborated the effectiveness of our technique as an accurate thymic output-related marker by correlating the peripheral sj/β-TREC ratio with a direct measurement of thymic function as the percentage of double positive thymocytes (r = 0.601, p < 0.001).

Introduction

Thymus is the major organ involved in the generation of mature T cells (Haynes et al., 2000). In humans its function is maximal at birth, but an age-related involution starts with puberty hormonal changes (Chiodi, 1940, Utsuyama and Hirokawa, 1989). As a consequence, lack of elderly thymic function had been assumed, but later studies suggested some degree of thymic function in adulthood (Jamieson et al., 1999, Shanker, 2004, Pawelec et al., 2006) and, recently, we have reported a wide range of thymic functions in elderly humans, with higher thymic capacity correlated with higher peripheral naive T-cell counts (Ferrando-Martinez et al., 2009). In addition, works focusing on clinical lymphopenia showed that adult thymus can partially reverse the atrophy process to better recover the immune space (Mackall et al., 1995, Franco et al., 2002, de la Rosa and Leal, 2003), enhancing the clinical relevance of monitoring the thymic function.

Since thymic biopsies are formally contraindicated, suitable tools for an indirect thymic output measurement in peripheral blood are needed. Characterization of the signal-joint T-cell receptor excision circles (sj-TRECs) was a milestone in thymic output quantification (Douek et al., 1998, Dion et al., 2007) in spite of its limitations. Nevertheless, the deep effect of peripheral proliferation of naive T cells in sj-TREC counts was quickly stated (Hazenberg et al., 2000, Harris et al., 2005), increasing the difficulty of interpretation. Subsequent mathematical models showed that, even in the steady state, the sj-TREC content is not a good measure of thymic function (Ribeiro and Perelson, 2007). Moreover, recent thymic emigrants (RTEs) are phenotipically indistinguishable from naive T cells and neither the study of the naive T cell nor the CD31+ TREC-rich naive T-cell subsets (Junge et al., 2007, Kohler and Thiel, 2009) are fitted approaches since they are not direct RTEs but RTE-rich subset markers.

In order to overcome the sj-TREC limitations Dion et al. (2004) described the sj/β-TREC ratio quantification. This elegant technique allows the measurement of two different TRECs: the DβJβ-TREC, product of the β chain TCR rearrangement at the most immature thymocyte subset, and the sj-TREC, product of the α chain TCR rearrangement, early at the double positive (DP) stage. The sj/β-TREC ratio is a direct quantification of the intrathymic proliferation step occurring between the β and α chain rearrangement and is not affected by peripheral T-cell dynamics, thus providing a potentially effective peripheral tool to measure thymic output (Dion et al., 2004). However, quantifying the sj/β-TREC ratio is difficult and time-consuming since 11 multiplex PCR reactions (10 × DβJβ + 1 × sj-TREC, all of them together with the amplification of the CD3 gene) with a two-step (nested) PCR strategy are needed for each point. When doing triplicate analysis thirty-three nested PCRs should be performed for each patient at each time point. Besides, in a single experiment a vast quantity of DNA, not always available, needs to be used (Dion et al., 2004). Probably due to these technical limitations, this powerful tool has only been used in a limited number of studies (Delobel et al., 2006, Dion et al., 2007, Gautier et al., 2007, Clave et al., 2009).

Accordingly, the aim of this study was to describe a simplified strategy to determine the sj/β-TREC ratio in PBMCs. In addition, we used simultaneous thymic tissue and peripheral blood samples to corroborate the sj/β-TREC ratio as a thymic function-surrogated marker using DP thymocytes as a direct measurement of human thymic function (Thoman, 1995, Almeida et al., 2001, Ferrando-Martinez et al., 2009).

Section snippets

Patients

Peripheral blood and thymic tissue samples were simultaneously collected from 50 consecutive cardiac surgery patients between February 2006 and August 2007 at Virgen del Rocio University Hospital in Seville, Spain. All patients underwent surgery for valvular repair or ischemic cardiopathy and all of them were, otherwise healthy adult individuals. Patients had not received any treatment that could influence their immune status such as radiation (Mackall et al., 1995), steroids (Cohen et al., 1992

Real-time PCR strategy for the sj/β-TREC ratio determination

In order to simplify the sj/β-TREC ratio quantification technique reported by Dion et al. (2004) we designed a nested, multiplex PCR procedure schematically represented in Fig. 1. As shown in Fig. 1, only two different PCR reactions are needed for the first round PCR: one amplifies the sj-TREC and the other one all the six DβJβ-TRECs, derived from the DβJβ cluster one, together. The six sense primers share the same antisense primer for the DβJβ-TRECs multiplex reaction. To improve the PCR

Discussion

Present study shows a simplified protocol to the sj/β-TREC ratio quantification. In addition, we show the strong correlation found between the sj/β-TREC ratio as a peripheral thymic function-related marker and the percentage of CD4+CD8+ DP thymocytes, standard of thymic function.

The study of the immune recovery on Hematopoietic stem cell transplant (HSCT) (Mackall et al., 1995) or HIV-infected patients under high activity antiretroviral therapy (HAART) (Franco et al., 2002, de la Rosa and Leal,

Acknowledgements

SFM and ERM have grants from the Fondo de Investigaciones Sanitarias (FIS06/00176 and CP08/00172 respectively). This study is supported by the Fundación la para la Investigación y la Prevención del SIDA en España (FIPSE, 12481/05, 36624/06), Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD06/0006/0021), Redes Temáticas de Cardiovascular (ISCIII RECAVA RD06/0014), Proyecto de Excelencia, Consejería de Innovación, Ciencia y Empresa (P06-CTS-01579) and Consejería de Salud, Servicio

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