Signal Joint T-cell Receptor Excision Research Articles

Selection of Stem Cells by Using Antibodies That Target Different CD34 Epitopes Yields Different Patterns of T-Cell Differentiation

The objective of this study was to compare the patterns of T-cell differentiation from CD34(+) human stem cells selected with different classes of antibody targeting the CD34 molecule. We compared signal-joint T-cell receptor excision circle (sjTREC) production in thymocytes selected with different classes of anti-CD34 antibody. Based on these results, we studied immune reconstitution in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice using human stem cells selected with the same antibodies that yielded variation in the thymocytes. Human CD34(+) stem cells were immunomagnetically selected using the class II QBEnd antibody (prevalent in clinical graft engineering) and the class III 8G12 antibody (common in diagnostic tests). Engraftment and T-cell reconstitution were examined after transplantation. Thymocytes selected with the 8G12 class III antibody have a higher TREC production than those selected with the QBEnd class II antibody. Of mice transplanted with cells selected using the 8G12 antibody, 50% had sjTREC production, compared with 14% of mice transplanted with cells selected using the clinically common antibody QBEnd. 8G12 thymic progenitors are characterized by higher quality in thymic distribution and higher activity in T-cell differentiation. Using class III antibody targeting the CD34 molecule resulted in increased T-cell reconstitution in the NOD/SCID mouse. Use of a single antibody epitope targeting the CD34 molecule may lead to loss of cells that might provide richer T-cell reconstitution. Use of different or multiple epitopes, targeting of alternate stem cell markers, or use of cell-depletion strategies might prevent this loss.

Evaluation of CD8+ T-cell and antibody responses following transient increased viraemia in rhesus macaques infected with live, attenuated simian immunodeficiency virus

In vivo depletion of CD8+ T cells results in an increase in viral load in macaques chronically infected with simian immunodeficiency virus (SIVmac239deltanef). Here, the cellular and humoral immune responses associated with this transient period of enhanced viraemia in macaques infected with SIVmac239deltanef were characterized. Fourteen days after in vivo CD8+ T-cell depletion, two of six macaques experienced a 1-2 log10 increase in anti-gp130 and p27 antibody titres and a three- to fivefold increase in gamma interferon-ecreting SIV-specific CD8+ T cells. Three other macaques had modest or no increase in anti-gp130 antibodies and significantly lower titres of anti-p27 antibodies, with minimal induction of functional CD8+ T cells. Four of the five CD8-depleted macaques experienced an increase in neutralizing antibody titres to SIVmac239. Induction of SIV-specific immune responses was associated with increases in CD8+ T-cell proliferation and fluctuations in the levels of signal-joint T-cell receptor excision circles in peripheral blood cells. Five months after CD8+ T-cell depletion, only the two high-responding macaques were protected from intravenous challenge with pathogenic SIV, whilst the remaining animals were unable to control replication of the challenge virus. Together, these findings suggest that a transient period of enhanced antigenaemia during chronic SIV infection may serve to augment virus-specific immunity in some, but not all, macaques. These findings have relevance for induction of human immunodeficiency virus (HIV)-specific immune responses during prophylactic and therapeutic vaccination and for immunological evaluation of structured treatment interruptions in patients chronically infected with HIV-1.

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.

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.

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.

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.

Hematopoietic stem cell dose correlates with the speed of immune reconstitution after stem cell transplantation

AbstractIn the current study, we tested whether higher numbers of hematopoietic stem cells correlate with the speed of immune reconstitution in a congenic transplantation model (C57BL/Ka, CD45.1, Thy1.1→C57BL/6, CD45.2, Thy1.2) using purified hematopoietic stem cells (c-Kit+Thy1.1lowLin-/lowSca-1+). There were 3 different doses of stem cells used (400, 1000, and 5000). Phenotypic analyses in peripheral blood and spleen demonstrated that higher numbers of infused stem cells are associated with more rapid regeneration of T cells (CD4+, CD8+, naive CD4+, naive CD8+) and B cells at early time points. The numbers of T and B cells eventually became equivalent between different dose groups at late time points. Production of interleukin-2 and inter-feron-γ per T cell was similar regardless of stem cell dose even when tested at the time when there were significant differences in peripheral T-cell counts. The improved immune recovery was attributed to a more rapid regeneration of donor-type immune cells. Higher numbers of total thymocytes and signal joint T-cell receptor excision circles were observed in the higher dose stem cell recipients, suggesting that accelerated regeneration of T cells was due to enhanced thymopoiesis. (Blood. 2004;103:4344-4352)

Thymic function and immunoglobulin mutation genotype in B-cell chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a profound dysregulation of the host's immune system at both the humoral and cellular level. We investigated to see if this dysregulation could be due partly to thymus dysfunction by quantifying the number of signal joint T-cell receptor excision circles (sjTRECs) in peripheral blood mononuclear cells of 30 untreated B-CLL patients at diagnosis and in age-matched healthy controls. sjTRECs were found decreased, normal and elevated in 19, 9 and 2 patients, respectively, in comparison to age-matched controls. We next speculated that sjTREC levels might be related to an accredited B-CLL prognostic marker represented by the somatic hypermutation (SHM) status of the variable heavy chain (V(H)) genes. Eight of 17 patients with SHMs had sjTREC levels in the range of or higher than normal donors, whereas only 3 of the 13 patients lacking SHMs had normal sjTREC levels. After a 5-year observation period in 16 patients for whom a clinical follow-up was available, only 2 of 10 patients with SHMs progressed vs. 5 of 6 patients without SHMs and 3 of 7 patients with normal or higher sjTREC levels progressed vs. 4 of 9 with low sjTREC levels. Our study demonstrates that sjTREC levels are decreased in >60% of B-CLL patients and suggests a potential role of thymus in the immune dysfunction of these patients.