Joint T-cell Receptor Rearrangement Excision Research Articles

A Blood-Based Molecular Clock for Biological Age Estimation.

In the last decade, extensive efforts have been made to identify biomarkers of biological age. DNA methylation levels of ELOVL fatty acid elongase 2 (ELOVL2) and the signal joint T-cell receptor rearrangement excision circles (sjTRECs) represent the most promising candidates. Although these two non-redundant biomarkers echo important biological aspects of the ageing process in humans, a well-validated molecular clock exploiting these powerful candidates has not yet been formulated. The present study aimed to develop a more accurate molecular clock in a sample of 194 Italian individuals by re-analyzing the previously obtained EVOLV2 methylation data together with the amount of sjTRECs in the same blood samples. The proposed model showed a high prediction accuracy both in younger individuals with an error of about 2.5 years and in older subjects where a relatively low error was observed if compared with those reported in previously published studies. In conclusion, an easy, cost-effective and reliable model to measure the individual rate and the quality of aging in human population has been proposed. Further studies are required to validate the model and to extend its use in an applicative context.

Telomere length and signal joint T-cell receptor rearrangement excision circles as biomarkers for chronological age estimation

Background Chronological age estimation is a challenging marker in the field of forensic medicine. The current study aimed to investigate the accuracy of signal joint T-cell receptor rearrangement excision circles (sjTRECs) quantification and telomere length measurement as methods for estimating chronological age. Methods Telomere length was estimated in the DNA derived from the buccal cells through estimating the telomeric restriction fragment (TRF) length using TeloTTAGGG Telomere Length Assay while the sjTRECs quantification was carried out on DNA isolated from the blood samples using qPCR. Results The TRF length was shortened with increased age (r = −0.722, p < 0.001). The sjTRECs were also decreased with increased age (r = −0.831, p < 0.001). Stronger coefficient and lower standard error of the estimate was obtained when multiple regression analysis for age prediction based on the values of both methods was applied (r = −0.876, p < 0.001).

QPCR Applications for the Determination of the Biological Age.

Individual age is a phenotypic trait that provides useful information not only in forensic investigations but also in the aging research which is becoming an urgent call due to the dramatic growth of the aging population worldwide.TaqMan quantification PCR (qPCR) can be successfully applied to biological age estimation, using method defined in Zubakov et al. (Curr Biol 20:R970-R971, 2010). Since levels of signal joint T-cell receptor rearrangement excision circle (sjTREC) in human lymphocytes are known to decrease with age increasing, the qPCR of sjTREC represents a simple and relatively reproducible technique which offers highly accurate age estimation results.

Biological Age versus Chronological Age in the Prevention of Age Associated Diseases

Aging is associated with an increasing incidence of major diseases among which cancer, cardiovascular, neurodegenerative, metabolic and autoimmune diseases. Primary prevention and early diagnosis of these diseases have a dramatic impact on incidence, outcome and quality of life and are commonly applied as age-dependent indications based on evidence of efficacy for specific groups of the aging population. They likely contribute to the observed increase in life expectancy through the reduction of incidence and the retardation of onset of age-associated diseases. In the present article, we develop the hypothesis that age-dependent preventative measures and diagnostic screenings might perform better if the biological age would be used instead of the chronological age for the definition of the population to be included. This is based on the observation that there are individual differences in the age-associated decline in performance that are reflected by measurable biological indicators, such as telomere length, signal joint T-cell receptor rearrangement excision circle, and specific DNA-methylation and gene expression events.

SjTREC quantification using SYBR quantitative PCR for age estimation of bloodstains in a Japanese population.

Individual age is a phenotypic trait that provides useful information in forensic investigations. Levels of signal joint T-cell receptor rearrangement excision circle (sjTREC) in human peripheral blood are known to decline with increasing age. The advantages of sjTREC quantification are the simple procedures and highly accurate age estimation results. Whereas TaqMan quantification PCR (qPCR) is widely used for sjTREC quantification, SYBR qPCR assay is not routinely used for evaluating ethnic data. Therefore, we focused on the advantages of the SYBR qPCR assay, which is cheaper and simpler to set up than the TaqMan probe assay. In this study, we developed a SYBR qPCR assay for sjTREC quantification from bloodstains from a Japanese population and evaluated the strength of correlation between sjTREC levels and actual age. The results were obtained from 194 individuals ranging from 18 to 81 years old, and showed a negative correlation between sjTREC level and individual age (r = -0.786). The equation for age estimation was Age = -6.27 dCt (CtTBP - CtsjTREC) - 25.841 with standard error ±8.0 years. Furthermore, this formula for the SYBR assay can be applied to not only fresh bloodstains, but also whole blood and bloodstains up to 1 month old. These results indicate that SYBR qPCR is an effective method for age estimation from bloodstains, and its practicality and affordability make it an attractive sjTREC quantification technique.

Predicting Human Age with Bloodstains by sjTREC Quantification

The age-related decline of signal joint T-cell receptor rearrangement excision circles (sjTRECs) in human peripheral blood has been demonstrated in our previous study and other reports. Until now, only a few studies on sjTREC detection in bloodstain samples were reported, which were based on a small sample of subjects of a limited age range, although bloodstains are much more frequently encountered in forensic practice. In this present study, we adopted the sensitive Taqman real-time quantitative polymerase chain reaction (qPCR) method to perform sjTREC quantification in bloodstains from individuals ranging from 0–86 years old (n = 264). The results revealed that sjTREC contents in human bloodstains were declined in an age-dependent manner (r = −0.8712). The formula of age estimation was Age = −7.1815Y−42.458±9.42 (Y dCtTBP-sjTREC; 9.42 standard error). Furthermore, we tested for the influence of short- or long- storage time by analyzing fresh and stored bloodstains from the same individuals. Remarkably, no statistically significant difference in sjTREC contents was found between the fresh and old DNA samples over a 4-week of storage time. However, significant loss (0.16–1.93 dCt) in sjTREC contents was detected after 1.5 years of storage in 31 samples. Moreover, preliminary sjTREC quantification from up to 20-year-old bloodstains showed that though the sjTREC contents were detectable in all samples and highly correlated with donor age, a time-dependent decrease in the correlation coefficient r was found, suggesting the predicting accuracy of this described assay would be deteriorated in aged samples. Our findings show that sjTREC quantification might be also suitable for age prediction in bloodstains, and future researches into the time-dependent or other potential impacts on sjTREC quantification might allow further improvement of the predicting accuracy.

Insufficient recovery of thymopoiesis predicts for opportunistic infections in allogeneic hematopoietic stem cell transplant recipients

Recovery of thymopoiesis after allogeneic hematopoietic stem cell transplantation is considered pivotal for full immune competence. However, it is still unclear to what extent insufficient recovery of thymopoiesis predicts for subsequent opportunistic infections and non-relapse mortality. A detailed survey of all post-engraftment infectious complications, non-relapse mortality and overall survival during long-term follow-up was performed in 83 recipients of allogeneic stem cell grafts after myeloablative conditioning. Recovery of thymopoiesis was assessed using analysis of signal joint T-cell receptor rearrangement excision circles. The impact of recovery of thymopoiesis at 2, 6, 9 and 12 months post-transplantation on clinical outcome beyond those time points was evaluated by univariate and multivariate Cox regression analyses. The cumulative incidence of severe infections at 12 months after transplantation was 66% with a median number of 1.64 severe infectious episodes per patient. Patients in whom thymopoiesis did not recover were at significantly higher risk of severe infections according to multivariable analysis. Hazard ratios indicated 3- and 9-fold increases in severe infections at 6 and 12 months, respectively. Impaired recovery of thymopoiesis also translated into a higher risk of non-relapse mortality and outweighed pre-transplant risk factors including age, donor type, and disease risk-status. These results indicate that patients who fail to recover thymopoiesis after allogeneic hematopoietic stem cell transplantation are at very high risk of severe infections and adverse clinical outcome.

Keratinocyte growth factor enhanced immune reconstitution in murine allogeneic umbilical cord blood cell transplant

Umbilical cord blood (UCB) is used increasingly as a source of hematopoietic cells because of a lower risk of graft-versus-host disease (GVHD). Myeloablative conditioning before allogeneic umbilical cord blood transplant (allo-UCBT) results in thymic epithelial cell injury and T-cell immune deficiency. Full-term fetal blood cells were used as hematopoietic cells in a previous murine allo-UCBT model with a limited number of mice surviving the myeloablative conditioning. We designed a viable murine allo-UCBT protocol with platelet concentrate support. Keratinocyte growth factor (KGF) is a mitogen of thymic epithelial cells that promotes recovery of thymic epithelium when given before total body irradiation (TBI)-containing conditioning in experimental murine models. We hypothesized that KGF pre-administration would improve post-allo-UCBT thymopoiesis. To test this hypothesis, allo-UCBT recipient mice were given KGF or control saline prior to UCBT. Platelet concentrate support significantly improved the survival rate of murine allo-UCBT recipients. KGF administration significantly increased donor-derived T and natural killer T (NKT) cells at day +35 in spleens of allo-UCBT recipients. KGF administration also improved thymic function after allo-UCBT, resulting in higher copies of signal joint T-cell receptor rearrangement excision circles (sjTRECs) in splenocytes. Finally, we found that KGF pre-administration could enhance the graft-versus-leukemia effect. In conclusion, KGF can be administered safely to recipients of allo-UCBT to enhance T-cell immune reconstitution.

Detection and quantification of the age-related sjTREC decline in human peripheral blood

The decline of signal joint T-cell receptor rearrangement excision circles (sjTRECs) in human peripheral blood has been demonstrated to be age-related, which can be a potential marker for individual age determination. However, little is known about the quantitative relationship between the levels of sjTREC and age. The aim of the present study was to investigate the levels of sjTREC in peripheral blood leukocytes (PBLs) among different age groups in Chinese population, so as to clarify whether it could serve as a suitable marker for biological age estimation in forensic practice. sjTREC levels were measured by real-time quantitative PCR analysis in peripheral blood samples from individuals of known age (n = 248). The quantification results showed that sjTREC declined in human PBLs in an age-dependent manner (r = -0.8177, P < 0.01). The formula for age estimation based on peripheral sjTREC decline was Y = -24.921x - 39.932 ± 10.47 (Y age, year; X log sjTREC/TBP; 10.47: standard error). Furthermore, there was no difference between males and females with regard to sjTREC levels. These results suggest that assessment of sjTREC in PBLs might be a valuable additional tool in age determination, especially in cases where traditional morphologic information is absent or inefficient in forensic practice.

T-Cell-Depleted CD34+ Cell Transplantation From an HLA-Mismatched Donor in a Low-Birthweight Infant With X-Linked Severe Combined Immunodeficiency

The best strategy of hematopoietic stem cell (HSC) transplantation for low-birthweight (LBW) infants with severe combined immunodeficiency (SCID) remains to be determined. To avoid the toxicity of drugs used for the transplantation and the risk of graft-versus-host disease (GVHD), the authors performed allogeneic bone marrow HSC transplantation with a combination of CD34 selection and T-cell depletion in a LBW infant with X-linked SCID. The authors analyzed the process of T-cell reconstitution after the transplantation in this patient. The patient was born at 30 weeks and 2 days' gestational age via cesarean section. He was diagnosed as having SCID at birth. The patient received a transplant of 1 million CD34+ cells/kg body weight. Immunologic reconstitution was investigated by means of phenotypic analysis of T cells and genetic analysis of coding joint T-cell receptor rearrangement excision circle expression. Increases in donor-derived NK cells and T cells were observed 2 and 3 months after the transplantation, respectively. The patient had no infectious complications or GVHD despite the presence of SCID and prematurity-associated immunodeficiency. Analysis of T-cell regeneration pathways revealed that T cells reconstituted mainly via the thymus-dependent pathway. T-cell-depleted CD34+ cell transplantation would be a safe and useful therapy for LBW infants with SCID.

Naïve T Cell Level and TCR Vβ Repertoire Usage in Patients with Chronic Myelogenous Leukemia.

During early T lymphopoiesis, rearrangement of the V, D and J segments of the TCR genes result in deletion of the intervening chromosomal DNA and formation of circular excision circles, the so-called signal joint T-cell receptor rearrangement excision circles (sjTRECs, TRECs). TRECs are assumed to have a high stability, and can not multiply and consequently are diluted during T cell proliferation. It has been used to evaluate thymic function in T-cell immune reconstruction after treatment in HTLV-I-infected or stem cell transplantation. Defects of cellular immunity, however, may also play a role in hematologic malignancies. On the other hand, examination of T cell receptor (TCR) gene repertoire is important to analysis the immune status of the patients with malignant neoplasms, because clonal expansion of T cells permit the identification of specific antigen response of T cells. Little is known about the feature of T-cell immune state in CML. In order to evaluate the thymic recent output function and the expansion feature of TCR V beta subfamily T cells in patients with chronic myelogenous leukemia (CML in chronic phase), TRECs level and TCR V beta repertoire usage and clonality were analyzed. Quantitative detection of TRECs in DNA of peripheral blood mononuclear cells from 20 cases with CML, purified CD4+ or CD8+ cells from 3 cases with CML were preformed by real-time PCR (TaqMan) analysis. And the TRECs-number was related to the number of T-cells by determination of the number of CD3-positive cells. The expression and cloanlity analysis of TCR V beta repertoire were detected by RT-PCR and genescan technique in PBMC from the 14 out of 20 patients. 9 normal individuals served as controls. A dramatic reduction of TRECs values in patients with CML was showed. The mean value of TRECs was 0.06±0.16 copies /1000 T cells in CML patients and 6.84±4.71 copies /1000 T cells in normal individuals, respectively (p<0.01). In 16 out of 20 CML cases no TRECs copies could be detected in peripheral blood, and lower value of TRECs could be identified in sorted CD4+ or CD8+ cells. The expression of 1–12 V beta subfamilies was found in samples from 14 patients. Clonal expanded T cells from 13 cases could be identified in some V beta subfamilies, which were preferentially used in V beta 3, V beta 10, V beta 19, V beta 21 and V beta 22 subfamilies. In conclusion, this is, to our knowledge, the first description of TRECs level in CML patients. These results showed a prominent reduction of TREC levels in CML. The predominant usage and clonal expansion of TCR Vβ subfamily T cells could be identified in CML patients, indicating the host could have the ability for specific immune response to leukemia associated antigen, in despite of T cell immunodeficiency was showed in patients.

Both age and gender affect thymic output: more recent thymic migrants in females than males as they age.

The thymus undergoes age-associated involution, with studies showing thymic size decreasing from birth at a rate of approximately 3% per year until middle age, and at a rate of 1% per year thereafter. The aim of this study was to determine the effect of thymic atrophy on T-lymphocyte production by the thymus, and to clarify the ongoing uncertainty regarding gender differences in thymic function. We quantified recent thymic emigrants (RTEs) in blood through the measurement of signal joint T-cell receptor rearrangement excision circles (sjTRECs), and showed that the decline in the number of RTEs in the blood with increasing age is gender-linked. Peripheral blood from females contained significantly higher levels of sjTRECs per CD3+ T cell than blood from males (P = 0.002), despite there being no significant gender difference in the absolute number of CD3+ T cells in the populations analysed (P > 0.10). Our findings suggest better thymic function in females compared with males, providing females with a higher number of recent thymic emigrants for longer periods of life. Such a finding provides a plausible explanation for the immunological gender differences observed in previous studies and possibly, for the general longer life expectancy in females compared with males.