ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans.

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.

Downmodulation of tumor suppressor p53 by T cell receptor signaling is critical for antigen-specific CD4(+) T cell responses.

Antigen specificity is critical in immune response and requires integration of antigen-specific signals with antigen-nonspecific signals such as those provided by cytokines. The mechanism integrating these pathways is incompletely understood. We report here that antigen-specific proliferative responses of CD4(+) T cells required downmodulation of tumor suppressor p53. In the absence of T cell receptor (TCR) signal, IL-2 induced sustained increase in p53 protein, which prevented proliferative responses despite strong signaling through the IL-2 receptor. In contrast, TCR signaling resulted in early termination of p53 protein expression by decreasing p53 mRNA as well as strong transcriptional induction of the p53-regulating protein Mdm2. Downmodulation of p53 in response to antigen stimulation was in fact critical for antigen-specific T cell proliferation, and preventing p53 degradation by inhibiting Mdm2 resulted in sustained p53 protein and prevented antigen-specific T cell proliferation. It is thus termination of p53 by TCR signaling that allows proliferative responses, enforcing antigen specificity.

ATM influences the efficiency of TCRß rearrangement, subsequent TCRß-dependent T cell development, and generation of the pre-selection TCRß CDR3 repertoire.

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRß recombination and in subsequent TCRß-dependent repertoire generation and thymocyte development. CD4(-)CD8(-) double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRß loci and reduced Vß-DJß rearrangement. Sequencing of TCRß complementarity-determining region 3 demonstrates that ATM-deficient CD4(+)CD8(+) double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRß rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRß repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the ß-selection checkpoint to become double negative stage 4 and CD4(+)CD8(+) thymocytes, resulting in reduced numbers of CD4(+)CD8(+) cells. Importantly, expression of a rearranged TCRß transgene substantially reverses this defect in CD4(+)CD8(+) cells, directly linking a requirement for ATM during endogenous TCRß rearrangement to subsequent TCRß-dependent stages of development. These results demonstrate that ATM plays an important role in TCRß rearrangement, generation of the TCRß CDR3 repertoire, and efficient TCRß-dependent T cell development.

The requirement for pre-TCR during thymic differentiation enforces a developmental pause that is essential for V-DJß rearrangement.

T cell development occurs in the thymus and is critically dependent on productive TCRß rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (ß checkpoint), which is uniquely permissive for V-DJß recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRß rearrangement and pre-TCR expression enforces suppression of TCRß rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRß product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the ß checkpoint, would suppress all V-DJß rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRß rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRß rearrangement occurs, V-DJß rearrangement never occurs, even in DP cells that are permissive for D-Jß and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJß suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRß rearrangement to occur.

Telomere length is inherited with resetting of the telomere set-point.

We have studied models of telomerase haploinsufficiency in humans and mice to analyze regulation of telomere length and the significance of "set points" in inheritance of telomere length. In three families with clinical syndromes associated with short telomeres resulting from haploinsufficient mutations in TERT, the gene encoding telomerase reverse transcriptase, we asked whether restoration of normal genotypes in offspring of affected individuals would elongate inherited short telomeres. Telomeres were shorter than normal in some but not all genotypically normal offspring of telomerase-mutant parents or grandparents. Analysis of these findings was complicated by heterogeneity of telomere length among individuals, as well as by the admixing of telomeres inherited from affected parents with those inherited from unaffected ("wild-type" TERT) parents. To understand further the inheritance of telomere length, we established a shortened-telomere mouse model. When Tert(+/-) heterozygous mice were successively cross-bred through 17 generations, telomere length shortened progressively. The late-generation Tert(+/-) mice were intercrossed to produce genotypically wild-type Tert(+/+) mice, for which telomere length was characterized. Strikingly, telomere length in these Tert(+/+) mice was not longer than that of their Tert(+/-) parents. Moreover, when successive crosses were carried out among these short-telomere Tert(+/+) offspring mice, telomere length was stable, with no elongation up to six generations. This breeding strategy therefore has established a mouse strain, B6.ST (short telomeres), with C57BL/6 genotype and stable short telomeres. These findings suggest that the set point of telomere lengths of offspring is determined by the telomere lengths of their parents in the presence of normal expression of telomerase.

Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence.

BACKGROUND: Cellular senescence is a state reached by normal mammalian cells after a finite number of cell divisions and is characterized by morphological and physiological changes including terminal cell-cycle arrest. The limits on cell division imposed by senescence may play an important role in both organismal aging and in preventing tumorigenesis. Cellular senescence and organismal aging are both accompanied by increased DNA damage, seen as the formation of gamma-H2AX foci (gamma-foci), which may be found on uncapped telomeres or at non-telomeric sites of DNA damage. However, the relative importance of telomere- and non-telomere-associated DNA damage to inducing senescence has never been demonstrated. Here we present a new approach to determine accurately the chromosomal location of gamma-foci and quantify the number of telomeric versus non-telomeric gamma-foci associated with senescence in both human and mouse cells. This approach enables researchers to obtain accurate values and to avoid various possible misestimates inherent in earlier methods. RESULTS: Using combined immunofluorescence and telomere fluorescence in situ hybridization on metaphase chromosomes, we show that human cellular senescence is not solely determined by telomeric DNA damage. In addition, mouse cellular senescence is not solely determined by non-telomeric DNA damage. By comparing cells from different generations of telomerase-null mice with human cells, we show that cells from late generation telomerase-null mice, which have substantially short telomeres, contain mostly telomeric gamma-foci. Most notably, we report that, as human and mouse cells approach senescence, all cells exhibit similar numbers of total gamma-foci per cell, irrespective of chromosomal locations. CONCLUSION: Our results suggest that the chromosome location of senescence-related gamma-foci is determined by the telomere length rather than species differences per se. In addition, our data indicate that both telomeric and non-telomeric DNA damage responses play equivalent roles in signaling the initiation of cellular senescence and organismal aging. These data have important implications in the study of mechanisms to induce or delay cellular senescence in different species.

Persistence of tumor infiltrating lymphocytes in adoptive immunotherapy correlates with telomere length.

Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.

Abnormal differentiation of memory T cells in systemic lupus erythematosus.

OBJECTIVE: The chemokine receptor CCR7 and the tumor necrosis factor receptor family member CD27 define 3 distinct, progressively more differentiated maturational stages of CD4 memory subpopulations in healthy individuals: the CCR7+, CD27+, the CCR7-, CD27+, and the CCR7-, CD27- populations. The goal of this study was to examine maturational disturbances in CD4 T cell differentiation in systemic lupus erythematosus (SLE), using these phenotypic markers. METHODS: Phenotypic analysis by flow cytometry, in vitro stimulation experiments, telomere length measurement, and determination of inducible telomerase were carried out. RESULTS. In SLE patients, significant increases of CCR7-, CD27- and CCR7-, CD27+ and a reduction of CCR7+, CD27+ CD4 memory T cells were found. In vitro stimulation of SLE T cells showed a stepwise differentiation from naive to CCR7+, CD27+ to CCR7-, CD27+ to CCR7-, CD27-; telomere length and inducible telomerase decreased in these subsets in the same progressive sequence. The in vitro proliferative response of these populations progressively declined as their susceptibility to apoptosis increased. Interestingly, a significant reduction in inducible telomerase was noted in SLE naive and CCR7+, CD27+ CD4+ memory T cells. Additionally, SLE CCR7-, CD27+ and CCR7-, CD27- CD4 memory T cells proliferated poorly in response to in vitro stimulation and underwent significantly more apoptosis than their normal counterparts. Finally, expression of CXCR4 was significantly reduced in all SLE subsets compared with normal. CONCLUSION: Together these data indicate an increased degree of in vivo T cell stimulation in SLE, resulting in the accumulation of terminally differentiated memory T cells with a decreased proliferative capacity and an increased tendency to undergo apoptosis upon stimulation.

Stepwise differentiation of CD4 memory T cells defined by expression of CCR7 and CD27.

To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7+CD27+, the CCR7-CD27+, and the CCR7-CD27- population. In vitro stimulation led to a stepwise differentiation from naive to CCR7+CD27+ to CCR7-CD27+ to CCR7-CD27-. Telomere length in these subsets differed significantly (CCR7+CD27+ > CCR7-CD27+ > CCR7-CD27-), suggesting that these subsets constituted a differentiative pathway with progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7+CD27+, whereas IFN-gamma was produced by CCR7-CD27+ and to a slightly lesser extent by CCR7-CD27- T cells. IL-4 secretion was predominantly conducted by CCR7-CD27- memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined.

Regulated costimulation in the thymus is critical for T cell development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint.

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.

In vivo regulation of telomerase activity and telomere length.

Telomeres are specialized DNA-protein structures found at the ends of all linear chromosomes. In mammalian cells, they consist of hexanucleotide (TTAGGG) repeats and multiple associated proteins. Telomeres protect the ends of chromosomes and prevent their recognition as DNA breaks. Loss of functional telomere length below a critical threshold can activate programs leading to cell senescence or death. Telomere length represents a balance between the loss of terminal telomeric repeats, which occurs during cell division with incomplete DNA replication, and the addition of telomeric repeats by the unique RNA-dependent DNA polymerase telomerase. Although most somatic cells do not express telomerase, telomerase is induced in lymphocytes at critical stages of development and activation. Telomerase expression thus may prolong the replicative capacity of lymphocytes and thereby enhance their function in immune responses. We have used murine model systems to address two broadly defined questions about lymphocyte telomere biology: how is telomerase physiologically regulated in T cells responding to antigen challenge, and what is the effect of transcriptionally altered telomerase expression on telomere length and, consequently, on immune function?

Telomere biology and immune system.

Extract: In the 1960s Hayflick reported that normal human somatic cells have a finite replicative capacity and that, after a limited number of cell divisions, cells no longer divide and enter a state termed senescence. Since this observation was made, considerable research energy has focused on identifying molecular pathways that regulate the proliferative capacity of normal somatic (body) cells. One molecular mechanism that has been implicated in the regulation of somatic cell proliferation is mediated by telomeres -- specialized DNA-protein structures that cap the ends of all linear chromosomes. In mammalian cells, telomeres are composed of hexanucleotide repeats (TTAGGG) and a variety of associated proteins. Although all mammalian telomeres are composed of these (TTAGGG) repeats, telomere length varies substantially between different species. The higher order chromatin structure formed by telomeres functions to protect chromosomes ends from degradation and activation of DNA-repair pathways. In the absence of compensatory mechanisms, telomeres shorten progressively with successive rounds of cell division as a result of incomplete replication of telomeric termini, until they reach a critically short length that is no longer protective. Cells that lack protective telomeres fail to proliferate, and they undergo senescence or apoptosis.

Expression of telomerase RNA template, but not telomerase reverse transcriptase, is limiting for telomere length maintenance in vivo.

Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT(+/-) heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR(+/-) heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

Analysis of telomere length and telomerase activity.

Telomeres are specialized DNA-protein structures present at the ends of all linear chromosomes and are characterized by (TTAGGG)(n) hexanucleotide repeats and associated proteins. Telomere length has been implicated in cell survival and replicative capacity of dividing somatic cells. In the absence of an active compensatory mechanism, telomere lengths shorten as a consequence of proliferation, both in vitro and in vivo. However, this loss of telomeric repeats can be compensated and telomere length maintained by the enzyme telomerase, which is capable of adding (TTAGGG) repeat sequences to the ends of telomeres. This unit describes methods that are used for the measurement of telomere length and telomerase activity in human and murine cells.

Induction of telomerase activity and maintenance of telomere length in virus-specific effector and memory CD8+ T cells.

Acute viral infections induce extensive proliferation and differentiation of virus-specific CD8+ T cells. One mechanism reported to regulate the proliferative capacity of activated lymphocytes is mediated by the effect of telomerase in maintaining the length of telomeres in proliferating cells. We examined the regulation of telomerase activity and telomere length in naive CD8+ T cells and in virus-specific CD8+ T cells isolated from mice infected with lymphocytic choriomeningitis virus. These studies reveal that, compared with naive CD8+ T cells, which express little or no telomerase activity, Ag-specific effector and long-lived memory CD8+ T cells express high levels of telomerase activity. Despite the extensive clonal expansion that occurs during acute lymphocytic choriomeningitis virus infection, telomere length is maintained in both effector and memory CD8+ T cells. These results suggest that induction of telomerase activity in Ag-specific effector and memory CD8+ T cells is important for the extensive clonal expansion of both primary and secondary effector cells and for the maintenance and longevity of the memory CD8+ T cell population.

Telomeres in T and B cells.

Telomeres are the structures at the ends of linear chromosomes. In mammalian cells, they consist of hexanucleotide (TTAGGG) repeats, together with many associated proteins. In the absence of a compensatory mechanism, dividing cells undergo gradual telomere erosion until a critical degree of shortening results in chromosomal abnormalities and cell death or senescence. For T and B cells, the ability to undergo extensive cell division and clonal expansion is crucial for effective immune function. This article describes our current understanding of telomere-length regulation in lymphocytes and its implications for immune function.

Haploinsufficiency of mTR results in defects in telomere elongation.

Telomeres are usually maintained about an equilibrium length, and the set point for this equilibrium differs between species and between strains of a given species. To examine the requirement for telomerase in mediating establishment of a new telomere length equilibrium, we generated interspecies crosses with telomerase mTR knockout mice. In crosses between C57BL/6J (B6) and either of two unrelated mouse species, CAST/Ei and SPRET/Ei, telomerase mediated establishment of a new telomere length equilibrium in wild-type mTR(+/+) mice. This new equilibrium was characterized by elongation of the short telomeres of CAST/Ei or SPRET/Ei origin. In contrast, mTR(-/-) offspring of interspecies crosses failed to elongate telomeres. Unexpectedly, haploinsufficiency was observed in mTR(+/-) heterozygous interspecies mice, which had an impaired ability to elongate short SPRET/Ei or CAST/Ei telomeres to the new equilibrium set point that was achieved in wild-type mTR(+/+) mice. These results demonstrate that elongation of telomeres to a new telomere set point requires telomerase and indicate that telomerase RNA may be limiting in vivo.