Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors

We have used T-cell receptor (TCR) transgenic mice to analyze the interaction of tumors with the immune system. We show that the tumor cell line Lewis lung-lymphocytic choriomeningitis virus (LL-LCMV), genetically manipulated to express an H-2 Db-restricted epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV33-41), can grow progressively in TCR transgenic mice, where approximately 50% of CD8+ T cells are specific for LCMV33-41. TCR transgenic T cells were not deleted in tumor-bearing mice, and their surface phenotype and cytokine secretion patterns remained typical of naive T cells. Also, TCR transgenic T cells from tumor-bearing mice had undiminished capacity to proliferate to antigen in vitro. Tumor-protective immune responses could be elicited in TCR transgenic mice by immunization with LCMV33-41 peptide-loaded dendritic cells. Tumor resistance correlated with the switch of TCR transgenic T cells from a CD44low to a CD44high phenotype and increased capacity to produce IFNgamma in vitro. Results similar to those obtained in TCR transgenic mice were also obtained using an adoptive transfer system, where small numbers of TCR transgenic T cells were injected into normal C57BL/6 hosts. These data indicate that even large tumors may not induce specific immunization, tolerance, or anergy to tumor antigens, and that high numbers of tumor-specific CTL precursors are not sufficient to provide tumor resistance.     

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.     

Anti-CD3 epsilon F(ab')2 fragments inhibit T cell expansion in vivo during graft-versus-host disease or the primary immune response to nominal antigen

This study was undertaken to distinguish between several mechanisms responsible for graft-vs-host disease (GVHD) protection in anti-CD3epsilonF(ab')2 fragment (Fr)-treated recipients: TCR down-modulation, deletion, failure of expansion, or anergy induction. To quantify alloreactive T cell expansion and function, thoracic duct lymphocytes (TDL) were analyzed. Sixfold fewer donor TDL T cells were recoverable from anti-CD3epsilonF(ab')2 Fr as compared with irrelevant F(ab')2 Fr-treated recipients at the time of peak T cell expansion in vivo. Kinetic analysis revealed that donor T cell expansion was inhibited and not simply delayed by anti-CD3epsilonF(ab')2 Fr. Similar proportions of TDL T cells in irrelevant and anti-CD3epsilonF(ab')2 Fr were undergoing apoptosis. Although TCR modulation was observed, donor TDL T cells had intact anti-host alloresponses as compared with irrelevant F(ab')2 Fr-treated recipients. Because donor CD4+ T cells are primarily responsible for GVHD in this model, an adoptive transfer system was used in which the function and kinetics of expansion of OVA-specific CD4+ TCR transgenic cells could be physically tracked. Relevant Fr severely blunted CD4+ TCR transgenic T cell clonal expansion after OVA administration. Nonviable transgenic and nontransgenic T cells were proportionally similar in OVA-pulsed recipients, regardless of whether relevant or irrelevant F(ab')2 Fr were given. After discontinuing Fr, transgenic T cells were found to have intact in vitro OVA-specific responses. Our current and previous results suggest that reduced donor T cell expansion and T cell depletion both contribute to GVHD protection by anti-CD3epsilonF(ab')2 Fr. These data have implications for designing therapeutic approaches directed toward TCR targeting in humans.     

Expression of two alpha chains on the surface of T cells in T cell receptor transgenic mice

CD8+ T cells taken directly from mice expressing a Kb-specific T cell receptor (TCR) transgene expressed the transgenic TCR in a bimodal profile as detected by flow cytometric analysis using a clonotype-specific monoclonal antibody. Those cells expressing the lower density of the transgenic TCR expressed the transgenic beta chain and two different alpha chains on their surface. One alpha chain was the product of the alpha transgene, whereas the other was derived by endogenous rearrangement. This report provides the first demonstration that T cells isolated directly from mice may express two different TCR clonotypes on their surface. The potential consequences of this finding for studies using TCR transgenic mice and for the induction of autoimmunity are discussed.     

Telomerase is activated in the prostate and seminal vesicles of the castrated rat

Telomeres, the repetitive non-coding DNA sequences found at the ends of all eukaryotic chromosomes, shorten with each cell division. It has been proposed that telomere shortening may be the counting element of a mitotic clock that keeps track of cell divisions; with shortening to a critical length acting as a senescence signal underlying cellular aging. The enzyme telomerase functions to maintain telomere length, thus allowing unlimited cell division, and has been associated with cellular immortalization and cancer. Stem cells have large, perhaps unlimited, replicative capacities. Since these cells are potentially immortal, we reasoned that they might posses active telomerase. We therefore assayed for telomerase activity in the stem cell enriched pools of the androgen-depleted sex accessory tissues in the castrated male rat. Following castration, the ventral prostate and seminal vesicles of the rat involute, losing approximately 90% of their cells by 21 days. These residual glands persist, and are enriched for stem cells, being capable of fully regenerating these glands if testosterone is re-introduced into the animal. We assayed telomerase activity in extracts from normal, involuted, and regenerating ventral prostate and seminal vesicles. Normal glands were found to be telomerase negative, whereas telomerase activity appeared as these glands involuted following castration. Conversely, telomerase activity disappeared during testosterone-induced regeneration of these residual glands. These results provide strong evidence for the ability of androgen to negatively-regulate telomerase activity in stem cell populations of the rat ventral prostate and seminal vesicles. and represent the first in vivo model system for the modulation of telomerase activity.     

The anti-emetic activity of GK-128 in Suncus murinus

We have studied memory in T cell receptor (TCR) transgenic mice expressing a Db-restricted TCR specific for the male peptide (H-Y). CD8+ T cells from female TCR transgenic C57BL/6 (B6) mice were activated by transferring them into X-irradiated male (B6 x bm12)F1 hybrid recipients. Subsequently, they were highly purified by cell sorting and transferred for various lengths of time into female B6 nu/nu recipient mice. Other nu/nu recipient mice received highly purified naive T cells expressing the transgenic TCR. The functional potential of naive and "memory" T cells was analyzed by stimulation with male cells in vivo. The results show that memory cells can be derived from activated T cells and persist in the absence of antigen for at least 13 weeks. Naive and memory T cells differ in that memory T cells give a more vigorous and sustained response than naive T cells.     

Kinetics of the response of naive and memory CD8 T cells to antigen: similarities and differences

We have studied the kinetics of the antigen induced response of naive and memory CD8 T cells expressing a transgenic T cell receptor (TCR) specific for the glycoprotein peptide amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV). Memory T cells were generated in vivo by adoptive transfer of LCMV TCR transgenic T cells into normal recipient mice, followed by LCMV infection. The results demonstrated that the cell cycle progression and kinetics of TCR down-modulation, CD25 and CD69 up-regulation were identical in naive and memory T cells after antigen recognition. Moreover, the two T cell populations did not differ in respect of activation thresholds and in their proliferative capacities neither in vitro nor in vivo. However, memory CD8 T cells could be more rapidly induced to become cytolytic and to secrete high levels of interleukin-2 and interferon-gamma than naive T cells. LCMV GP33-specific CD8 memory T cells were only slightly more efficient in reducing LCMV titers in the spleen but were far more effective than naive LCMV GP33-specific T cells in controlling subcutaneous tumor growth of B16.F10 melanoma cells which expressed the LCMV GP33 epitope as tumor-associated antigen. Thus, in our experiments the main difference between CD8 memory T cells and naive cells is the ability of the former to rapidly acquire effector cell functions.     

The avidity spectrum of T cell receptor interactions accounts for T cell anergy in a double transgenic model

The mechanism of self-tolerance in the CD4(+) T cell compartment was examined in a double transgenic (Tg) model in which T cell receptor (TCR)-alpha/beta Tg mice with specificity for the COOH-terminal peptide of moth cytochrome c in association with I-Ek were crossed with antigen Tg mice. Partial deletion of cytochrome-reactive T cells in the thymus allowed some self-specific CD4(+) T cells to be selected into the peripheral T cell pool. Upon restimulation with peptide in vitro, these cells upregulated interleukin (IL)-2 receptor but showed substantially lower cytokine production and proliferation than cells from TCR Tg controls. Proliferation and cytokine production were restored to control levels by addition of saturating concentrations of IL-2, consistent with the original in vitro definition of T cell anergy. However, the response of double Tg cells to superantigen stimulation in the absence of exogenous IL-2 was indistinguishable from that of TCR Tg controls, indicating that these self-reactive cells were not intrinsically hyporesponsive. Measurement of surface expression of Tg-encoded TCR alpha and beta chains revealed that cells from double Tg mice expressed the same amount of TCR-beta as cells from TCR Tg controls, but only 50% of TCR-alpha, implying expression of more than one alpha chain. Naive CD4(+) T cells expressing both Tg-encoded and endogenous alpha chains also manifested an anergic phenotype upon primary stimulation with cytochrome c in vitro, suggesting that low avidity for antigen can produce an anergic phenotype in naive cells. The carboxyfluorescein diacetate succinimidyl ester cell division profiles in response to titered peptide +/- IL-2 indicated that expression of IL-2 receptor correlated with peptide concentration but not TCR level, whereas IL-2 production was profoundly affected by the twofold decrease in specific TCR expression. Addition of exogenous IL-2 recruited double Tg cells into division, resulting in a pattern of cell division indistinguishable from that of controls. Thus, in this experimental model, cells expressing more than one alpha chain escaped negative selection to a soluble self-protein in the thymus and had an anergic phenotype indistinguishable from that of low avidity naive cells. The data are consistent with the notion that avidity-mediated selection for self-reactivity in the thymus may lead to the appearance of anergy within the peripheral, self-reactive T cell repertoire, without invoking the induction of hyporesponsiveness to TCR-mediated signals.     

Overexpression of natural killer T cells protects Valpha14- Jalpha281 transgenic nonobese diabetic mice against diabetes

Progression to destructive insulitis in nonobese diabetic (NOD) mice is linked to the failure of regulatory cells, possibly involving T helper type 2 (Th2) cells. Natural killer (NK) T cells might be involved in diabetes, given their deficiency in NOD mice and the prevention of diabetes by adoptive transfer of alpha/beta double-negative thymocytes. Here, we evaluated the role of NK T cells in diabetes by using transgenic NOD mice expressing the T cell antigen receptor (TCR) alpha chain Valpha14-Jalpha281 characteristic of NK T cells. Precise identification of NK1.1(+) T cells was based on out-cross with congenic NK1.1 NOD mice. All six transgenic lines showed, to various degrees, elevated numbers of NK1.1(+) T cells, enhanced production of interleukin (IL)-4, and increased levels of serum immunoglobulin E. Only the transgenic lines with the largest numbers of NK T cells and the most vigorous burst of IL-4 production were protected from diabetes. Transfer and cotransfer experiments with transgenic splenocytes demonstrated that Valpha14-Jalpha281 transgenic NOD mice, although protected from overt diabetes, developed a diabetogenic T cell repertoire, and that NK T cells actively inhibited the pathogenic action of T cells. These results indicate that the number of NK T cells strongly influences the development of diabetes.     

Cellular mechanisms involved in protection against influenza virus infection in transgenic mice expressing a TCR receptor specific for class II hemagglutinin peptide in CD4+ and CD8+ T cells

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.     

Separately expressed T cell receptor alpha and beta chain transgenes exert opposite effects on T cell differentiation and neoplastic transformation

Two aspects of T cell differentiation in T cell receptor (TCR)-transgenic mice, the generation of an unusual population of CD4-CD8-TCR+ thymocytes and the absence of gamma delta cells, have been the focus of extensive investigation. To examine the basis for these phenomena, we investigated the effects of separate expression of a transgenic TCR alpha chain and a transgenic TCR beta chain on thymocyte differentiation. Our data indicate that expression of a transgenic TCR alpha chain causes thymocytes to differentiate into a CD4-CD8-TCR+ lineage at an early developmental stage, depleting the number of thymocytes that differentiate into the alpha beta lineage. Surprisingly, expression of the TCR alpha chain transgene is also associated with the development of T cell lymphosarcoma. In contrast, expression of the transgenic TCR beta chain causes immature T cells to accelerate differentiation into the alpha beta lineage and thus inhibits the generation of gamma delta cells. Our observations provide a model for understanding T cell differentiation in TCR-transgenic mice.     

Telomerase in cancer: clinical applications

The biology of telomeres and telomerase has been the subject of intensive investigative effort since it became evident that they play a significant role in two important biological processes, the loss of cellular replicative capacity inherent to organismal ageing and the unrestricted cell proliferation characteristic of carcinogenesis. Telomere shortening in normal cells is a result of DNA replication events, and reduction beyond a critical length is a signal for cellular senescence. One of the cellular mechanisms used to overcome proliferative restriction is the activation of the enzyme telomerase, which replaces the loss of telomeric DNA that occurs at each cell division. Studies have demonstrated that tumours have shorter telomeres than normal tissue and that telomerase is activated in up to 90% of all human cancers while it is present only in a limited range of normal adult tissues. The role of telomerase in the extension of the cellular replicative lifespan has recently been shown by ectopic expression of the enzyme, being consistent with the oncogenesis model whereby the acquisition of an 'immortal' phenotype is a requirement for advanced tumour progression. In this article we review the present knowledge of telomeres and telomerase in cancer and discuss the potential use of this enzyme as a diagnostic and prognostic tumour marker and as a target for cancer therapy.     

Telomerase activity in human acute myelogenous leukemia: inhibition of telomerase activity by differentiation-inducing agents

The terminal regions of human chromosomes, the telomeres, shorten with each cell division in most normal somatic cells. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is activated in germline cells and almost all tumor cells. Telomerase activity maintains the stability of telomere length, resulting in indefinite cellular proliferation (immortality). In the present study, telomerase activity was analyzed in leukemic mononuclear blood cells obtained from 56 patients with acute myelogenous leukemia (AML) with known cytogenetic alterations. Heterogenous levels of telomerase activity were observed and generally correlated with cytogenetic status. Patients with 11q abnormalities and -5/-7 (unfavorable cytogenetics) tended to have high telomerase activity compared with cells obtained from AML patients with other types of cytogenetics. Additional studies with a larger cohort of patients will determine whether these differences are statistically significant. Chemotherapy agents that result in differentiation of leukemic cells also resulted in inhibition of telomerase activity. Knowledge of telomerase activity in patients with AML, before and throughout therapy, may have clinical utility for following disease progression and may predict early cancer relapse.     

Effect of oncogene expression on telomerase activation and telomere length in human endothelial, fibroblast and prostate epithelial cells

Although strong evidence is mounting that telomerase reactivation and the thereof resulting stabilization of telomeres is a major mechanism for human cells to overcome replicative senescence, a causal relationship linking telomerase activation conclusively to tumorigenesis remains to be established. Thus, the possibility exists that telomerase activation is passively co-selected as tumors develop. To elucidate the function of telomerase during tumorigenesis, we followed telomerase reactivation during immortalization of human primary cell types with in vitro transforming agents and determined the tumorigenic potential of these cells at various stages of transformation. The effects of SV40, v-Ki-ras, HPV-18 and HPV-16 E6/E7 oncoproteins on telomerase expression was examined in primary and immortalized human prostate epithelial (HPE), human prostate fibroblast (HPF), and umbilical vein endothelial cells (HUVEC). All of five SV40-transformed HPE and HPF lines were telomerase positive and had shorter telomeres than primary cells. The two HPV-18 immortalized HPE cell lines also expressed telomerase activity. In contrast, E6 or E7 alone could not produce immortalized HUVEC and did not reactivate telomerase. Life-span, however, was extended. The E6/E7 immortalized HUVEC had telomerase activity and short but stable telomeres. HPE, HPF or HUVEC cells which had been transformed by one oncoprotein alone were not tumorigenic although they had overcome cellular senescence and re-activated telomerase. However, if these cells were transformed by a second agent, either infection with v-Ki-ras or X-ray treatment, they were able to form tumors in nude mice. This suggests that tumorigenesis is a multistep process and that telomerase activation alone is not sufficient for malignant transformation in human cells.     

Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin

Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis in mice is an autoimmune disease model of rheumatoid arthritis, which is MHC class II restricted and CD4 T cell dependent. To better understand the fundamental role of T cells in arthritis, we have generated a transgenic mouse carrying the rearranged Valpha11.1 and Vbeta8.2 TCR chain genes isolated from a type II collagen (CII)-specific T cell hybridoma. Cell surface analysis indicated that Vbeta8.2 chain was expressed on the surface of nearly all peripheral T cells. Analysis of T cell subsets in transgenic mice revealed a profound skewing in peripheral T cells towards the CD4 population. Although peripheral T cells were not tolerant to CII and responded to CII stimulation in vitro, transgenic mice did not develop spontaneous arthritis. However, a rapid onset of arthritis with severe clinical signs was detected in transgenic mice after immunization with CII in complete Freund's adjuvant. Histological analysis of inflamed joints showed a great resemblance to arthritic joints in man. This unique transgenic mouse model provides valuable insights into the mechanism of arthritis and into potential specific immune interventions.     

T cell receptor beta chain dimers on immature thymocytes from normal mice

During T cell development the T cell receptor (TCR) beta chain is expressed before the TCR alpha chain. Experiments in TCR beta transgenic severe combined immune deficiency (SCID) mice have shown that the TCR beta protein can be expressed on the cell surface of immature thymocytes in the absence of the TCR alpha chain and that the TCR beta protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCR beta chain. Subsequent experiments have shown that on the surface of thymocytes from TCR beta transgenic SCID mice the TCR beta protein can be expressed in a monomeric and dimeric form whereas only the dimeric form was found on the surface of a TCR beta-transfected, immature T cell line. The results presented here show that normal thymocytes from 16-day-old fetuses likewise express only the dimeric form and that the monomeric form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCR beta dimer can be expressed on the cell surface without the TCR alpha chain.     

Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.     

CD8 inhibits signal transduction through the T cell receptor in CD4-CD8- thymocytes from T cell receptor transgenic mice reconstituted with a transgenic CD8 alpha molecule

T cell repertoire selection processes involve intracellular signaling events generated through the TCR. The CD4 and CD8 coreceptor molecules can act as positive regulators of TCR signal transduction during these developmental processes. In this report, we have used TCR transgenic mice to determine whether TCR signaling can be modulated by the CD8 coreceptor molecule. These mice express on the majority of their T cells a TCR specific for the male (H-Y) Ag presented by the H-2Db MHC class I molecule. We show that CD4-CD8-, but not CD4-CD8+, thymocytes expressing the H-Y TCR responded with high intracellular calcium fluxes to TCR/CD3 stimulation without extensive receptor cross-linking. To examine the effects of CD8 expression on intracellular signaling responses in the CD4-CD8- cells, the H-Y TCR transgenic mice were mated with transgenic mice that constitutively expressed the CD8 alpha molecule on all T cells. The expression of the CD8 alpha alpha homodimer in the CD4-CD8-thymocytes led to impaired intracellular calcium responses and less efficient protein tyrosine phosphorylation of substrates after TCR engagement. In male H-2b H-Y transgenic mice, the majority of thymocytes have been deleted with the surviving cells expressing a high density of the transgenic TCR and exhibiting either a CD4-CD8- or CD4-CD8lo phenotype. It has been postulated that these cells escaped deletion by down-regulating the CD8 molecule. In the H-Y TCR/CD8 alpha double transgenic male mice, the CD4-CD8lo cells were completely eliminated as a result of CD8 alpha expression. However, the CD4-CD8- T cells were not deleted despite normal levels of the CD8 alpha transgene expression. These results suggest that the CD4-CD8- thymocytes may not be susceptible to the same deletional mechanisms as other thymocytes expressing TCR-alpha beta.     

Regulation of telomerase activity in immortal cell lines

Telomerase is a ribonucleoprotein whose activity has been detected in germ line cells, immortal cells, and most cancer cells. Except in stem cells, which have a low level of telomerase activity, its activity is absent from normal somatic tissues. Understanding the regulation of telomerase activity is critical for the development of potential tools for the diagnosis and treatment of cancer. Using the telomeric repeat amplification protocol, we found that immortal, telomerase-positive, pseudodiploid human cells (HT1080 and HL60 cells) sorted by flow repressed in quiescent cells. This was true whether quiescence was induced by contact inhibition (NIH 3T3 mouse cells), growth factor removal (bromodeoxyuridine-blocked mouse myoblasts), reexpression of cellular senescence (the reversibly immortalized IDH4 cells), or irreversible cell differentiation (HL60 promyelocytic leukemia cells and C2C12 mouse myoblasts). Taken together, these results indicate that telomerase is active throughout the cell in dividing, immortal cells but that its activity is repressed in cells that exit the cell cycle. This suggests that quiescent stem cells that have the potential to express telomerase may remain unaffected by potential antitelomerase cancer therapies.