T-cell Receptor Excision Circle Content Research Articles

Determining T and B Cell development by TREC/KREC analysis in primary immunodeficiency patients and healthy controls.

T cell receptor excision circles (TRECs) and kappa-deleting excision circles (KRECs) are DNA fragments potentially indicative of T and B cell development, respectively. Recent thymic emigrants (RTEs) are a subset of peripheral cells that may also represent thymic function. Here, we investigated TREC/KREC copy numbers by quantitative real-time PCR in the peripheral blood of patients with primary immunodeficiencies (PIDs, n=145) and that of healthy controls (HCs, n=86) and assessed the correlation between RTEs and TREC copy numbers. We found that TREC copy numbers were significantly lower in children and adults with PIDs (P<.0001 and P<.002, respectively) as compared with their respective age-matched HCs. A moderate correlation was observed between TREC copies and RTE numbers among children with PID (r=.5114, P<.01), whereas no significant correlation was detected between RTE values and TREC content in the HCs (r=.0205, P=.9208). Additionally, we determined TREC and KREC copy numbers in DNA isolated from the Guthrie cards of 200 newborns and showed that this method is applicable to DNA isolated from both peripheral blood samples and dried blood spots, with the two sample types showing comparable TREC and KREC values. We further showed that RTE values are not always reliable markers of T cell output. Although additional confirmatory studies with larger cohorts are needed, our results provide thresholds for TREC/KREC copy numbers for different age groups.

Increased Naive, Stem Cell Memory and Terminally Differentiated CD8+ T Cells in Chronic Graft Versus Host Disease

BACKGROUND AND AIMSDespite improved clinical outcomes of patients undergoing Hematopoietic Stem Cell Transplantation (HSCT) in recent years, chronic GVHD (cGVHD) and its associated complications have a significant impact on non-relapse mortality and quality of life. Loss of immune tolerance in the post-transplant period plays an important role in the emergence of cGVHD, and while imbalances in CD4+ T cell subsets, such as regulatory T cells, are known to contribute to its pathobiology, the role of different subsets of CD8+ T cells is poorly characterized.PATIENTS AND METHODSWe prospectively monitored a group of 56 patients who underwent unrelated donor HSCT after reduced intensity conditioning containing ATG. GVHD prophylaxis consisted of cyclosporine plus mycophenolate mofetil for all patients. 15 patients were excluded due to early disease relapse and/or death secondary to infection or acute GVHD in the first 9 months post-HSCT. cGVHD diagnosis and staging was performed according to the 2014 NIH criteria. Patients were divided into 2 groups according to cGVHD status: 17 patients (41%) with cGVHD (cGVHD+) and 24 patients (59%) without cGVHD (cGVHD-).Peripheral blood was analyzed by flow cytometry to characterize T cell subsets at fixed time points over a 2 year period. CD8+ T subsets were identified as follows: True Naïve cells as CD45RA+CD62L+CD95- (TN), Stem Cell Memory cells as CD45RA+CD62L+CD95+ (SCM), Central Memory cells as CD45RA-CD62L+ (CM), Effector Memory cells as CD45RA-CD62L- (EM) and terminally differentiated effector cells as CD45RA+CD62L- (EMRA).Frozen PBMC from patients at months 9 and 12 post-HSCT were used to purify CD8+ T cells by cell sorting to quantify Signal-Joint T Cell Receptor Excision Circles (TREC) by Real-Time PCR and to evaluate TCR diversity by TCR Vbeta complementarity-determining region 3 spectratyping.RESULTSPatients with cGVHD had significantly increased percentages and absolute counts of TN CD8 in the first 6 months post-HSCT compared with cGVHD- patients (p≤0.04 at months 1 and 3 for percentages; p≤0.02 for months 1-6 in absolute counts).Furthermore, cGVHD+ patients had increased SCM CD8 cells throughout the post-HSCT period (significances of p<0.05 at months 6 and 12 for percentages and 18 and 24 for counts). The increased prevalence of these two subsets was not associated with increased proliferation as assessed by Ki67, which was reduced in cGVHD+ in the total CD45RA+CD62L+ gate (TN plus SCM cells) (p≤0.04 at months 6, 12 and 18). No significant differences were observed in susceptibility to apoptosis as measured by BCL2 or CD95 expression levels.CM and EM subset counts were reduced in cGVHD+ as compared to cGVHD- (p=0.04 and 0.02 at month 12). EM CD8 were the dominant population in cGVHD- patients throughout the follow up. Significant increases in the percentage of EM CD8 cells in cGVHD- were observed at months 2, 9, 12 and 18 (p≤0.04).cGVHD+ patients had increased percentages of EMRA (p<0.04 at months 9, 12 and 18) and absolute counts (p<0.04 in months 2, 9 and 24). This was not correlated to altered Ki67, Bcl-2 or CD95 levels.The increase in TN in cGVHD+ patients was not associated with increased TREC numbers within CD8+ T cells. TREC content was significantly reduced in both patient groups at month 9 as compared healthy controls (HC) (p<0.05). This could be due to the low prevalence of these cells and/or to rapid differentiation into other memory subsets such as the SCM and EMRA. Despite the differences in subset prevalence in the two patient groups, we did not observe any significant differences in TCR Vbeta diversity in CD8+T cells. Reduced TCR diversity was only observed when patients were compared to HC (p=0.0001).DISCUSSIONOur observations of increased TN, SCM and EMRA CD8 subsets very early after HSCT in patients who subsequently develop cGVHD suggest the possibility that these may be relevant biomarkers for cGVHD development. SCM T cells have previously been shown to cause lethal GVHD in mouse models and to represent a reservoir of antigen-specific T cells capable of differentiating in vivo into other memory subsets with effector function. We speculate that thymic tissue damage resulting from conditioning regimens and/or acute GVHD, together with other yet unidentified factors, may lead to the emergence of self-reactive naïve CD8+ T cells that differentiate to SCM and later to EMRA cells potentially mediating cGVHD. DisclosuresNo relevant conflicts of interest to declare.

Naive and Stem Cell Memory T Cell Subset Recovery Reveals Opposing Reconstitution Patterns in CD4 and CD8 T Cells in Chronic Graft vs. Host Disease.

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of hematological malignancies remains hampered by life-threatening chronic graft vs. host disease (cGVHD). Although multifactorial in nature, cGVHD has been associated with imbalances between effector and regulatory T cells (Treg). To further elucidate this issue, we performed a prospective analysis of patients undergoing unrelated donor allo-HSCT after a reduced intensity conditioning (RIC) regimen containing anti-thymocyte globulin (ATG) and the same GVHD prophylaxis, at a single institution. We studied T cell subset homeostasis over a 24-month follow-up after HSCT in a comparative analysis of patients with and without cGVHD. We also quantified naive and memory T cell subsets, proliferation and expression of the apoptosis-related proteins Bcl-2 and CD95. Finally, we assessed thymic function by T cell receptor excision circle (TREC) quantification and T cell receptor (TCR) diversity by TCRVβ spectratyping. While the total number of conventional CD4 (Tcon) and CD8 T cells was similar between patient groups, Treg were decreased in cGVHD patients. Interestingly, we also observed divergent patterns of Naive and Stem Cell Memory (SCM) subset recovery in Treg and Tcon compared to CD8. Patients with cGVHD showed impaired recovery of Naive and SCM Tcon and Treg, but significantly increased frequencies and absolute numbers of Naive and SCM were observed in the CD8 pool. Markedly increased EMRA CD8 T cells were also noted in cGVHD. Taken together, these results suggest that Naive, SCM and EMRA CD8 play a role in the emergence of cGHVD. Reduced Naive and recent thymic emigrant Tcon and Treg in cGVHD was likely due to impaired thymic output, as it was accompanied by decreased CD4 TREC and TCR diversity. On the other hand, CD8 TCR diversity was similar between patient groups. Furthermore, no correlation was observed between CD8 TREC content and Naive CD8 numbers, suggesting limited thymic production of Naive CD8 T cells in patients after transplant, especially in those developing cGVHD. The mechanisms behind the opposing patterns of CD4 and CD8 subset cell recovery in cGVHD remain elusive, but may be linked to thymic damage associated with the conditioning regimen and/or acute GVHD.

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

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

T-Cell Activation Independently Associates With Immune Senescence in HIV-Infected Recipients of Long-term Antiretroviral Treatment.

Aging-associated noncommunicable comorbidities are more prevalent among human immunodeficiency virus type 1 (HIV)-infected individuals than among HIV-uninfected individuals. Residual HIV-related chronic immune activation and senescence may increase the risk of developing comorbidities. Immune phenotyping, thymic output, and telomere length were assessed in 94 HIV-infected individuals who were aged >45 years and receiving antiretroviral therapy (ART; cases) and 95 age-matched uninfected controls. Cases had lower CD4(+) T-cell counts, higher CD8(+) T-cell counts, and increased levels of immune activation (ie, increased soluble CD14 [sCD14] level and increased percentages of CD38(+)HLA-DR(+) cells among both CD4(+) and CD8(+) T cells), regulatory T cells, and percentage of programmed cell death 1 (PD-1)-expressing cells among CD4(+) T cells. Immune senescence levels (ie, percentages of CD27(-)CD28(-) cells or CD57(+) cells) were comparable between cases and controls. Peripheral blood mononuclear cells from cases had shorter telomeres but increased single-joint T-cell receptor excision circle content and CD31(+) naive CD4(+) T cells. Although cytomegalovirus (CMV) antibody titers were higher in cases, CMV-specific T-cell responses were comparable between cases and controls. T-cell senescence in cases was independently associated with T-cell activation but not with CMV-specific immune responses. Despite long-term receipt of ART, HIV-infected adults had higher levels of immune activation, regulatory T cells, and PD-1-expressing CD4(+) cells and shorter telomeres. The increased soluble CD14 levels and percentage of CD38(+)HLA-DR(+) cells among CD4(+) T cells correlated with shorter telomeres and increased regulatory T-cell levels. This suggests that HIV influences immune function irreversibly, with several pathways that are persistently abnormal during effective ART. Therapies aimed at improving immune health during ART are needed.

Primary Cytomegalovirus Infection Significantly Impacts Circulating T Cells in Kidney Transplant Recipients.

Cytomegalovirus (CMV) infection profoundly affects the T cell compartment and is associated with alterations in T cell aging parameters and generation of cytotoxic CD4(+) CD28null T cells. Hence, the effect of a primary CMV infection post-kidney transplantation (KT) on the peripheral T cell compartment was examined. As aging parameters, we determined the T cell differentiation status, T cell receptor excision circle (TREC) content, CD31(+) naïve T cell numbers and relative telomere length (RTL) pre-KT and 12 months post-KT. CMV-seronegative KT recipients, receiving a kidney from a CMV-seropositive donor (D+/R-) were compared to D+/R+ KT recipients. Eleven out of the 22 D+/R- KT recipients had CMV viremia post-KT. They developed CMV-specific CD4(+) and CD8(+) T cells and their T cell compartment shifted towards a more differentiated memory phenotype with expansion of CD4(+) CD28null and CD8(+) CD28null cells. One year post-KT, the CD8(+) T cell count was almost doubled compared to nonviremic D+/R- and D+/R+ KT recipients. In addition, the RTL of the CD8(+) T cell was significantly lower and both the TREC content and CD31(+) naïve T cell numbers significantly decreased. Moreover, primary CMV infection was associated with a negative impact on glomerular filtration rate. In conclusion, primary CMV infection has a substantial impact on the number and phenotype of peripheral T cells and may negatively affect renal allograft function.

TREC Based Newborn Screening for Severe Combined Immunodeficiency Disease: A Systematic Review.

BackgroundNewborn screening (NBS) by quantifying T cell receptor excision circles (TRECs) in neonatal dried blood spots (DBS) enables early diagnosis of severe combined immunodeficiency disease (SCID). In recent years, different screening algorithms for TREC based SCID screening were reported.PurposeTo systematically review the diagnostic performance of published algorithms for TREC based NBS for SCID.MethodsPubMed, EMBASE and the Cochrane Library were systematically searched for case series and prospective cohort studies describing TREC based NBS for SCID. We extracted TREC content and cut-off values, number of retests, repeat DBS and referrals, and type and number of typical SCID and other T cell lymphopenia (TCL) cases. We calculated positive predictive value (PPV), test sensitivity and SCID incidence.ResultsThirteen studies were included, re-confirming 89 known SCID cases in case series and reporting 53 new SCID cases in 3.15 million newborns. In case series, the sensitivity for typical SCID was 100 %. In the prospective cohort studies, SCID incidence was ~1.7:100,000, re-test rate was 0.20–3.26 %, repeat DBS rate 0.0–0.41 % and referral rate 0.01–1.35 %. PPV within the five largest cohorts was 0.8–11.2 % for SCID and 18.3–81.0 % for TCL. Individual TREC contents in all SCID patients was <25 TRECs/μl (except in those evaluated with the New York State assay).ConclusionsThe sensitivity of TREC based NBS for typical SCID was 100 %. The TREC cut-off score determines the percentage of non-SCID TCL cases detected in newborn screening for TCL. Adapting the screening algorithm for pre-term/ill infants reduces the amount of false positive test results.Electronic supplementary materialThe online version of this article (doi:10.1007/s10875-015-0152-6) contains supplementary material, which is available to authorized users.

Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.

Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.

Uremia-associated immunological aging is stably imprinted in the T-cell system and not reversed by kidney transplantation.

The uremia-induced inflammatory environment in end-stage renal disease (ESRD) patients is associated with premature T-cell aging resulting in a defective T-cell immunity. As kidney transplantation (KTx) reduces the pro-inflammatory environment, we hypothesized that KTx would rejuvenate the aged T-cell system. As aging parameters, we determined in 70 KTx recipients the differentiation status by immunophenotyping, thymic output by the T-cell receptor excision circle (TREC) content together with CD31(+) naïve T-cell numbers and the relative telomere length (RTL) as a measure for proliferative history at pre-KTx, 3, 6 and 12months post-KTx. In addition, T-cell function was determined by measuring the proliferative capacity and percentages of cytokine-producing cells. Directly post-KTx, memory T-cell numbers were diminished but restored to pre-KTx values at 12months, except for CD4(+) EM T cells. The RTL of (memory) CD4(+) and CD8(+) T cells did not change. In contrast, TREC content and CD31(+) naïve T-cell numbers were stable post-KTx although the RTL of naïve CD4(+) and CD8(+) T cells decreased implying homeostatic proliferation of naïve cells, in response to a temporary decrease in memory cells. The T-cell function was not improved post-KTx. Our findings demonstrate that the uremia-associated aged phenotype is stably imprinted in the T-cell system and not reversed by KTx.

Bayesian immunological model development from the literature: Example investigation of recent thymic emigrants

Bayesian estimation techniques offer a systematic and quantitative approach for synthesizing data drawn from the literature to model immunological systems. As detailed here, the practitioner begins with a theoretical model and then sequentially draws information from source data sets and/or published findings to inform estimation of model parameters. Options are available to weigh these various sources of information differentially per objective measures of their corresponding scientific strengths. This approach is illustrated in depth through a carefully worked example for a model of decline in T-cell receptor excision circle content of peripheral T cells during development and aging. Estimates from this model indicate that 21years of age is plausible for the developmental timing of mean age of onset of decline in T-cell receptor excision circle content of peripheral T cells.

Modified interferon-α subtypes production and chemokine networks in the thymus during acute simian immunodeficiency virus infection, impact on thymopoiesis

Thymus dysfunction characterizes human/simian immunodeficiency virus (SIV) infections and contributes to physiopathology. However, both the mechanisms involved in thymic dysfunction and its precise timing remain unknown. We here analyzed thymic function during acute SIV infection in rhesus macaques. Rhesus macaques were intravenously infected with SIVmac251 and bled every 2/3 days or necropsied at different early time points postinfection. Naive T-cell counts were followed by flow cytometry and their T-cell receptor excision circle content evaluated by qPCR. Thymic chemokines were quantified by reverse transcription-qPCR and localized by in-situ hybridization in thymuses collected at necropsy. Thymic interferon alpha (IFN-α) subtype production was quantified by reverse transcription-qPCR combined to heteroduplex tracking assay. The effect of thymic IFN-α subtypes was tested on sorted triple negative thymocytes cultured on OP9-hDL1 cells. A reduced intrathymic proliferation history characterizes T cells produced during the first weeks of infection. Moreover, we evidenced a profound alteration of both chemokines and IFN-α subtypes transcriptional patterns in SIV-infected thymuses. Finally, we showed that IFN-α subtypes produced in the infected thymuses inhibit thymocyte proliferation, still preserving their differentiation capacity. Thymopoiesis is deeply impacted from the first days of SIV infection. Reduced thymocyte proliferation - a time-consuming process - together with modified chemokine networks is consistent with thymocyte differentiation speed-up. This may transiently enhance thymic output, thus increasing naive T-cell counts and diversity and the immune competence of the host. Nonetheless, long-lasting modification of thymic physiology may lead to thymic exhaustion, as observed in late primary HIV infection.

Uremia causes premature ageing of the T cell compartment in end-stage renal disease patients

BackgroundEnd-stage renal disease (ESRD) patients treated with renal replacement therapy (RRT) have premature immunologically aged T cells which may underlie uremia-associated immune dysfunction. The aim of this study was to investigate whether uremia was able to induce premature ageing of the T cell compartment. For this purpose, we examined the degree of premature immunological T cell ageing by examining the T cell differentiation status, thymic output via T cell receptor excision circle (TREC) content and proliferative history via relative telomere length in ESRD patients not on RRT.ResultsCompared to healthy controls, these patients already had a lower TREC content and an increased T cell differentiation accompanied by shorter telomeres. RRT was able to enhance CD8+ T cell differentiation and to reduce CD8+ T cell telomere length in young dialysis patients. An increased differentiation status of memory CD4+ T cells was also noted in young dialysis patients.ConclusionBased on these results we can conclude that uremia already causes premature immunological ageing of the T cell system and RRT further increases immunological ageing of the CD8+ T cell compartment in particular in young ESRD patients.

The Editors’ Choice

The Editors’ Choice

CD31 (PECAM‐1) is a marker of recent thymic emigrants among CD4+ T‐cells, but not CD8+ T‐cells or γδ T‐cells, in HIV patients responding to ART

Some severely immunodeficient HIV patients experience poor recovery of CD4(+) T-cell counts on antiretroviral therapy (ART). Evaluation of the function of thymopoiesis in T-cell production in individual patients requires a simple marker of T-cells that have recently emigrated from the thymus. Here, we address whether expression of CD31 on CD4(+) T-cells, CD8(+) T-cells, regulatory T-cells and gammadelta T-cells correlates with other indicators of thymus function. Adult HIV-1 patients (n=27) with nadir CD4(+) T-cell counts <100 per mul and a sustained virological response to ART and healthy controls (n=23) were studied. CD31 expression was assessed by flow cytometry, T-cell receptor excision circles content by real-time PCR and thymic volume by spiral computed tomography. Proportions of CD4(+) T-cells expressing CD45RA and CD31 declined with age in HIV patients (P=0.03) and healthy controls (P<0.0001), and correlated directly with other markers of thymus function. In controls, proportions of CD8(+) T-cells expressing CD45RA and CD31 declined with age (P=0.003) and correlated directly with some markers of thymus function, but this was not seen in HIV patients. Proportions of CD45RA(+) CD31(+) gammadelta T-cells were higher in patients than controls (P=0.007) and did not correlate with thymus volume. In controls, proportion of gammadelta T-cells co-expressing CD45RA and CD31 increased with age (P=0.002). These data support the use of CD31 as a marker of recent thymic origin in CD4(+) T-cells, but not CD8(+) T-cells in HIV patients receiving ART. In such patients, CD31 expression is unlikely to indicate thymic origin in gammadelta T-cells.

Elevated Proportions of Recent Thymic Emigrants in Children and Adolescents with Type 1 Diabetes

It is unclear, whether pediatric patients with type 1 diabetes (T1DM) show immunological alterations typically found in autoimmune conditions resembling immune dysfunction of the thymus, such as decrease of naïve T cells, lower T cell receptor excision circle (TREC) numbers, telomeric erosion, and diminished interleukin-7 (IL-7) levels. Furthermore, it is unknown, whether long-term therapy with insulin, a thymic growth factor, interferes with these changes. Therefore, the aim of this study was to analyze the quantity of the naïve T cell subset and its TREC content, relative telomere length (RTL) of naïve T cells, and peripheral IL-7 levels in patients with recent-onset T1DM (n = 5), long-standing T1DM (n = 33), and age-matched healthy donors (HD) (n = 37). In long-standing T1DM, TREC numbers/CD8+CD45RA+ T cells were enhanced (p < 0.01) compared to HD and correlated with disease duration (p < 0.02), an independent factor for increased thymic output (p < 0.01), and insulin dosage at blood withdrawal (p < 0.05). IL-7 serum levels were elevated in long-standing T1DM (p < 0.001) and positively correlated with TREC numbers (p < 0.01) and disease duration (p < 0.0001). RTLs in CD8+CD45RA+ T cells were significantly increased compared to HD (p < 0.02). Our data suggest that longterm insulin therapy may serve as a driving factor for thymic function and rejuvenation of the naïve T cell compartment. The ability of the immune system to reconstitute the naïve T cell compartment under well-adjusted insulin therapy may be of major importance for recognition of new antigens, response to vaccinations, and defense of infectious complications.

T-cell Receptor Excision Circles (TREC) in CD4+ and CD8+ T-cell Subpopulations in Atopic Dermatitis and Psoriasis Show Major Differences in the Emission of Recent Thymic Emigrants

We used T-cell receptor excision circles (TREC) to evaluate thymic function in adult patients with atopic dermatitis and psoriasis. We observed that men, but not women, with atopic dermatitis had a significantly faster decline in TREC content with increasing age compared with healthy men. In contrast, both men and women with psoriasis had significantly reduced TREC levels, which were, on average, only 30% of that of healthy persons. In atopic dermatitis the levels of TREC declined with increasing levels of IgE, disease intensity and extent of eczema. Furthermore, patients with atopic dermatitis showed signs of altered thymus function, as they had a significantly greater variation in TREC content measured over time than healthy controls, especially within the CD8+ T-cell subpopulation. Because both atopic dermatitis and psoriasis patients have an increased number of T-cells, this indicates that atopic dermatitis patients can have compensatory emissions of thymic emigrants, whereas psoriatic patients do not, thus supporting different thymic function in these two diseases.

The contribution of the thymus to the recovery of peripheral naive T-cell numbers during antiretroviral treatment for HIV infection.

The quantitative contribution of the thymus to the maintenance of peripheral populations of naive T cells is poorly understood. Several new lines of evidence indicate that thymic activity continues into adulthood, albeit at lower levels than in early life, and that this is important for a range of lymphopenic disorders. A measure of thymic activity that is often used is the quantification of T-cell receptor excision circles (TRECs). It has been shown that TREC levels decline after infection with HIV-1 and that they recover to above normal levels after antiretroviral treatment. The reasons for the latter observation are unknown. Here we quantitatively explore different possible causes for supranormal levels of TREC per cell and show that the small total number of cells involved in reconstituting the TREC+ T-cell pool of HIV-1-infected patients suffices to explain the observation. Even the expected small thymic outputs into a strongly depleted naive T-cell peripheral pool lead to a slow transient of elevated levels of TREC per cell. The main biological lesson from our quantitative modeling approach is that middle-aged human thymi continue to produce naive T cells and that this production can be demonstrated by tracking the increase of total TREC numbers (rather than the TREC content).

T cell receptor excision circles (TRECs) analysis during acute intrarectal infection of cynomolgus monkeys with pathogenic chimeric simian human immunodeficiency virus

Several studies have shown the importance of evaluating Recent Thymic Emigrants (RTEs) by quantification of T cell receptor-rearrangement excision circles (TRECs), as a measure of de novo T cell generation during human immunodeficiency virus-1 (HIV-1) infection. To determine whether acute viral infection may have an impact on TRECs, cynomolgus monkeys ( Macaca fascicularis) were infected intrarectally with simian human immunodeficiency virus (SHIV) 89.6P cy11 and the number of signal-joint (sj) TRECs was determined in purified CD4 + and CD8 + populations for up to 28 weeks post-infection. Four weeks after infection, TRECs levels significantly decreased in both CD3 +CD4 + and in CD3 +CD8 + T lymphocytes of infected monkeys, whereas they remained unchanged in uninfected animals. This reduction was followed by a progressive TRECs number recovery in CD3 +CD4 + T lymphocytes that positively correlated with changes in the levels of circulating CD3 +CD4 + T cells. In the CD3 +CD8 + T cell subset, TRECs number remained significantly low and inversely correlated with the increase in the percentages of CD3 +CD8 + T cells. These data suggest that SHIV89.6P cy11 intrarectal infection of cynomolgus monkeys differently affects TRECs content in CD3 +CD4 + and CD3 +CD8 + T cell subsets.

Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C

AbstractHepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4+CD25+ T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-γ production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3+CD4+CD25+ regulatory T cells (TRegs) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3+CD4+CD25+ TRegs suppressed IFN-γ production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, TReg cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3+CD4+CD25+ TReg cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3+CD4+CD25+TReg cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3+CD4+CD25+ TReg cells.

Maintenance of Telomere Length in Peripheral Blood CD4+CD25+ Regulatory T-Cells of Cancer Patients Despite Active Proliferation.

CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the up-regulation of telomerase activity, whose regulation also remains unknown for Treg. We therefore isolated Treg and the respective CD4+CD25− control T-cell population from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17). Analysis of their content of T-cell receptor excision circles (TREC) revealed that the observed increase of Treg frequencies in peripheral blood is due to active cycling rather than to redistribution from other compartments (i.e. secondary lymphoid organs or bone-marrow), as Treg from cancer patients are characterized by a significant decrease of TREC content when compared to TREC content of Treg isolated from healthy age-matched controls. Surprisingly, despite their proven in vivo proliferation, telomere length is not further shortened in Treg from peripheral blood of cancer patients as shown by Flow-Fish, Real-Time PCR and Southern Blotting. Accodingly, telomerase activity of Treg was readily inducible in vitro by OKT3 together with IL-2. Notably, sorting of in vitro proliferating Treg using dilution of CFSE revealed a significant telomere shortening in Treg with high proliferative capacity (i.e. CFSElow fraction) under conditions of strong in vitro stimulatory growth conditions despite a high telomerase activity. Thus, under conditions of strong in vitro stimulation induction of telomerase seems to be insufficient to avoid progressive telomere shortening. In contrast, in actively proliferating peripheral blood Treg from patients with epithelial malignancies induction of telomerase activity is likely to compensate for further telomere erosion.