Signal-joint T-cell Receptor Excision Circles Research Articles

Keratinocyte growth factor impairs human thymic recovery from lymphopenia.

BACKGROUNDThe lymphocyte-depleting antibody alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis (RRMS); however, 50% of patients develop novel autoimmunity after treatment. Most at risk are individuals who reconstitute their T cell pool by proliferating residual cells, rather than producing new T cells in the thymus, raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in nonhuman primates.METHODSFollowing a dose tolerability substudy, individuals with RRMS (duration ≤10 years; expanded disability status scale ≤5.0, with ≥2 relapses in the previous 2 years) were randomized to placebo or 180 μg/kg/d palifermin, given for 3 days immediately before and after each cycle of alemtuzumab, with repeat doses at month 1 (M1) and M3. The interim primary endpoint was naive CD4+ T cell count at M6. Exploratory endpoints included number of recent thymic emigrants (RTEs) and signal joint T cell receptor excision circles/ml (sjTRECs/ml) of blood. The trial’s primary endpoint was incidence of autoimmunity at M30.RESULTSAt M6, individuals receiving palifermin had fewer naive CD4+ T cells (2.229 × 107/l vs. 7.733 × 107/l; P = 0.007), RTEs (16% vs. 34%), and sjTRECs/ml (1100 vs. 3396), leading to protocol-defined termination of recruitment. No difference was observed in the rate of autoimmunity between the 2 groups.CONCLUSIONIn contrast with animal studies, palifermin reduced thymopoiesis in our patients. These results offer a note of caution to those using palifermin to promote thymopoiesis in other settings, particularly in the oncology/hematology setting, where alemtuzumab is often used as part of the conditioning regime.TRIAL REGISTRATIONClinicalTrials.gov NCT01712945.FUNDINGMRC and Moulton Charitable Trust.

Maintained thymic output of conventional and regulatory T cells during human pregnancy

Maintained thymic output of conventional and regulatory T cells during human pregnancy

Independent validation of DNA-based approaches for age prediction in blood

Numerous molecular biomarkers have been proposed as predictors of chronological age. Among them, T-cell specific DNA rearrangement and DNA methylation markers have been introduced as forensic age predictors in blood because of their high prediction accuracy. These markers appear highly promising, but for better application to forensic casework sample analysis the proposed markers and genotyping methods must be tested further. In the current study, signal-joint T-cell receptor excision circles (sjTRECs) and DNA methylation markers located in the ELOVL2, C1orf132, TRIM59, KLF14, and FHL2 genes were reanalyzed in 100 Korean blood samples to test their associations with chronological age, using the same analysis platform used in previous reports. Our study replicated the age association test for sjTREC and DNA methylation markers in the 5 genes in an independent validation set of 100 Koreans, and proved that the age predictive performance of the previous models is relatively consistent across different population groups. However, the extent of age association at certain CpG loci was not identical in the Korean and Polish populations; therefore, several age predictive models were retrained with the data obtained here. All of the 3 models retrained with DNA methylation and/or sjTREC data have a CpG site each from the ELOVL2 and FHL2 genes in common, and produced better prediction accuracy than previously reported models. This is attributable to the fact that the retrained model better fits the existing data and that the calculated prediction accuracy could be higher when the training data and the test data are the same. However, it is notable that the combination of different types of markers, i.e., sjTREC and DNA methylation, improved prediction accuracy in the eldest group. Our study demonstrates the usefulness of the proposed markers and the genotyping method in an independent dataset, and suggests the possibility of combining different types of DNA markers to improve prediction accuracy.

High pre-transplant TREC levels indicate good prognosis after hematopoietic stem cell transplantation

BackgroundThymus-dependent T-cell reconstitution plays a role in immune recovery after stem cell transplantation (HSCT). High pre-HCST thymic function has been associated with higher survival, lower incidence of acute and chronic graft versus host disease (GVHD) and lower incidence of infections. The aim of this study was to analyze the relationship between pre-HSCT peripheral blood levels of T-cell receptor excision circles (TREC) and post-HSCT clinical events in recipients of HLA-identical hematopoietic stem cell transplants.MethodDelta deletion signal joint TRECs (sjTRECs) formed by the dREC-yJa rearrangement were quantified by real time PCR in peripheral blood lymphocytes of 62 HSCT recipients.ResultsUnivariate analysis revealed an association between low TREC levels and a higher incidence of grade II-IV acute GVHD (p=0.026), bacterial infection (p=0.005) and cytomegalovirus infection (p=0.033), whereas high TREC levels were associated with higher overall survival (p=0.028). In the multivariate analysis, low pre-HSCT TREC levels remained independently associated with lower survival (p=0.032; RR 2.6), occurrence of grade II-IV acute GVHD (p=0.031; RR: 2.5), bacterial infection (p=0.006, RR: 6.6) and cytomegalovirus infection (p=0.039; RR:2.8).ConclusionOur results corroborate the concept that pre-HSCT recipient´s thymic function is an important predictor of risk for acute grade II-IV GVHD and infection.

Thymic emigration patterns in patients with type 2 diabetes treated with metformin.

Recent data suggest that thymic output, which provides the naive T cells necessary for the normal functioning of T-cell-dependent immunosurveillance cellular immunity including anti-cancer protection, can be disturbed in the course of type 2 diabetes. Metformin, an anti-diabetic drug commonly confirmed as an agent with many potential anti-cancer activities, might be helpful in this immune correction. The profile of thymic output was evaluated in the current study on the basis of the signal-joint T-cell receptor excision circle (sjTREC) concentration in peripheral blood polymorphonuclear cells and thymic emigrant content in peripheral blood evaluated from CD127 and/or CD132 antigen expression. It was revealed that recent thymic emigrants and more differentiated CD127(+) CD132(+) cell populations were decreased among naive T cells and CD8(+) T cells, whereas RTE count was increased in CD4(+) T cells, and the CD127(+) CD132(+) cell population was less numerous than in non-diabetic participants. Terminally differentiated thymic emigrants, i.e. CD127(-) CD132(+) cells, were increased in naive T cells and in CD8(+) T cells. Metformin affects mainly the early phases of thymic export, increasing CD127(+) CD132(-) and CD127(+) CD132(+) cell populations in naive T cells and the CD127(+) CD132(-) population in CD4(+) T lymphocytes. It could be concluded that type 2 diabetes deteriorates thymic immunostasis. The decreased thymic output could be compensated by metformin, especially with regard to CD4(+) naive T cells. It is the first time that therapy with metformin has been documented by us as particularly useful in the control and normalization of thymus function, regarding correction of early populations of thymic emigrants.

T-Cell Receptor Excision Circle Levels After Allogeneic Stem Cell Transplantation Are Predictive of Relapse in Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

In this retrospective study, 209 patients with malignant disease were analyzed for levels of T-cell receptor excision circles (TRECs) for the first 24 months after allogeneic stem cell transplantation. CD3(+) cells were separated by direct antibody-coupled magnetic beads, followed by DNA extraction according to a standard protocol. The δRec-ψJα signal joint TREC was measured with real-time quantitative PCR. Patients were grouped based on malignant disease: chronic myeloid leukemia, chronic lymphatic leukemia, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and myelodysplastic syndrome (MDS). Patients were further subdivided based on TREC levels below (low-TREC) or above (high-TREC) median at each time point. TREC levels were then correlated to relapse incidence and relapse-free survival (RFS). For patients with AML, low TREC levels 2 months post-transplantation were correlated to high relapse incidence at 5 years (P<0.05). In patients with chronic leukemia, high TREC levels were correlated with improved RFS (P<0.05). For patients with MDS, high TREC levels at 9 months post-transplantation were associated with higher RFS at 5 years (P<0.02) and lower relapse incidence (P<0.02). This study shows the potential use of TREC measurement in blood to predict relapse in patients with AML and MDS after allogeneic hematopoietic stem cell transplantation.

Chimerism in Children With Primary Immunodeficiencies is Influenced by Number of Activating Killer Cell Immunoglobulin-Like Receptor Genes in the Donor and/or Killer Cell Immunoglobulin-Like Receptor Ligand Mismatch

BackgroundStem cell transplantation (SCT) is a curative treatment for children with primary immunodeficiencies. MethodsThe present retrospective analysis describes the long-term outcomes at a median follow-up of 9 years of 29 patients with immunodeficiency after SCT; 5 sibling and 24 alternative donor transplantations. T-cell engraftment emphazed on thymic dependent signal-joint T-cell receptor excision circles (sjTREC) generation and donor chimerism in relation to killer cell immunoglobulin-like receptor genes and their ligands. ResultsAll children except two were reconstituted successfully from grafted material, including 9 and 18 cases of mixed chimerism (MC) and complete chimerism (CC), respectively. Univariate analyses showed that the number of activating KIR genes or HLA-C1/C2 ligand mismatches (P = .048) and possibly transplantation from an alternative donor (P = .054) facilitated CC development. Multivariate analysis showed that the presence of donor KIR haplotype B or incompatibility within C1/C2 ligands (relative risk, 6.1; 95% confidence interval, 1.08–34.69; P = .025) were significantly associated with the development of CC. ConclusionsThese results suggested that the donor-activating KIR gene repertoire affected successful engraftment.

Thymopoiesis and regulatory T cells in healthy children and adolescents

OBJECTIVES:The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages.MATERIALS AND METHODS:The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry.RESULTS:Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095±36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p<0.001) as well as between the proportion of CD4+CD25+Foxp3+ T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4+CD25+Foxp3+ T cells and the amount of T-cell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02).CONCLUSIONS:In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.

Progressive Activation of CD127+132− Recent Thymic Emigrants into Terminally Differentiated CD127−132+ T-Cells in HIV-1 Infection

AimHIV infection is associated with distortion of T-cell homeostasis and the IL-7/IL7R axis. Progressive infection results in loss of CD127+132− and gains in CD127−132+ CD4+ and CD8+ T-cells. We investigated the correlates of loss of CD127 from the T-cell surface to understand mechanisms underlying this homeostatic dysregulation.MethodsPeripheral and cord blood mononuclear cells (PBMCs; CBMC) from healthy volunteers and PBMC from patients with HIV infection were studied. CD127+132−, CD127+132+ and CD127−132+ T-cells were phenotyped by activation, differentiation, proliferation and survival markers. Cellular HIV-DNA content and signal-joint T-cell receptor excision circles (sjTRECs) were measured.ResultsCD127+132− T-cells were enriched for naïve cells while CD127−132+ T-cells were enriched for activated/terminally differentiated T-cells in CD4+ and CD8+ subsets in health and HIV infection. HIV was associated with increased proportions of activated/terminally differentiated CD127−132+ T-cells. In contrast to CD127+132− T-cells, CD127−132+ T-cells were Ki-67+Bcl-2low and contained increased levels of HIV-DNA. Naïve CD127+132− T-cells contained a higher proportion of sjTRECs.ConclusionThe loss of CD127 from the T-cell surface in HIV infection is driven by activation of CD127+132− recent thymic emigrants into CD127−132+ activated/terminally differentiated cells. This process likely results in an irreversible loss of CD127 and permanent distortion of T-cell homeostasis.

Signal Joint T-Cell Receptor Excision Circle Assay in Miniature Swine

Assays for T cell receptor excision circles (TREC) have been utilized in human, primate, and mouse models as a measure of thymic activity, but no comparable assay has been described in artiodactyls. We describe the development of the porcine signal joint (sj) TREC assay, and provide a likely reason for previous difficulties in its identification in artiodactyls. Utilizing the homology between the known genomic sequences in sjTREC in human and mouse, polymerase chain reaction (PCR) primers were derived for the putative porcine sjTREC. Primers from the ψJα side of the sjTREC were derived from the known porcine sequence. The sjTREC in two artiodactyls, swine and sheep, was identified using forward primers from the ψJα region, and reverse primers from the putative δ-rec region. Unlike in the detection of primate TRECs, initially the use of similar primers close to the δ-rec failed to yield the sjTREC product. Marching about 800 basepairs into δ-rec, primers derived from a homology region between human and mouse led to the detection of sjTREC. Comparing sjTREC amongst the species revealed highest homology between the two artiodactyls. A quantitative PCR (QPCR) assay of porcine sjTREC was also developed. Identification and analysis of the sjTREC sequences in two artiodactyls suggested why previous attempts at cloning the pig TREC using known sjTREC sequences were unsuccessful. The development of the porcine signal joint TREC assay should enable a more direct quantification of thymic activity in porcine models of transplant biology.

Thymic recovery after allogeneic hematopoietic cell transplantation with non-myeloablative conditioning is limited to patients younger than 60 years of age

Long-term immune recovery in older patients given hematopoietic cell transplantation after non-myeloablative conditioning remains poorly understood. This prompted us to investigate long-term lymphocyte reconstitution and thymic function in 80 patients given allogeneic peripheral blood stem cells after non-myeloablative conditioning. Median age at transplant was 57 years (range 10-71). Conditioning regimen consisted of 2 Gy total body irradiation (TBI) with (n=46) or without (n=20) added fludarabine, 4 Gy TBI with fludarabine (n=6), or cyclophosphamide plus fludarabine (n=8). Stem cell sources were unmanipulated (n=56), CD8-depleted (n=19), or CD34-selected (n=5) peripheral blood stem cells. Immune recovery was assessed by signal-joint T-cell receptor excision circle quantification and flow cytometry. Signal-joint T-cell receptor excision circle levels increased from day 100 to one and two years after transplantation in patients under 50 years of age (n=23; P=0.02 and P=0.04, respectively), and in those aged 51-60 years (n=35; P=0.17 and P=0.06, respectively), but not in patients aged over 60 (n=22; P=0.3 and P=0.3, respectively). Similarly, CD4(+)CD45RA(+) (naïve) T-cell counts increased from day 100 to one and two years after transplantation in patients aged 50 years and under 50 (P=0.002 and P=0.02, respectively), and in those aged 51-60 (P=0.4 and P=0.001, respectively), but less so in patients aged over 60 (P=0.3 and P=0.06, respectively). In multivariate analyses, older patient age (P<0.001), extensive chronic GVHD (P<0.001), and prior (resolved) extensive chronic graft-versus-host disease (P=0.008) were associated with low signal-joint T-cell receptor excision circle levels one year or more after HCT. In summary, our data suggest that thymic neo-generation of T cells occurred from day 100 onwards in patients under 60 while signal-joint T-cell receptor excision circle levels remained low for patients aged over 60. Further, chronic graft-versus-host disease had a dramatic impact on thymic function, as observed previously in patients given grafts after myeloablative conditioning.

Thymic Recovery After Allogeneic Hematopoietic Cell Transplantation with Nonmyeloablative Conditioning Might Be Limited to Patients Younger Than 60 Years of Age.

Abstract 1149Poster Board I-171 BackgroundNonmyeloablative conditioning followed by allogeneic hematopoietic cell transplantation (HCT) has been increasingly used as treatment for elderly patients with hematologic malignancies. It has been suspected that T cell reconstitution would be impaired in elderly patients given nonmyeloablative conditioning because of age-related thymic atrophy. Here, we investigated long term lymphocyte reconstitution and thymic function in 80 patients given allogeneic peripheral blood stem cells (PBSC) after nonmyeloablative conditioning. Patients and MethodsMedian age at transplant was 57 years (range 10-71). Conditioning regimen consisted of 2 Gy total body irradiation (TBI) with (n=46) or without (n=20) added fludarabine, 4 Gy TBI with fludarabine (n=6), or cyclophosphamide plus fludarabine (n=8). Thirty-three of the 80 patients received grafts from HLA-matched related donors, 22 from HLA-matched unrelated donors, and 25 from HLA-mismatched related or unrelated donors. PBSC were unmanipulated in 56 patients, CD8-depleted in 19 others, and CD34-selected in the remaining 5 patients. GVHD prophylaxis consisted of mycophenolate mofetil and cyclosporine or tacrolimus. Immune recovery was assessed between 1 and 8.5 years after HCT by signal-joint T-cell receptor excision circle (sjTREC) quantification (221 samples), and flow cytometry. Further, in order to demonstrate a potential thymic recovery, sjTREC level changes from day 100 to days 365 and 730 were also assessed by using Wilcoxon signed rank tests. ResultsThere was a close correlation between sjTREC levels and naive CD4+ T cells (defined as CD4+CD45RA+) counts (P<0.0001). An inverse correlation was observed between the levels of sjTREC/ml and the recipient's age (R=-0.41, p<0.0001). Interestingly, sjTREC levels increased from day 100 to 1 and 2 years after transplantation in patients ≤ 50 (n=23; P=0.02 and P=0.04, respectively), and in those 51-60 years of age (n=35; P=0.17 and P=0.06, respectively), but not in patients >60 (n=22; P=0.3 and P=0.3, respectively) (Figure 1). Similarly, naïve CD4 T cell counts increased from day 100 to 1 and 2 years after transplantation in patients ≤50 (n=23; P=0.01 and P=0.3, respectively), and in those 51-60 years of age (n=35; P=0.5 and P<0.001, respectively), but not in patients >60 (n=22; P=0.9 and P=1.0, respectively). In multivariate analyses, older patient age (P<0.001), extensive chronic GVHD (P<0.001), and prior (resolved) extensive chronic GVHD (P=0.008) were associated with low sjTREC levels, while older patient age (P<0.001), and extensive chronic GVHD (P<0.001) were associated with low naïve CD4 T cell counts. ConclusionsOur data suggest that thymic neo-generation of T cells occurred from day 100 onwards in patients under 60. However, the levels of sjTREC remained low for patients above 60. Further, chronic GVHD has a dramatic impact on thymic function, as observed after myeloablative conditioning. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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

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).

Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation

AbstractLong-term T-cell reconstitution after hematopoietic stem cell transplantation (HSCT) is dependent on patient thymic function and affected by graft-versus-host disease (GVHD). To assess the impact of acute GVHD (aGVHD) on thymic function, we followed a cohort of 93 patients who received HSCT from a human histocompatibility leukocyte antigen-identical sibling, mainly for hematologic malignancies. Thymic output was measured by signal-joint T-cell receptor excision circles (sjTREC) real-time polymerase chain reaction. Absolute sjTREC number was lower at 6 months in patients with aGVHD (P = .014), associated with lower absolute counts of naive CD4 T cells at 6 and 12 months (P = .04 and .02), and persistent abnormalities in T-cell repertoire diversity. Age and aGVHD affected thymic function independently in multivariate analysis. In patients less than 25 years of age, thymic function recovered almost totally at 1 year. As a marker of thymocyte proliferation, we quantified the βTREC generated during the T-cell receptor β-chain recombination, in a group of 20 age-matched patients. Mean βTREC level was reduced at 6 months in patients with aGVHD, indicating an impact on early thymic differentiation rather than on intrathymic proliferation. These data show that aGVHD or its treatment has a transient impact on thymic function in younger patients in the first months after HSCT.

Impact of Growth Hormone (GH) Deficiency and GH Replacement upon Thymus Function in Adult Patients

BackgroundDespite age-related adipose involution, T cell generation in the thymus (thymopoiesis) is maintained beyond puberty in adults. In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues reverse age-related changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels.Methodology/Principal FindingsTwenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/β TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were decreased (p<0.001). Decreases in IGF-1 and sjTREC levels were correlated (r = 0.61, p<0.01). There was also a decrease in intrathymic T cell proliferation as indicated by the reduced sj/β TREC ratio (p<0.01). One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency regained a level equivalent to the one before GH withdrawal. The sj/β TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal.ConclusionsIn patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymus function are completely or partially restored one month after GH resumption. These data indicate that the functional integrity of the somatotrope GH/IGF-1 axis is important for the maintenance of a normal thymus function in human adults.Trial RegistrationClinicalTrials.gov NTC00601419

Impact of Growth Hormone (GH) Deficiency and GH Replacement upon Thymus Function in Adult Patients (138.26)

Abstract Context: In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues (such as ghrelin) reverse age-related changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). Objectives: The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels. Design: Twenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/ß TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. Results: After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were significantly decreased. Decreases in IGF-1 and sjTREC levels were significantly correlated. There was also a significant decrease in intrathymic T cell proliferation as indicated by the reduced sj/ß TREC ratio. One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency were restored at the level before arrest of GH treatment. The sj/ß TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal. Conclusions: In patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymopoiesis are completely or partially restored one month after GH resumption.

Evidence for neo-generation of T cells by the thymus after non-myeloablative conditioning

Background and objective. We investigated immune recovery in 50 patients given either unmanipulated or CD8-depleted allogeneic peripheral blood stem cells after non-myeloablative conditioning. Fifty patients were randomized to receive either CD8-depleted (n=22) or non-manipulated (n=28) peripheral blood stem cells. The median patients age was 57 (range 36-69) years. The conditioning regimen consisted of 2 Gy total body irradiation with or without added fludarabine. Twenty patients received grafts from related donors, 14 from 10/10 HLA-allele matched unrelated donors, and 16 from HLA-mismatched unrelated donors. Graft-versus-host disease pro-phylaxis consisted of mycophenolate mofetil and cyclosporine. Immune recovery during the first year after hematopoietic cell transplantation was assessed by flow cytometry phenotyping, analyses of the diversity of the TCRBV repertoire, and quantification of signal-joint T-cell receptor excision circles (sjTREC). CD8-depletion of the graft reduced the recovery of CD8(+) T-cell counts in the first 6 months following transplantation (p<0.0001) but had no significant impact on the restoration of other T-cell subsets. Both sjTREC concentration and CD3(+) T-cell counts increased significantly between day 100 and 365 (p=0.010 and p=0.0488, respectively) demonstrating neo-production of T cells by the thymus. Factors associated with high sjTREC concentration 1 year after transplantation included an HLA-matched unrelated donor (p=0.029), a high content of T cells in the graft (p=0.002), and the absence of chronic graft-versus-host disease (p<0.0001). Our data suggest that while immune recovery is mainly driven by peripheral expansion of the graft-contained mature T cells during the first months after non-myeloablative transplantation, T-cell neo-generation by the thymus plays an important role in long term immune reconstitution in transplanted patients.

Evaluation of Pre-Transplantation T-Cell Receptor Excision Circles (TREC) in Reduced-Intensity Stem Cell Transplantation (RIST) Recipients.

〈Background and Methods〉: Previous studies have shown that the recipient's thymic function before hematopoietic stem cell transplantation (HSCT) is critical for subsequent immune reconstitution and that the value of pre-transplantation T-cell receptor excision circles (TREC) is related to the incidence of graft-versus-host disease (GVHD), infection and survival. However, these observations have been mainly based on younger patients, who were eligible for myeloablative HSCT. In contrast, little is known about thymic function in elderly patients and its influence on the outcome of reduced-intensity stem cell transplantation (RIST). In this study, TREC values were measured in a total of 180 HSCT recipients who received myeloablative HSCT (n=60: mean age, 34 y) or RIST (n=120, 51 y). Quantification of thymic signal joint TREC was performed by real-time quantitative PCR and TREC values were calculated as TREC copies per 10 ng DNA obtained from peripheral blood mononuclear cells.〈Results〉: The mean and median pre-transplantation TREC values were 3.9 and 1.3, respectively, and these showed a significant inverse correlation with patient age (p=0.0002). The mean TREC value was 3.0 in males and 5.1 in females (p=0.22), and was significantly lower in 19 patients who had received previous radiation therapy involving the mediastinum (0.96, p=0.003). The TREC value was 3.7 in 99 patients who had a disease duration of longer than 365 days, and 4.2 in 81 patients with a shorter duration (p=0.61). The TREC value was 5.5 for solid tumor (n=10), 5.0 for CML (n=10), 4.8 for AML (n=52), 3.7 for MDS (n=23), 4.0 for ALL (n=23), 2.8 for lymphoma (n=60) and 0.4 for myelofibrosis (n=2), with no difference between those in complete remission and others (both 3.9). The clinical outcome in relation to the TREC value was analyzed in 98 patients who received RIST from an HLA-identical donor. A comparison of 51 patients with lower pre-transplant TREC values (<1.3) and 47 patients with higher values (>1.3) disclosed that the incidence of acute GVHD was 64% vs 57% (p=0.52), that of chronic GVHD was 69% vs 77% (p=0.56), CMV reactivation was 72% vs 60% (p=0.20) and progression-free survival at day 100 was 72% vs 100% (p=0.0001).〈Conclusion〉: Pre-transplant TREC values showed an inverse correlation with age, and were significantly lower among patients who received radiotherapy involving the thymus. Although these differences were not statistically significant, patients with lymphoid malignancy, patients with longer disease duration and male patients tended to have lower TREC values, while the disease status did not influence the TREC value. This study provides the baseline reference data regarding the thymic function in patients undergoing RIST, although further study is required to evaluate the significance of TREC on the clinical outcome of RIST.

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.