Recent Thymic Output Function Research Articles

Age-Related Immune Profile of the T Cell Receptor Repertoire, Thymic Recent Output Function, and miRNAs.

Background T cell immunity plays a central role in the body's defense system, including maintaining homeostasis and preventing tumorigenesis and viral infection. Immune system functions degenerate with age, leading to immune senescence. Physiologically, immune senescence is characterized by a decrease in T cell receptor diversity, naive T cell deficiency, and alterations in T cell immune-related miRNAs. However, little is known about the characteristics of T cell immunosenescence in Chinese individuals. Results A significant decrease in the miR-17, miR-92a, and miR-181a levels in PBMCs was detected with age. The miR-92a and miR-181a levels were upregulated in CBMCs when comparing healthy individuals to group I (0~9 years), whereas miR-17 was downregulated. The sjTREC level in PBMCs was negatively correlated with age, and a sharp decrease in sjTRECs was found between groups I and II (10~19 years). Twenty-four TCR Vβ subfamilies could be detected in most samples, and most displayed polyclonality, while skewed expression of the Vβ subfamilies as well as an increased oligoclonal tendency was found with age. Similarly, the frequencies of the TCR Vγ and Vδ subfamilies decreased with age, and the alteration in clonality appeared to be stable at different ages. Conclusion We made the novel observation of T cell immunosenescence with age in Chinese individuals, which may provide information for immune targets to enhance the T cell immune response in immunotherapy settings for elderly patients.

The feature of clonal expansion of TCR Vβ repertoire, thymic recent output function and TCRζ chain expression in patients with immune thrombocytopenic purpura

In order to identify the feature of T cells immune status in idiopathic/immune thrombocytopenic purpura (ITP) patients, TCR Vbeta repertoire, T-cell receptor (TCR) excision DNA circles (TRECs) and TCRzeta chain gene expression were examined. Reverse-transcriptase polymerase chain reaction, genescan and real-time PCR techniques were used to analyze. DNA and cDNA from peripheral blood mononuclear cells (PBMCs) of 18 ITP patients were investigated and 25 normal individuals served as control. The results showed that only 4-11 Vbeta subfamilies could be detected in each ITP case. The most frequently expressed Vbeta subfamilies were Vbeta2 and Vbeta3, while 11 Vbeta subfamilies were absent. Clonal expansion of Vbeta T cells were found in all patients. The most frequent clonal expansion T cell was Vbeta21. The number of TRECs in ITP patients (2.60 +/- 2.99 copies/1000 PBMCs) was not significant different, compared with control (3.76 +/- 3.42 copies/1000 PBMCs). The expression level of TCRzeta gene was significantly lower in ITP samples than those in control (P = 0.017). In summary, T cells in ITP patients were characterized by restricted expression of TCR Vbeta subfamilies and clonal expansion predominant in Vbeta21. The alteration of peripheral TCR Vbeta repertoire pattern may not relate to the thymic, recent output function in ITP patients. Our report is the first description of decreased TCRzeta mRNA level in the majority of ITP patients.

Decreased T‐cell receptor excision DNA circles in peripheral blood mononuclear cells among benzene‐exposed workers

Benzene is a volatile aromatic hydrocarbon solvent which is widely used in many industries. The chronic exposure of humans to benzene in the workplace has been associated with blood disorders, as well as toxicity in lymphopoiesis, including aplastic anaemia and leukaemia. However, the mechanisms of benzene-induced haematotoxicity and leukaemogenesis remain unclear. In this study, we investigated the level of T-cell receptor excision DNA circles (TRECs) in peripheral blood mononuclear cells (PBMCs) in benzene-exposed workers. This would therefore be considered as a potential marker for estimates of thymic output and an evaluation of the content of naïve T-cells. It is hoped that the data will bring a comprehensive understanding on the influence of benzene exposure in the host T-cell immune function. Quantitative detection of TRECs in DNA of PBMCs from benzene-exposed workers was preformed by real-time polymerase chain reaction using the TaqMan technique. The benzene-exposed workers were divided into four groups, and 27 normal individuals were served as controls. The result indicated that the TRECs levels of all benzene-exposed groups were significantly decreased as compared with those of controls. In conclusion, the recent thymic output function and the T-cell immune function were apparently impaired in workers after benzene exposure.

The Feature of TCR Vβ Repertoire, Thymic Recent Output Function and TCR-zeta Chain Expression in Patients with Immune Thrombocytopenic Purpura.

Chronic idiopathic/immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet antibodies induce destruction of platelets. T cells may play a critical role in controlling the synthesis of autoantibodies, and may relate to initiation of ITP. In order to identify the feature of T cells immune state in patients with ITP, TCR Vβ repertoire which can identify T cell proliferation in response to antigenic stimulation; signal joint T-cell receptor excision DNA circles (TRECs) which can use to estimate the thymic recent naïve T cells output function and the expression level of TCR ζ chain gene which defined as an important factor in T cells signaling. Specific Vβ family primers, RT-PCR and genescan technique were use to analyze the expression of TCR Vβ subfamily and the clonality of TCR Vβ T cells in cDNA form peripheral blood mononuclear cells (PBMCs) of 5 ITP patients; quantitative analysis of TRECs in DNA of PBMCs from 15 cases were preformed by real-time PCR (TaqMan); real-time PCR with SYBR Green I technique was used for detecting TCR ζ gene expression level in cDNA of PBMCs from of 18 patients, β2 -microglobulin gene was used as an endogenous reference, relative changes in TCR ζ chain gene expression level were used by the 2− ΔCt method. 25 normal individuals served as control. The results showed that1.only 4–11 Vβ subfamily T cells could be identified in ITP cases. The frequent expression Vβ subfamilies were Vβ2 (100%), Vβ3 (100%), Vβ19 (80%) and Vβ21 (80%), while it could not detected the expression of Vβ4, Vβ6–12, Vβ17, Vβ20 and Vβ24. However, all 24 Vβ subfamilies could be detected in samples from normal individuals and all of them displayed polyclonality, clonal expansion Vβ T cells could be found in some subfamilies in every patient. The frequent clonal expansion T cells were Vβ21 subfamily which could be identified in 4 out of 5 cases.2.The mean value of TRECs was 2.60±2.99 copies/1000 PBMCs in ITP patients, it was not significantly difference (p>0.05) in comparison with normal group (3.76±3.42 copies/1000 PBMCs), although the TRECs level displayed changefully in different patients with ITP. In four out of fifteen ITP cases no TRECs copies could be detected in more than 10000 PBMCs. The higher frequency of TRECs was found in female group, however it was not statistical significance in comparison with male group.3.The expression of TCR ζ chain gene could be detected in all of 25 normal samples, and TCR ζ gene was found in 17 out of 18 cases with ITP.However, the expression level of TCR ζ gene was lower in ITP samples than those from normal individuals (P=0.017). The expression level of TCR ζ gene is nonsignificat age-associated or gender-link in ITP patients. In conclusions, the restricted expression of TCR Vb subfamilies and clonal expanded Vβ T cells are the T-cell immune feature in ITP, and the frequent presentation of clonal expanded Vβ21 T cells may relate to the disorder of cellular immune function in ITP. At least, in most cases with ITP, the normal level of naïve T cells and thymic recent output function were detected. It is, to our knowledge, the first description in the expression feature of TCR ζ gene in ITP patients, which displayed down expression, it might be a feature in autoimmune disease.

The Molecular Characteristics in CDR3 of TCR Vα and Vβ Genes Associated with cGVHD in Patients after Allogeneic Hematopoietic Stem Cell Transplantation.

Successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) requires reconstitution of normal T-cell immunity. Chronic graft-versus-host disease (cGVHD) is one of the major complications following allo-HSCT. The poor reconstitution of T-cell immunity (including the reconstitution of recent thymic output function and T-cell receptor (TCR) repertoire) was associated with cGVHD. In the previous study, we found that cGVHD predicted low TCR rearrangement excision circles (TRECs) levels and slow naïve T-cell recovery. Because GVHD displayed as clonal proliferation of special T-cells clones, which was triggered by donor T cells to recognize the host's allogene antigen, in the present study we analyzed the TCR Vα and Vβ repertoire and cloanlity in patients with cGVHD, in order to find the special T-cell clones associated with cGVHD and evaluate the molecular characteristics of the CDR3 of TCR Vα and Vβ repertoire of GVHD-associated T-cell clones. Peripheral blood mononuclear cells (PBMNCs) were obtained from 5 leukemia patients with cGVHD after allo-HSCT. The expression and cloanlity analysis of TCR Vα and Vβ repertoire were detected by RT-PCR and genescan technique. Six donors served as controls. Almost all of TCR Vα and Vβ repertoire with polyclonal pattern were identified in normal controls. However, the skew expression pattern of TCR Vα and Vβ repertoire could be detected in patients with cGVHD even more than 4 year after allo-HSCT. Among 29 Vα and 24 Vβ subfamilies, there were only 4∼12 Vα and 4∼11 Vβ subfamilies expressed in patients with cGVHD. Oligoclonal or monoclonal expanded T cells were identified in TCR Vα 2, 3, 6, 10, 12, 14, 15, 25, 26 and TCR Vβ 1, 3, 7∼9, 13, 17, 19, 20 subfamilies respectively. The CDR3 sequences were further analyzed and all the sequences were blasted by internet (http://www.ncbi.nlm.nih.gov) and confirmed that it belonged to specific TCR Vα or Vβ gene rearrangement. The lengths of CDR3 were ranged from 12 to 15 amino acids. The molecular characteristics of the CDR3 of TCR Vα and Vβ genes rearrangement were TCRVα 3 (new name: Vα 17*01)-N-Jα 48*01-Cα (motif: CATEVDFGNEKLIF), TCRVα 2 (new name: Vα 12–2*01)-N-Jα 20*01-Cα (motif: CAVNLNDYKLIF), TCRVβ 1 (new name: Vβ 9*01)-N-Dβ 2*01-N-Jβ 2–1*01-Cβ 2 (motif: CASSDPPETYNEQFF), TCRVβ 7 (new name: Vβ 4–3*01)-N-Dβ 1*01-Jβ 1–1*01-Cβ 1 (motif: CASSHESGNTEAFF). Some TCR subfamily genes shared similarity in CDR3 amino acid motif. The role of specific sequences of CDR3 of TCR Vα and Vβ repertoire and T-cell clones will be confirmed in vivo by animal models.

TRAV and TRBV repertoire, clonality and the proliferative history of umbilical cord blood T-cells

Umbilical cord blood (CB) has been used successfully as a source of hematopoietic stem cells for transplantation. But the distribution and clonality of T-cell receptor alpha variable region ( TRAV) and T-cell receptor beta variable region ( TRBV) subfamily T-cells, the naïve T-cells level and the diversity of thymic recent output function in CB has not been yet clearly defined. In order to characterize the repertoire of CB T-cells, the CDR3 of 29 TRAV and 24 TRBV subfamily genes were analyzed in T-cells from 12 cord blood samples, using RT-PCR and genescan technique. To determine the proliferative history of CB T-cells, quantitative analysis of δRec-ψJα signal joint T-cell receptor excision circles (sjTRECs) was performed in mononuclear cells, CD3+, CD4+ and CD8+ T-cells from 20 CB samples by real-time PCR. In addition the analysis of 23 TRBV– TRBD1 sjTRECs in 10 cases of CB CD4+ T-cells and CB CD8+ T-cells was performed by semi-nested PCR. We found a marked restriction of TRBV expression pattern in CBMCs compared to peripheral blood mononuclear cells (PBMC), which expressed all 24 TRBV genes. All PCR products of TRAV and TRBV subfamilies from CB, except for 3 cases, displayed polyclonal rearrangement pattern. The δRec-ψJα sjTRECs counts were significantly higher in CB, than in PB samples. Also the number of detectable TRBV sjTRECs was higher in CB than in peripheral blood. In conclusion, our results indicate polyclonal but restricted repertoire and a very short proliferative history of CB T-cells. The incomplete repertoire and naivety of CB T-cells might be the reason that CB hematopoietic stem cells transplant recipients are less likely to develop graft vs host disease.

Effects of lead exposure on thymic output naive T cells function

To investigate the levels of T cell receptor rearrangement excision DNA circles (TRECs) within peripheral blood from workers exposed to lead, and thereby to evaluate the number of naive T cells and recent thymic output function. Quantitative detection of TRECs in peripheral blood mononuclear cells (PBMNC) from 10 cases of workers exposed to lead was performed by real time PCR analysis. 11 workers without exposure to lead served as unexposed controls. In addition, the relationship between TRECs, age, length of service, blood lead, urea lead, blood ZPP and urea delta-ALA was investigated. The mean value of TRECs in workers exposed to lead was (2.44 +/- 1.87)/1000 PBMC, significantly under (5.60 +/- 3.96)/1000 PBMC in unexposed controls. A significant negative correlation was found between the TRECs and urea-ALA. But there was no significant correlation between them after controlling for blood lead, urea lead. Lead exposure may damage thymic output naive T cells function. Furthermore, low-level exposure to lead may damage immune system and earlier than expected.

The Feature of δRec-ψJα sjTRECs Level and Frequency of 23 TCR Vβ-Dβ1 sjTRECs in Mononuclear Cells, CD4+ and CD8+ T Cells from Cord Blood and Peripheral Blood of Normal Individuals.

Thymic recent output function is characterized its importance of thymus to T-cell diversity in the periphery of both children and adults. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCR α and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs), which is considered as a very valuable tool to estimate thymic function. Quantitative of δRec-ψJα sjTRECs can direct evaluate the recent thymic output function, but it is unable to analyze the particular thymic output function of different TCR Vβ subfamily naive T cells. The complexity of TCR Vβ repertoire is an important factor for immune reconstitution, quantitative analysis of series TCR Vβ-Dβ sjTRECs could be used to evaluate the levels of different Vβ subfamily naive T cells.In the present study, quantitative analysis of δRec-ψJα sjTRECs was performed in mononuclear cells, CD3+, CD4+ and CD8+T cells from peripheral blood of normal individuals and cord blood by real-time PCR(TaqMan). And the analysis of 23 TCR Vβ-Dβ1 sjTRECs was performed by semi-nested PCR. Different amounts of DNA (corresponding to 2*105, 5*104, 1*104 and 1*103 cells respectively) from all samples were amplified to estimate the frequency of TCR Vβ-Dβ sjTRECs.The mean value of δRec-ψJα sjTRECs was detected in 4.10±3.65/1000 PBMCs, 6.37±5.28/1000 CD3+cells, 3.28±1.24/1000 CD4+cells, 4.67±3.63/1000 CD8+cells from normal individuals (n=14) and 35.59±47.56/1000 CBMC, 71.48±86.42/1000 CD3+cells, 41.02±32.9/1000 CD4+ cells, 52.05±52.32/1000 CD8+cells from cord blood (n=9) (p=0.0208, p=0.0096, p=0.0003, p=0.0026, respectively).A part of Vβ subfamily sjTRECs could be detected in all samples from cord blood (Vβ2, 3, 4, 5, 10, 13, 14, 15, 19 and 22) and peripheral blood (Vβ10, 13 and 14) at 5*104 cells level, some of Vβ subfamily sjTRECs could be detected in 1*103 cells level. The frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration. The number of detectable Vβ subfamily sjTRECs was 22.00±0.94/2×105, 18.8±1.87/5×104, 10.40±2.99/1×104 and 0.78±1.39/1×103 CBMCs, as compared with 18.70±2.45/2×105 (p=0.002), 13.7±2.67/5×104 (p<0.001), 5.5±2.07/1×104 (p=0.001) and 0.50±0.71/1×103 (p=0.739) in PBMCs from normal individuals. Similar results were found in CD4+ and CD8+ T cells which were sorted from both CBMCs and PBMCs, the number of detectable Vβ subfamily sjTRECs was 13.90±2.38/1×104 CD4+cells, 11.5±1.96/1×104CD8+cells from cord blood and 5.6±2.68/1×104 CD4+cells (p<0.001) and 8.2±2.57/1×104CD8+cells (p>0.005) from normal individuals. The results indicate that the number of detectable sjTRECs of Vβ subfamilies and the frequencies of most Vβ-Dβ1 sjTRECs in normal PBMCs, CD4+ and CD8+T cells were obviously lower than those in cord blood.In conclusions, the results provide the base data of naïve T cells levels and thymic recent output function in cord blood and peripheral blood of normail individuals in chinese.

Analysis of the Recent Thymic Output Function of 23 TCR Vβ Subfamily Naïve T Cells in Patients with AML.

Regeneration of the T-cell population proceeds normally along two different pathways, in which the thymic-dependent pathway accounts for the more durable reconstitution of the T-cell compartment, and generates a more diverse TCR repertoire. Thus thymus function serves as a direct index to understand cellular immune function and potential of long-term TCR Vβ repertoire reconstitution. The complexity of TCR repertoire is generated in the thymus by regular recombination of series of gene fragments of TCR α or β chains. During these process, by-products of rearrangements are generated in the form of signal joint T-cell receptor excision DNA circles(sjTRECs), as sjTRECs are stable extrachromosomal annular DNA, are not replicated while cell dividing, and their existence suggests functional TCR generation. Thus thymic function can be evaluated by measuring sjTRECs in peripheral blood. At present, the total recent thymic output function is evaluated by quantitating δRec-ψJα sjTRECs, but it can not evaluate particular thymic emigrants of different Vα or Vβ subfamily naïve T cells. As TCR Vα and Vβ naïve T cells include many subfamilies which play different immune roles, and the complexity of TCR repertoire is an important factor for cellular immune reconstitution.Our previous studies had showed that the recent thymic output function in patients with AML was significant decrease by quantitative detection of δRec-ψJα sjTRECs. To further estimate the recent thymic emigrants of different TCR Vβ subfamily naïve T cells, this study was designed to detect the existence of 23 TCR Vβ subfamily sjTRECs in peripheral blood mononuclear cells (PBMCs) by using 23 Vβ subfamily special primers (including 2Dβ1 sense primers and 23 Vβ subfamily antisense primers). TCR 23 Vβ-Dβ 1 sjTRECs were separately amplified in genomic DNA from 5×104 and 1×104 PBMCs of samples (10 cases of normal individuals and 32 cases of the different FAB subtypes of AML patients) to estimate the frequencies of TCR 23 Vβ-Dβ1 sjTRECs by using semi-nest PCR.The results indicated that the frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration, and the higher cellular concentration the higher frequency of Vβ subfamily sjTRECs. The number of detectable Vβ subfamily sjTRECs was (5.09±3.28) and (2.59±2.06) in 5×104 and 1×104 PBMCs from AML patients, as compared with (13.7±2.67) and (5.50±2.07) from normal individuals, and the differences were significant (both p=0.000). About 5/23(22%) of Vβ-Dβ1 sjTRECs were detected in 5×104 PBMCs from AML patients, and the frequencies of 13 Vβ subfamily sjTRECs (including Vβ1,Vββ,Vβ3,Vβ4,Vβ5, Vβ9,Vβ10,Vβ12,Vβ13,Vβ14,Vβ17,Vβ22,Vβ24-Dβ1 sjTRECs) were significantly lower than those from normal individuals, among which the lowest were Vβ10 and Vβ14-Dβ1 sjTRECs, the most frequency was Vβ21. But the difference was not significant within the different FAB subtypes of AML patients. It was negative correlation between age and the number of detectable Vβ subfamily sjTRECs in patients with AML, and patients who were < 30 years tended to be higher number of detectable Vβ subfamily sjTRECs than those ≥ 30 years, which became significant at 5×104 PBMCs level (r=−0.481, p=0.005). Taken together, the recent thymic emigrants of 23 Vβ subfamily naïve T cells were absent to a different extent or lower level among patients with AML. These results suggested that AML patients had severe cellular immunodeficiency and the capacity and potential of long-term TCR Vβ repertoire reconstitution were dramatically lowered.

Changes in Thymic Recent Output Function in Patients with B-Cell Lymphocytic Malignancy.

Since 1998 the use of signal joint T cell receptor excision circles (sjTRECs) to study changes in the frequency of recent thymic emigrants was reported, this study drew attention to the fact that the level of sjTRECs can be used to estimate the recent thymic output function. Therefore, sjTRECs was used as a new marker for analysis of thymic output function in different immunodeficiency diseases, immune reconstitution in patients after stem cell transplantation. Defects of cellular immunity, however, may also play a role in hematologic malignancies. Little is known about the feature of T-cell immune state in B-cell lymphocytic malignancy. In order to identify number of naïve T-cells in B-cell lymphocytic malignancy, sjTRECs-content was determined in the mononuclear cell fraction and the sjTRECs-number was related to the number of T-cells (according to the number of CD3-positive cells). Quantitative analysis of sjTRECs in DNA of peripheral blood T cells from 45 cases with B-cell lymphocytic malignancy (including 17 cases with ALL, 4 cases with CLL, 15 cases with B-NHL and 6 cases with MM) were preformed by real-time PCR and TaqMan analysis, and 14 normal individuals as controls. The results showed a dramatic reduction of sjTRECs values in patients. Some cases no sjTRECs copies could be detected in 40000 T cells. The mean value of sjTRECs was 0.39±0.75 copies/1000 PBMCs in ALL, 0.11±0.15 copies/1000 PBMCs in B-CLL, 0.67±1.39 copies/1000 PBMCs in B-NHL, 0.91±1.16 copies/1000 PBMCs in MM patients, as compared with 4.1±3.65 copies/1000 PBMCs in normal individuals, the sjTRECs level in all goups of B-cells lymphocytic malignancy were significant decrease (p=0.0001, p=0.048, p=0.002) except MM group (p=0.053). However, when comparison of the sjTRECs value in CD3+ compartments, it was not statistically different from the sjTRECs counts between patients with ALL (3.91±7.20 copies/1000 CD3+cells) and normal individuals (6.36±5.28 copies/1000 CD3+cells) (p=0.2139), suggesting that the reduction of sjTRECs level in patients wit ALL may due to lower numbers of T cells in peripheral blood. In conclusions, the results suggest that some thymic dysfunction in T cell generation was detected at least in most patient with B-cell lymphocytic malignancy. However, whether this is due to clonal expansion of T-cells to antigens, or reflects impairment of immune function associated to the malignancy, remains an open question. In a prospective study sjTRECs levels will be determined in different T- cell compartments including CD45RA+, CD4+ and CD8+ cells and so on.

The Recent Thymic Output Function Might Relate with BCR-ABL mRNA Level in Patients with CML.

Objective In order to analyze the relationship between the content of T-cell receptor excision DNA circles (TRECs) and BCR-ABL mRNA levels, evaluating the prognostic significance of thymic recent output function monitoring in patients with chronic myelogenous leukemia (CML).Methods Quantitative detection of TRECs and BCR-ABL fusion gene transcripts in peripheral blood from 15 CML patients were preformed using real-time PCR technique. And the TREC-number was related to the number of T-cells by determination of the number of CD3-positive cells. The change of BCR-ABL level in 6 CML patients was followed-up for two years.Results There was no significant correlation between TRECs and BCR-ABL mRNA in peripheral blood from CML patients at the first diagnose. Patients who had higher TRECs at diagnosis had a larger reduction of BCR-ABL level after 2 years of follow-up. While in 2 patients who underwent haemopoietic stem cell transplantation(HSCT), BCR-ABL in one patient with higher pre-transplantation TRECs value became undetectable with three consecutive detections during the first year post transplantation, but low level of BCR-ABL could be identified in the other patient.Conclusion High thymic output function in CML patients could be helpful for anti-residual CML cells.

Detection of sjTRECs within Peripheral Blood T Cells Evaluation of Recent Thymic Output Function in Myelodysplastic Syndrome.

ObjectiveThe myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders, while, immunological abnormalities are frequently observed in patients with MDS. Several reports revealed that about 10% of MDS patients have clinical autoimmune disorders like skin vasculitis, rheumatic disease, or autoimmune hemolytic anemia. Furthermore, serological immunological abnormalities like hyper- or hypogammaglobulinemia, positivities of antinuclear antibody, positivities of direct Coombs test, or inverted CD4/8 ratios were found in 18–65% of patients with MDS. Recently immunosuppressive therapies including prednisolone, antithymocyte globulin, and cyclosporin A (CsA) are used to treat cytopenia in some patients with MDS. But it isn't very clear the immunosuppressive mechanism in MDS and the value of the treatment. To analyze the content of signal joint Tcell receptor excision DNA circles signal joint T cell receptor excision DNA circles(sjTRECs) within peripheral blood mononuclear cells (PBMCs), thereby to infer the level of naive T cells and the recent thymic output function in patients with myelodyspoastic syndrom. Methods Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individuals and 8 patienets were performed by real-time polymerase chain reaction (PCR) and TaqMan technique.ResultsThe median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal individuals whereas it was1.07 ±1.40 copies P1 000 PBMCs in myelodysplastic syndrom patients (P <0. 05). Conclusions MDS Patients decrease in recent thymic output function.

T Cell Receptor Excision Circles (TRECs) Decrease in Patients after Allogeneic Stem Cell Transplantation.

Human thymus is required for establishment of a T-cell pool in fetal life, T-cell emigration from thymus (thymic recent emigrants [TRECs]) is a continuous thymic-dependent process. To analyze the content of signal joint Tcell receptor excision DNA circles signal joint T cell receptor excision DNA circles(sjTRECs) within peripheral blood mononuclear cells (PBMCs), thereby to infer the level of naive T cells and the recent thymic output function in patients with allogeneic stem cell transplantation. We used real-time polymerase chain reaction (PCR) to quantify SjTRECs in 5 patients with chronic myeloid leukemia-chronic phase. Five patients(4 males, 1 females; median age 37 years,) who underwent HLA-matching sibling BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department. Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individuals. The median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal individuals whereas it was 0.57±0.51 copies P1 000 PBMCs in patients at least two years after allogeneic stem cell transplantation (P < 0. 03). We conclude that these Patients decrease in recent thymic output function,. Therefore, measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution after transplantation.

Detection of 24 TCR Vβ-Dβ1 sjTRECs in T Cells from Cord Blood, Peripheral Blood of Normal Individuals and Patients with AML-M2.

Thymic function is characterized its importance of thymus to T-cell diversity in the periphery of both children and adults during both health and disease. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCRα and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs). As sjTRECs are stable extrachromosomal DNA fragments, are not replicated during mitosis and thus diluted with each round of cell division, and are therefore most frequent in naïve T cells that have recently left the thymus, their quantification is actually considered as a very valuable tool to estimate thymic function. Quantitative of δRec-ψJα sjTRECs can direct evaluate the recent thymic output function, but it is unable to analyze the particular thymic output function of different TCR Vβ subfamily naive T cells. The complexity of TCR Vβ repertoire is an important factor for immune reconstitution, it should be established the quantitative analysis of series TCR Vβ-Dβ sjTRECs to evaluate the levels of different Vβ subfamily naive T cells. In the present study, analysis of 24 TCR Vβ-Dβ sjTRECs was established by semi-nested PCR using 24 Vβ subfamily antisense primers and 2 Dβ1 sense primers. TCR Vβ-Dβ sjTRECs were amplified in genomic DNA from mononuclear cells of 10 cord blood samples, 10 cases of peripheral blood from normol individuals and 11 cases with AML-M2. Different amounts of DNA (corresponding to 2*105, 5*104, 1*104 and 1*103 cells respectively) from all samples were amplified to estimate the frequency of TCR Vβ-Dβ sjTRECs. The results showed that most Vβ subfamily sjTRECs could be detected in all samples from cord blood and peripheral blood at 2*105 or 5*104 cells level, some of Vβ subfamily sjTRECs could be detected in 1*103 cells level. Whereas the frequency of Vβ subfamily sjTRECs were lower in peripheral blood T cells from patients with AML-M2 than in normal individuals (p<0.05). Vβ19, Vβ23 and Vβ24 subfamily sjTRECs could not be detected in all samples at 5*104 cells level. The results indicated that Vβ subfamily naive T cells could be detected with different frequency in peripheral blood of normaol individuals as well as in some patients with AML-M2. Lower frequency of Vβ subfamily naive T cells was found in most AML-M2 patients.

Recent thymic output function in patients with hematological malignancy

Thymic function is important for the generation of T-cell diversity in the periphery of both children and adults during both health and disease. Until recently, thymic function could not be monitored, as a consequence of the absence of adequate technology to differentiate recent thymic emigrants (RTEs) from naïve T cells. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCR α and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs). As sjTRECs are stable extrachromosomal DNA fragments, they are not replicated during mitosis and thus diluted with each round of cell division, and are therefore most frequent in naïve T cells that have recently left the thymus, their quantification is actually considered as a valuable tool to estimate thymic function. Therefore, quantitative sjTRECs content have been recently used to assess thymic output during both health and disease. In this review, we summarize recent data on the recent thymic output function feature in patients with hematological malignancy and the immune reconstitution after stem cell transplantation and we also characterize factors that may improve the thymic output function.

The Significant Decrease of Recent Thymic Output Function in Patients with Benzene-Poisoned Aplastic Anemia.

It has been known for many years that benzene causes hematotoxicity, as well as the toxicity in lymphopoiesis, and it associated with aplastic anemia and leukemia. The aim of the present study was to investigate the level of T-cell receptor excision DNA circles (TRECs) within peripheral blood mononuclear cells (PBMCs) in patients with benzene-poisoned aplastic anemia (AA), thereby to evaluate the content of naive T cells and the recent thymic output function. Quantitative detection of TRECs in DNA of PBMCs from 16 normal individuals and 7 cases with benzene-poisoned AA was preformed by real-time PCR using TaqMan technique. The results showed that TRECs level was 6.69±4.79/1000 PBMCs in normal individuals, it was significant decrease in patients with benzene-poisoned AA (2.24±1.57/1000 PBMCs, p<0.05). The TRECs levels in 2 cases with benzene-poisoned AA were followed up in different time points up to 55 weeks. The TRECs level was persistent decrease after diagnosis of benzene-poisoned and leaving the benzene exposure workplaces and undergoing clinic specific treatment, even if peripheral blood cell counts were became normal levels. The TRECs levels were 1.35±0.87/1000 PBMCs (at 5 time points) and 0.61±0.45/1000 PBMCs (at 4 time pionts) in two cases respectively. The results indicated that the recent thymic output function was remarkable decrease in patients with benzene-poisoned AA. It may obviously damage the T cell immune function in benzene poisoning.

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.