Associations of Arsenic Exposure With Telomere Length and Naïve T Cells in Childhood-A Birth Cohort Study.

There is limited knowledge of association between arsenic exposure and telomere length (TL) and signal joint T-cell receptor excision circle (sjTREC) that are potential biomarkers of immune senescence and disease susceptibility. We aimed to clarify whether long-term inorganic arsenic exposure influences TL and sjTRECs in childhood. Children born in a longitudinal mother-child cohort were followed-up at 4.5 (n = 275) and 9 years (n = 351) of age. Arsenic exposure was assessed by metabolite concentrations in urine (U-As) from mothers at gestational week 8 (prenatal) and their children at 4.5 and 9 years. TL and sjTRECs were determined in blood cells using quantitative PCR. The oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) in plasma was measured by ELISA. In multivariable-adjusted spline regression analyses, both prenatal and childhood arsenic exposure above U-As of 45 µg/l were significantly inversely associated with TL and sjTRECs at 9 years. Fraction of monomethylarsonic acid (MMA) above spline knot 7% were significantly inversely associated with both TL and sjTRECs reflecting increased toxicity due to less-efficient arsenic metabolism in 9--year-old children. Prenatal and childhood arsenic exposure were positively associated with 8-OHdG at 9 years which in turn was inversely associated with sjTRECs at 9 years. However, adjustment with 8-OHdG did not change the estimates of the association of U-As with sjTRECs reflecting little contribution from 8-OHdG-induced oxidative stress. Our findings suggest that chronic arsenic exposure from early life can result in TL attrition and lower production of naïve T cells potentially leading to immunosenescence and immunodeficiency.

Minimal residual disease (MRD) detection using rearrangement of immunoglobulin/T cell receptor genes in adult patients with acute lymphoblastic leukemia (ALL).

MRD detection with allele-specific oligonucleotide-quantitative polymerase chain reaction (ASO-qPCR) and using clone-specific immunoglobulin/T cell receptor rearrangements is considered as a powerful prognostic factor in acute lymphoblastic leukemia (ALL). In the present study, we evaluated an ASO-qPCR assay for MRD quantification in peripheral blood (PB) samples of adult patients with ALL. DNA was isolated from PB samples of patients with newly diagnosed ALL. They were first investigated by multiplex-PCR assay to identify V/J usage. An ASO-qPCR technique was then applied for 2.5-year monthly MRD quantification for detection of patient-specific Ig/TCR receptor rearrangements as a molecular target. From 98 patients who were diagnosed as ALL, 72 (73.5%) were enrolled in the present study for MRD detection. MRD was successfully quantified in patients with 1-month interval time. MRD level at the end of induction therapy up to day 88 was the only significant prognostic factor. Regarding MRD level, patients were categorized into two groups of low and high-risk. 2.5-year OS in all three time points (days 28, 58 and 88) were significantly lower in high-risk group (P < 0.008). The results of the 2.5-year MRD detection indicate that MRD level at the end of induction up to about 6 months after the first diagnosis was associated with clinical outcome. This study may highlight the usefulness of PB and the definitions of cut-off level for early prediction of relapse and for stratifying ALL patients. Short-interval time points and frequent PB sampling to monitor MRD level is suggested for early clinical relapse prediction and clinical management of the disease.

Vitamin C promotes maturation of T-cells.

AIMS: Vitamin C (ascorbic acid) is thought to enhance immune function, but the mechanisms involved are obscure. We utilized an in vitro model of T-cell maturation to evaluate the role of ascorbic acid in lymphocyte development. RESULTS: Ascorbic acid was essential for the developmental progression of mouse bone marrow-derived progenitor cells to functional T-lymphocytes in vitro and also played a role in vivo. Ascorbate-mediated enhancement of T-cell development was lymphoid cell-intrinsic and independent of T-cell receptor (TCR) rearrangement. Analysis of TCR rearrangements demonstrated that ascorbic acid enhanced the selection of functional TCRαß after the stage of ß-selection. Genes encoding the coreceptor CD8 as well as the kinase ZAP70 were upregulated by ascorbic acid. Pharmacologic inhibition of methylation marks on DNA and histones enhanced ascorbate-mediated differentiation, suggesting an epigenetic mechanism of Cd8 gene regulation via active demethylation by ascorbate-dependent Fe(2+) and 2-oxoglutarate-dependent dioxygenases. INNOVATION: We speculate that one aspect of gene regulation mediated by ascorbate occurs at the level of chromatin demethylation, mediated by Jumonji C (JmjC) domain enzymes that are known to be reliant upon ascorbate as a cofactor. JmjC domain enzymes are also known to regulate transcription factor activity. These two mechanisms are likely to play key roles in the modulation of immune development and function by ascorbic acid. CONCLUSION: Our results provide strong experimental evidence supporting a role for ascorbic acid in T-cell maturation as well as insight into the mechanism of ascorbate-mediated enhancement of immune function.

An in vitro model of T cell receptor revision in mature human CD8+ T cells.

V(D)J recombination is a mechanism peculiar to the somatic rearrangement of antigen receptor genes. It requires both expression of the RAG-1 and RAG-2 recombinases and accessibility of the substrate to its recombinase and post-cleavage/DNA repair stage. TCR revision is a genetic correction mechanism that changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. This process is now well described in both normal or pathological murine and human settings. Many of its features, such as the question of whether it occurs in truly mature T cells, remain to be elucidated. Its occurrence in human CD8+ T cells is also an open question. We have therefore established an in vitro model of TCR revision in mature human CD8+ T cells to determine whether down-regulation of the TCR/CD3 complex from the cell surface in the presence of IL7 as a factor favouring chromatin remodelling initiates a TCR revision pathway. Only mature CD8+ T cells carrying already-formed antigen receptors were used. CD8+ T cells treated with anti-CD3 and IL7 showed rearrangement intermediates and expressed new Vbeta-chains on their surface. Investigation of the molecular pathway thus induced disclosed up-regulation of the RAG-2 transcript, but absence of the 'canonical' RAG-1 mRNA. A surprising finding was the demonstration of alternative splice forms of this mRNA, already expressed in untreated CD8+ T cells, encoding for the full-length RAG-1 protein, which was increased three-fold in the treated cells. All the V(D)J requirements were thus fulfilled when mature human CD8+ T cells were stimulated with anti-CD3 and IL7. Induction of TCR revision in vitro in mature T cells is an easily controllable system that could be employed in further studies to elucidate the molecular pathways involved in secondary V(D)J rearrangements in peripheral cells.

[Effects of lead exposure on thymic output naive T cells function].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Lead exposure may damage thymic output naive T cells function. Furthermore, low-level exposure to lead may damage immune system and earlier than expected.

Different thresholds of Notch signaling bias human precursor cells toward B-, NK-, monocytic/dendritic-, or T-cell lineage in thymus microenvironment.

Notch receptors are involved in lineage decisions in multiple developmental scenarios, including hematopoiesis. Here, we treated hybrid human-mouse fetal thymus organ culture with the gamma-secretase inhibitor 7 (N-[N-(3,5-difluorophenyl)-l-alanyl]-S-phenyl-glycine t-butyl ester) (DAPT) to establish the role of Notch signaling in human hematopoietic lineage decisions. The effect of inhibition of Notch signaling was studied starting from cord blood CD34(+) or thymic CD34(+)CD1(-), CD34(+)CD1(+), or CD4ISP progenitors. Treatment of cord blood CD34(+) cells with low DAPT concentrations results in aberrant CD4ISP and CD4/CD8 double-positive (DP) thymocytes, which are negative for intracellular T-cell receptor beta (TCRbeta). On culture with intermediate and high DAPT concentrations, thymic CD34(+)CD1(-) cells still generate aberrant intracellular TCRbeta(-) DP cells that have undergone DJ but not VDJ recombination. Inhibition of Notch signaling shifts differentiation into non-T cells in a thymic microenvironment, depending on the starting progenitor cells: thymic CD34(+)CD1(+) cells do not generate non-T cells, thymic CD34(+)CD1(-) cells generate NK cells and monocytic/dendritic cells, and cord blood CD34(+)Lin(-) cells generate B, NK, and monocytic/dendritic cells in the presence of DAPT. Our data indicate that Notch signaling is crucial to direct human progenitor cells into the T-cell lineage, whereas it has a negative impact on B, NK, and monocytic/dendritic cell generation in a dose-dependent fashion.

Delayed expansion of a restricted T cell repertoire by low-density TCR ligands.

The role of TCR ligand density (i.e. the number of antigen-MHC complexes) in modulating the diversity of a T cell response selected from a pool of naive precursors remains largely undefined. By measuring early-activation markers up-regulation and proliferation following stimulation with staphylococcal enterotoxin A (SEA), we demonstrate that decreasing the ligand dose below an optimal concentration leads to the delayed activation of a restricted set of TCRVbeta-bearing T cells, with the specific, non-stochastic exclusion of some TCRVbeta+ T cells from the activated pool. Our results suggest that the failure of these TCRVbeta-bearing T cells to reach the activation threshold at sub-optimal ligand concentration is due to the inefficiency of TCR engagement, as measured by TCR internalization, and does not correlate with the relative precursor frequency in the non-immune repertoire. Moreover, even at SEA concentrations that lead to the simultaneous proliferation of all SEA-reactive T cells, we observe marked differences in the ability to secrete cytokines among the different responsive TCRVbeta-bearing T cells. Altogether, our results indicate that the development of a T cell response to a scarce display of ligand significantly narrows TCR repertoire diversity by mechanisms that involve focusing of the repertoire on the expansion of those T cells with the highest avidity of TCR engagement.

Increased peripheral expansion of naive CD4+ T cells in vivo after IL-2 treatment of patients with HIV infection.

Intermittent interleukin-2 (IL-2) therapy has been shown to increase the number of CD4+ T cells, preferentially cells with a naive phenotype, in patients with HIV infection. For this report we investigated the mechanism underlying this expansion by studying the relative roles of peripheral expansion and thymic output. In a cohort of six patients receiving IL-2 over a period of 1 year, the mean number of naive CD4+ T cells increased from 139 to 387 cells per microl while levels of T cell receptor rearrangement excision circles (TRECs) declined from 47,946 to 26,510 copies per 10(6) naive T cells, thus making it unlikely that the CD4+ T cell count increases were secondary to increase in thymic output. To examine directly the impact of IL-2 on peripheral expansion, peripheral blood mature, naive CD4+ T cells were labeled ex vivo with 5-bromodeoxyuridine as well as stained directly for Ki67. These studies revealed a 7-fold increase in the percentage of 5-bromodeoxyuridine-positive cells and a 20-40-fold increase in Ki67 staining in the naive CD4+ T cell pool in the setting of IL-2 administration. This degree of increase in mature CD4+ T cell turnover induced by IL-2 does not compromise the future replicative potential of these cells, because longitudinal measurements of telomere length went from 6,981 to 7,153 bp after 1 year of IL-2 therapy. These data strongly suggest that much of the increase in CD4+ cells associated with IL-2 treatment is caused by peripheral expansion of existing naive CD4+ T cells rather than increased thymic output and that these increases occur without compromising the potential of these cells for further cell division.

Effects of exogenous interleukin-7 on human thymus function.

Immune reconstitution is a critical component of recovery after treatment of human immunodeficiency virus (HIV) infection, cancer chemotherapy, and hematopoietic stem cell transplantation. The ability to enhance T-cell production would benefit such treatment. We examined the effects of exogenous interleukin-7 (IL-7) on apoptosis, proliferation, and the generation of T-cell receptor rearrangement excision circles (TRECs) in human thymus. Quantitative polymerase chain reaction demonstrated that the highest level of TRECs (14 692 copies/10 000 cells) was present in the CD1a(+)CD3(-)CD4(+)CD8(+) stage in native thymus, suggesting that TREC generation occurred following the cellular division in this subpopulation. In a thymic organ culture system, exogenous IL-7 increased the TREC frequency in fetal as well as infant thymus, indicating increased T-cell receptor (TCR) rearrangement. Although this increase could be due to the effect of IL-7 to increase thymocyte proliferation and decrease apoptosis of immature CD3(-) cells, the in vivo experiments using NOD/LtSz-scid mice given transplants of human fetal thymus and liver suggested that IL-7 can also directly enhance TREC generation. Our results provide compelling evidence that IL-7 has a direct effect on increasing TCR-alphabeta rearrangement and indicate the potential use of IL-7 for enhancing de novo naïve T-cell generation in immunocompromised patients.

Biomarker correlations of urinary 2,4-D levels in foresters: genomic instability and endocrine disruption.

Forest pesticide applicators constitute a unique pesticide use group. Aerial, mechanical-ground, and focal weed control by application of herbicides, in particular chlorophenoxy herbicides, yield diverse exposure scenarios. In the present work, we analyzed aberrations in G-banded chromosomes, reproductive hormone levels, and polymerase chain reaction-based V(D)J rearrangement frequencies in applicators whose exposures were mostly limited to chlorophenoxy herbicides. Data from appliers where chlorophenoxy use was less frequent were also examined. The biomarker outcome data were compared to urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) obtained at the time of maximum 2,4-D use. Further comparisons of outcome data were made to the total volume of herbicides applied during the entire pesticide-use season.Twenty-four applicators and 15 minimally exposed foresters (control) subjects were studied. Categorized by applicator method, men who used a hand-held, backpack sprayer in their applications showed the highest average level (453.6 ppb) of 2,4-D in urine. Serum luteinizing hormone (LH) values were correlated with urinary 2,4-D levels, but follicle-stimulating hormone and free and total testosterone were not. At the height of the application season; 6/7 backpack sprayers, 3/4 applicators who used multinozzle mechanical (boom) sprayers, 4/8 aerial applicators, and 2/5 skidder-radiarc (closed cab) appliers had two or more V(D)J region rearrangements per microgram of DNA. Only 5 of 15 minimally exposed (control) foresters had two or more rearrangements, and 3 of these 5 subjects demonstrated detectable levels of 2,4-D in the urine. Only 8/24 DNA samples obtained from the exposed group 10 months or more after their last chlorophenoxy use had two rearrangements per microgram of DNA, suggesting that the exposure-related effects observed were reversible and temporary. Although urinary 2,4-D levels were not correlated with chromosome aberration frequency, chromosome aberration frequencies were correlated with the total volume of herbicides applied, including products other than 2,4-D. In summary, herbicide applicators with high urinary levels of 2,4-D (backpack and boom spray applications) exhibited elevated LH levels. They also exhibited altered genomic stability as measured by V(D)J rearrangement frequency, which appears reversible months after peak exposure. Though highly detailed, the limited sample size warrants cautious interpretation of the data.

Biomarkers in long survivors of pediatric acute lymphoblastic leukemia patients: late effects of cancer chemotherapy.

In order to elucidate the late effects of cancer chemotherapy, mutant frequencies (Mfs) at the hypoxanthine phosphoribosyl transferase (hprt) locus were evaluated in pediatric patients with early pre-B acute lymphoblastic leukemia (ALL). Hprt-Mfs were measured at least 2 years after completion of chemotherapy. Ten out of 15 patients were found to have hprt-Mfs exceeding the 99% confidence limits as calculated from observations of healthy controls. Although there was some intraindividual variation, serial measurements of hprt-Mfs with intervals of more than 6 months revealed that hprt-Mfs were fairly stable. Patients with high Mfs tended to have sibling clones as detected by clonality analysis using the T-cell receptor (TCR) rearrangement pattern, but clonality did not have a major effect on the Mfs. On the other hand, Mfs at the TCR locus and sister chromatid exchange frequency were within the normal range in all patients. These data suggest that chemotherapy can cause persistent genotoxicity in vivo in a subset of pediatric ALL patients and that the hprt-Mf is a useful method for measuring such an effect.

Il-7 supports D-J but not V-DJ rearrangement of TCR-beta gene in fetal liver progenitor cells.

The rearrangement of TCR-beta gene, one of the earliest events in T cell development, consists of two consecutive steps: D-J rearrangement and V-DJ rearrangement. The present study examined the signals supporting D-J beta and V-DJ beta rearrangements during early T cell development from progenitor cells that reside in fetal liver. We have found that there is an interval of 1 to 2 days between D-J beta and V-DJ beta rearrangements during the early T cell development from fetal liver progenitor cells in deoxyguanosine-treated thymus lobes. We have also found that IL-7, a cytokine expressed in the subcapsular area of the thymus, can promote D-J beta rearrangement of fetal liver progenitor cells, and that anti-IL-7 and anti-IL-7R Abs inhibit the D-J beta rearrangement and further T cell development of fetal liver progenitor cells in the thymus environment. Interestingly, unlike the thymus environment, IL-7 alone was not capable of supporting V-DJ beta rearrangement in the fetal liver cell cultures. These results indicate that D-J beta rearrangement during fetal liver-derived early T cell development is supported in the thymus by IL-7. Furthermore, the present results demonstrate that IL-7, supporting D-J beta rearrangement, does not promote V-DJ beta rearrangement of fetal liver progenitor cells, suggesting that intrathymic molecules promoting V-DJ beta rearrangement are distinct from IL-7 that supports the D-J beta rearrangement.

Role of IL-7 in the shaping of the pulmonary gamma delta T cell repertoire.

Gamma delta T cells bearing the canonical fetal-type V gamma 6/V delta 1 rearrangements are the predominant gamma delta T cells in the lungs of adult mice. In contrast, these V gamma 6/V delta 1 T cells are virtually absent in the pulmonary epithelia of nude mice. The intraepithelial dominance of gamma delta T cells that express this particular TCR is thought to result from a preferred thymic pathway of gene rearrangement and not from TCR-mediated positive selection. We now show that gamma delta T cell precursors in the lung epithelium of both euthymic and athymic neonatal mice generate this rearrangement in situ. In athymic mice, these clonotypes do not survive, but can be rescued in vitro and in vivo by the lymphokine IL-7.

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation.

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR beta and gamma genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mean MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR gamma along with an apparent germline TCR beta configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR gamma and beta genes. Possible explanations for these findings are discussed.

Large granular lymphocytosis terminating in a polymorphous B-lymphocytic proliferation after low-dose cyclophosphamide therapy: a case report with necropsy findings.

A 70-year-old man presented with clonal large granular lymphocytosis of T-suppressor/cytotoxic immunophenotype, neutropenia, paraproteinemia, and proneness to infection. The patient became severely leukopenic during 14 days of chemotherapy with low-dose cyclophosphamide, and remained so after discontinuation of the drug. Clinically, he was thought to have prolonged chemotherapy-induced marrow hypoplasia. At death, 16 days after the last dose of chemotherapy, autopsy confirmed bone marrow hypoplasia and revealed that well-differentiated, polymorphous, and (immunophenotypically and genotypically) polyclonal B-lymphocytes predominated in normal hematopoietic and lymphoid organs. A similar lymphoid infiltrate was intimately associated with multiple ulcers and smooth muscle necrosis in the stomach. These terminal findings resemble B-lymphoproliferative conditions described in certain forms of immune deficiency.

Use of immunoglobulin gene rearrangements to show clonal lymphoproliferation in hyper-reactive malarial splenomegaly.

In Africa, hyper-reactive malarial splenomegaly (HMS), which is also known as tropical splenomegaly syndrome, can be associated with a prominent lymphocytosis in blood and bone marrow that is difficult to distinguish clinically from chronic lymphocytic leukaemia (CLL). The observation that some patients with HMS become resistant to treatment with anti-malarial drugs has led to the suggestion that HMS may evolve into a malignant lymphoproliferative disorder. To test this hypothesis, 22 Ghanaian patients with HMS and/or lymphocytosis were categorised by degree of response to proguanil according to standard clinical criteria, and DNA was extracted from peripheral blood cells and screened for rearrangements of the Jh region of the immunoglobulin gene with a DNA probe. Clonal rearrangements of the Jh region were found in all 3 patients with no response, in none of 13 patients with sustained response, and in 2 of 6 patients with moderate response or relapse on proguanil therapy. The detection of such rearrangements, and hence clonal lymphoproliferation in individuals with clinical features intermediate between HMS and CLL, supports the hypothesis that HMS may evolve into a malignant lymphoproliferative disorder.