Karen S. Hathcock

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.

Inhibition Of Transplant Rejection Following Treatment With Anti-b7-2 And Anti-b7-1 Antibodies

Antigen-specific T cell activation depends initially on the interaction of the T cell receptor (TCR) with peptide/MHC. In addition, a costimulatory signal, mediated by distinct cell surface accessory molecules, is required for complete T cell activation leading to lymphokine production and proliferation. CD28 has been implicated as the major receptor on T cells responsible for delivering the costimulatory signal. Although two distinct ligands for CD28, B7-1 and B7-2, have been identified on antigen-presenting cells (APC), the co-stimulatory role of each molecule during a physiological immune response remains unresolved. In the present study, the relative roles of B7-1 and B7-2 interactions were evaluated in an allogeneic pancreatic islet transplant setting. In isolation, anti-B7-2 mAbs and, to a much lesser degree, anti-B7-1 mAbs suppressed T cell proliferative responses to allogeneic islets or splenic APC in vitro. Maximal inhibition of the allogeneic response was observed using a combination of the anti-B7-1 and anti-B7-2 mAbs. Administration of anti-B7-2 but not anti-B7-1 mAbs prolonged C3H allograft survival in B6 recipients, with a combination of both mAbs significantly prolonging rejection beyond either mAb alone. The immunosuppressive effects of the in vivo mAb treatment were not manifested in in vitro analyses as T cells isolated from suppressed mice responded normally to allogeneic stimuli in terms of both proliferation and lymphokine production. However, combined mAb therapy in vivo selectively delayed CD4+ T lymphocyte infiltration into the graft. These data suggest that both B7-1 and B7-2 costimulatory molecules are active in vivo, although B7-2 plays a clearly dominant role in this allograft model. The mechanism of immune suppression in vivo remains unresolved but may occur at sites distinct from the allograft.

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-γ 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.

Regulation of Telomere Length and Telomerase in T and B Cells: A Mechanism for Maintaining Replicative Potential

Recent studies have established that telomere length is altered during differentiation of both T and B lymphocytes. There exists a clear and strong correlation of replicative capacity with telomere length in normal somatic cells. In human T cells, this correlation extends to differences in telomere length and replicative capacity in subsets such as naive and memory CD4 cells, or CD28−positive and negative CD8 T cells. In addition, telomerase, a unique reverse transcriptase that is capable of extending telomeric length, is highly regulated during development and activation of both T and B lymphocytes. Together, these findings have provided a basis for hypotheses linking telomere length regulation to a functional role in sustaining the capacity for extensive clonal expansion in antigen-specific lymphocytes. In addition to providing insights into basic immune function, manipulation of telomere length has potential therapeutic applications as well. For example, the ability to extend the replicative capacity of cells such as hematopoietic stem cells or mature lymphocytes through telomerase induction by transfection could be critical to therapeutic approaches to adoptive cell transfer or reconstitution. In assessing the feasibility of such approaches, it will be critical not only to measure extension of the capacity for cell division but also to consider other possible consequences such as enhanced susceptibility to malignant transformation through dysregulated telomerase activity. Conversely, the proposed use of telomerase inhibition as a modality for anticancer therapy should consider the possible impact of such intervention on any telomerase-dependent aspects of immune function. The rapid pace of gene discovery and genetic engineering, in combination with a richness of available systems for studying immune cell biology, should allow vigorous pursuit of these remaining questions.‡To whom correspondence should be addressed (e-mail: Richard_Hodes@nih.gov).

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.

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.

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

BackgroundCellular 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 γ-H2AX foci (γ-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 γ-foci and quantify the number of telomeric versus non-telomeric γ-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.ResultsUsing 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 γ-foci. Most notably, we report that, as human and mouse cells approach senescence, all cells exhibit similar numbers of total γ-foci per cell, irrespective of chromosomal locations.ConclusionOur results suggest that the chromosome location of senescence-related γ-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.

Expression of Telomerase RNA Template, but Not Telomerase Reverse Transcriptase, Is Limiting for Telomere Length Maintenance In Vivo

ABSTRACT 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.

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.

Distinct B cell subpopulations differ in their genetic requirements for activation by T helper cells.

Is the activation of B cells by T-helper (TH) cells genetically restricted by products of the MHC or not? Although this would appear to be a straightforward question, attempts to answer it have resulted in one of the most persistent and perplexing controversies in modern cellular immunology (Katz et al. 1973, Sprent 1978a, 1978b,Swierkoszetal. 1978, Yamashita&Shevach 1978, McDougal & Cort 1978, Erb et al. 1979, Singer et al. 1979,1980, Shih et al. 1980). A general concensus exists that TH cell-macrophage (M0) interactions are always MHC restricted (Rosenthal & Shevach 1973). In contrast, it has become increasingly apparent that TH cell-B cell interactions are MHC restricted under some experimental circumstances but are not MHC restricted under other experimental circumstances. However, it has not been obvious what distinguishes those circumstances in which TH-B interactions are genetically restricted from those in which they are not. One possibility which we considered and will detail in this review is that there exist two distinct subpopulations of B cells which differ in their genetic requirements for activation by TH cells. B cells from normal adult mice can be separated into two subpopulations of approximately equal size based on their expression of the differentiation antigen Lyb5, such that one subpopulation is Lyb5* and one subpopulation is LybS (Ahmed et al. 1977, Ahmed & Scher 1979). The Lyb5 determinant itself is encoded by a single locus with two allelic forms, Lyb5.1 and Lyb5.2. B cells that do not express the Lyb5 determinant (i.e. Lyb5 B cells) are present at birth while Lyb5̂ B cells do not appear in the spleens of normal mice until 2-3 weeks of age.