Cord blood T cells retain early differentiation phenotype suitable for immunotherapy after TCR gene transfer to confer EBV specificity.

Adoptive T cell therapy can be effective for Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia.

To test the role of telomere biology in T-cell prolymphocytic leukemia (T-PLL), a rare aggressive disease characterized by the expansion of a T-cell clone derived from immuno-competent post-thymic T-lymphocytes, we analyzed telomere length and telomerase activity in subsets of peripheral blood leukocytes from 11 newly diagnosed or relapsed patients with sporadic T-PLL. Telomere length values of the leukemic T cells (mean+/-s.d.: 1.53+/-0.65 kb) were all below the 1st percentile of telomere length values observed in T cells from healthy age-matched controls whereas telomere length of normal T- and B cells fell between the 1st and 99th percentile of the normal distribution. Leukemic T cells exhibited high levels of telomerase and were sensitive to the telomerase inhibitor BIBR1532 at doses that showed no effect on normal, unstimulated T cells. Targeting the short telomeres and telomerase activity in T-PLL seems an attractive strategy for the future treatment of this devastating disease.

Dissociation of telomerase activity and telomere length maintenance in primitive human hematopoietic cells.

Primitive human hematopoietic cells have low endogenous telomerase activity, yet telomeres are not maintained. In contrast, ectopic telomerase expression in fibroblasts and other cells leads to telomere length maintenance or elongation. It is unclear whether this disparity can be attributed to telomerase level or stems from fundamentally different telomere biology. Here, we show that telomerase overexpression does not prevent proliferation-associated telomere shortening in human hematopoietic cells, pointing to the existence of cell type-specific differences in telomere dynamics. Furthermore, we observed eventual stabilization of telomere length without detectable changes in telomerase activity during establishment of two leukemic cell lines from normal cord blood cells, indicating that additional cooperating events are required for telomere maintenance in immortalized human hematopoietic cells.

Telomerase is limiting the growth of acute myeloid leukemia cells.

Telomeres play an important role in the proliferation and senescence of normal and malignant cells. To test the role of telomerase in acute myeloid leukemia (AML), we expressed the telomerase reverse transcriptase (hTERT) gene, a dominant-negative hTERT (DN-hTERT) (D868A, D869A) gene, or a gene encoding green fluorescence protein (GFP) in the leukemia cell line K562 and in primary AML cells from different patients, using retroviral vectors. Cells transduced with hTERT exhibited elevated levels of telomerase activity compared to GFP controls, whereas cells expressing DN-hTERT had decreased telomerase activity. K562 populations transduced with DN-hTERT showed reduced clonogenicity, telomere dysfunction and increased numbers of apoptotic cells compared to GFP- or hTERT-transduced cells. Two of four clones transduced with DN-hTERT died after 30 and 53 population doublings, respectively. Transduced AML cells were tested in primary colony-forming unit (CFU) and suspension culture assays. Relative to hTERT- and GFP-transduced controls, AML cells transfected with DN-hTERT produced fewer CFU and showed lower engraftment after transplantation into sublethally irradiated beta(2)-m(-/-) nonobese diabetic/severe combined immunodeficient mice. We conclude that telomerase is limiting the growth of the leukemic cell line K562 and primary AML progenitor cells. Our data warrant further studies of the therapeutic use of telomerase inhibitors in AML.

Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells.

DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ.

Limited telomere shortening in hematopoietic stem cells after transplantation.

The number of cell divisions in hematopoietic stem cells (HSCs) following transplantation of bone marrow or mobilized peripheral blood into myelo-ablated recipients is unknown. This number is expected to depend primarily on the number of transplanted stem cells, assuming that stem cells do not differ in engraftment potential and other functional properties. In a previous study, we found that the telomere length in circulating granulocytes in normal individuals shows a biphasic decline with age, most likely reflecting age-related changes in the turnover of HSCs. In order to study HSCs' proliferation kinetics following stem cells transplantation, we analyzed the telomere length in donor-derived nucleated blood cells in four HLA-matched bone marrow transplant recipients relative to comparable cells from the sibling donors. In each case, the telomeres in granulocytes were shorter in the recipient than in the donor. This difference was established in the first year post transplantation and did not change after that. The telomere length in naïve and memory T cells showed marked differences after transplantation, complicating the interpretation of telomere length data using unseparated nucleated blood cells. Interestingly, the telomere length in naïve T cells that were first observed six months post transplantation was very similar in donor and recipient pairs. Our observations are compatible with a limited number of additional cell divisions in stem cell populations after bone marrow transplantations and support the idea that different populations of stem cells contribute to short-term myeloid and long-term lympho myeloid hematopoiesis.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential.

In most human somatic cells telomeres progressively shorten with each cell division eventually leading to chromosomal instability and cell senescence. The loss of telomere repeats with cell divisions may also limit the replicative life span of antigen-specific T lymphocytes. Recent studies have shown that the replicative life span of various primary human cells can be prolonged by induced expression of the telomerase reverse transcriptase (hTERT) gene. To test whether introduction of hTERT can extend the life span of primary human T lymphocytes, naive CD8(+) T lymphocytes were transfected with retroviral vectors containing the hTERT gene. Transduced T-cell clones expressed high levels of telomerase and either maintained or elongated their telomere lengths upon culture for extended periods of time. Two of the transduced subclones retained a normal cloning efficiency for more than 170 population doublings (PDs). In contrast, T-cell clones transfected with control vectors exhibited progressive telomere length shortening and stopped proliferation at around 108 PDs. Telomerase-positive T clones had a normal 46,XY karyotype, maintained their cytotoxic properties, and showed very little staining for the apoptotic marker annexin-V. These results indicate that ectopic hTERT gene expression is capable of extending the replicative life span of primary human CD8(+) cytotoxic T lymphocytes. (Blood. 2001;98:597-603)

CD8+ T cells in large granular lymphocyte leukemia are not defective in activation- and replication-related apoptosis.

Persistent lymphocytosis in large granular lymphocyte leukemia (LGL) may result from defects in activation- or Fas crosslinking-induced cell death. Here we show that Fas crosslinking and CD3 activation causes apoptosis of in vitro activated CD8 T cells, but not of freshly isolated CD8 T cells. Death was partially blocked by a neutralizing antibody to FasL. Inhibition of metalloproteinase-mediated FasL solubilization significantly potentiated induction of cell death. Furthermore, CD3 plus CD28 stimulation resulted in telomeric erosion in LGL cells, and ultimately proliferation ceased. Together, these data indicate that activation- and proliferation-related cell death mechanisms are functional in LGL cells.

DNA strand break-sensing molecule poly(ADP-Ribose) polymerase cooperates with p53 in telomere function, chromosome stability, and tumor suppression.

Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP(-/-) cells showed telomere shortening and p53(-/-) cells showed normal telomere length, inactivation of PARP in p53(-/-) cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.

Extra-chromosomal telomeric DNA in cells from Atm(-/-) mice and patients with ataxia-telangiectasia.

Ataxia-telangiectasia (AT) is an autosomally recessive human genetic disease with pleiotropic defects such as neurological degeneration, immunodeficiency, chromosomal instability, cancer susceptibility and premature aging. Cells derived from AT patients and ataxia-telangiectasia mutated (ATM)-deficient mice show slow growth in culture and premature senescence. ATM, which belongs to the PI3 kinase family along with DNA-PK, plays a major role in signaling the p53 response to DNA strand breaks. Telomere maintenance is perturbed in yeast strains lacking genes homologous to ATM and cells from patients with AT have short telomeres. We examined the length of individual telomeres in cells from ATM(-/-) mice by fluorescence in situ hybridization. Telomeres were extensively shortened in multiple tissues of ATM(-/-) mice. More than the expected number of telomere signals was observed in interphase nuclei of ATM(-/-) mouse fibroblasts. Signals corresponding to 5-25 kb of telomeric DNA that were not associated with chromosomes were also noticed in ATM(-/-) metaphase spreads. Extrachromosomal telomeric DNA was also detected in fibroblasts from AT patients and may represent fragmented telomeres or by-products of defective replication of telomeric DNA. These results suggest a role of ATM in telomere maintenance and replication, which may contribute to the poor growth of ATM(-/-) cells and increased tumor incidence in both AT patients and ATM(-/-) mice.

Telomere length in leukocyte subpopulations of patients with aplastic anemia.

In most human cells, the average length of telomere repeats at the ends of chromosomes provides indirect information about their mitotic history. To study the turnover of stem cells in patients with bone marrow failure syndromes, the telomere length in peripheral blood granulocytes and lymphocytes from patients with aplastic anemia (AA, n = 56) and hemolytic paroxysmal nocturnal hemoglobinuria (n = 6) was analyzed relative to age-matched controls by means of fluorescence in situ hybridization and flow cytometry. The telomere lengths in granulocytes from patients with AA were found to be significantly shorter than those in age-adjusted controls (P =.001). However, surprisingly, telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia showed marked and significant telomere shortening. These results support extensive proliferation of hematopoietic stem cells in subgroups of AA patients. Because normal individuals show significant variation in telomere length, individual measurements in blood cells from AA patients may be of limited value. Whether sequential telomere length measurements can be used as a prognostic tool in this group of disorders remains to be clarified. (Blood. 2001;97:895-900)

Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation.

Using quantitative fluorescence in situ hybridization and flow cytometry, the telomere length of telomere repeat sequences after stem cell transplantation (SCT) were measured. The study included the telomeres of peripheral blood monocytes that should reflect the length of telomeres in stem cells and the telomeres of T lymphocytes that could shorten as a result of peripheral expansion. The loss of telomeres in monocytes and in memory T cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. In addition, the telomere length in the naive T cells that were produced by the thymus was comparable to the telomere length in the naive T cells of the donor. Compared to the total length of telomeres available, the loss of telomere repeats in leukocytes after SCT resembles the accelerated shortening seen in early childhood and remains, therefore, relatively insignificant.

Multicolor fluorescence in situ hybridization with peptide nucleic acid probes for enumeration of specific chromosomes in human cells.

In previous studies, we showed that peptide nucleic acid (PNA) probes have significant advantages over conventional synthetic RNA or DNA probes in FISH procedures for detecting telomeric and trinucleotide repeat sequences. Here, we report that directly labeled PNA probes recognizing chromosome-specific repeat sequences are also powerful tools for detecting and enumerating specific chromosomes in interphase and metaphase cells. This is illustrated by multicolor FISH experiments with cells from normal individuals and patients with numerical sex chromosome aberrations.

The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo.

Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.

Polyclonal hematopoiesis with variable telomere shortening in human long-term allogeneic marrow graft recipients.

Donor-derived hematopoiesis was assessed in 17 patients who received allogeneic marrow grafts from HLA-matched siblings between 1971 and 1980. Complete blood counts were normal or near normal in all patients except one. Chimerism analyses, using either dual-color XY-chromosome fluorescence in situ hybridization (FISH) or analysis of variable number tandem repeat loci, indicated that 15 out of 16 patients had greater than 97% donor-derived hematopoiesis, whereas 1 patient had indeterminate chimerism. All 12 recipients of grafts from female donors exhibited polyclonal hematopoiesis by X-linked clonal analysis with the use of molecular probes. Of the 17 recipients, 9 exhibited a less than 1.0-kilobase shortening of granulocyte telomere length compared with their respective donors, according to terminal restriction fragment analysis or flow-FISH with a fluorescein-labeled peptide nucleic acid probe. These data suggest that under standard transplantation conditions, the stem cell proliferative potential is not compromised during hematopoietic reconstitution. (Blood. 2000;96:3991-3994)