In vitro senescence models for human T lymphocytes

Vaccine 18 (2000) 1666±1674

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In vitro senescence models for human T lymphocytes Graham Pawelec*, Medi Adibzadeh, Arnika Rehbein, Karin HaÈhnel, Wolfgang Wagner, Andrea Engel TuÈbingen Ageing and Tumour Immunology (TATI) Group, Section for Transplantation Immunology and Immunohaematology, Second Department of Internal Medicine, Medizinische UniversitaÈtsklinik und Poliklinik, Otfried-MuÈller-Str. 10, D-72076, TuÈbingen, Germany

Abstract Immunosenescence is an age-associated dysregulation of immune function which may contribute to the increased susceptibility of the elderly to infectious disease. Although age-associated changes are measurable in the innate immune system, it is the adaptive arm of the immune system which is particularly susceptible to the deleterious e€ects of ageing, especially the T cell compartment. In this review, the characteristics of longitudinal ageing in cultured monoclonal human T cell populations will be summarized. It will be argued that parallels between this in vitro model and T cell senescence in vivo suggest the use of such models to screen for interventions ameliorating immunosenescence in vivo. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Immunosenescence; T cells; Intervention

1. Introduction: adaptive immune responses Adaptive immune responses are mediated by T and B cells. The humoral immune system depends on the adequate function of the T cell system, which in turn depends upon fully functional innate immunity. Although ageing a€ects the innate immune system in many ways that are just beginning to be explored [1], on the whole, antigen presentation functions seem to be fairly adequately maintained in the elderly [2]. Alterations in humoral immunity appear to be mostly consequent to alterations in T cell immunity. Therefore, this review will con®ne itself to age-associated alterations in human T cells. Successful immune responses depend on the initial activation of very small numbers of antigen-speci®c T cells and their clonal expansion to sucient numbers for e€ective immunity. The precursor frequency for antigen-speci®c T cells is something like 1 in 10,000± * Corresponding author. Tel.:+49-7071-298-2805; fax: +49-7071294464. E-mail address: [email protected] (G. Pawelec).

100,000. In acute viral infection, at least, the immune response seems to depend on only a relatively small number of di€erent T cell clones, but each has to expand to a clone size of 108 or more [3]. This implies that around 28 population doublings (PD) would be required of each T cell clone. After elimination of antigen, 99% of these cells die by apoptosis and the remainder survive as memory cells (by analogy to the results from transgenic mice, Ref. [4]). On re-exposure to the same antigen, these memory cells would again have to divide to generate sucient cells once more. Given the ®nite lifespans of somatic cells, such extensive clonal proliferation might begin to confront the reacting T cell clones with a limit to their proliferative capacity [5]. To investigate T cell ageing in a model system, and to quantify precisely (for the ®rst time) the average and maximum lifespans of human T lymphocytes, we have elected to study monoclonal populations. This is because only in clonal populations is it possible to follow longitudinally the behaviour of a single cell type and be certain that changes observed are not merely a consequence of alterations in the proportions of the di€erent cells present. In addition, although at ®rst sight paradoxical, the calculation of

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Table 1 T cell longevity in culturea Originb

CE (%)c

Percentage of clones reaching population size: n, clones/experiments 20 PD (106) 30 PD (109) 40 PD (1012)

CD34+ CD3ÿCD7+ CD3+ (young, HS) CD3+ (old, HS) CD3+ (young, X-V) CD3+ (old, X-V)

59 16 47 0 86 52

581/7 108/8 1250/14 0/6 96/1 116/2

32 15 46 0 32 55

18 9 23

7% 3 16

nytd nyt

Longest-lived 60 PD 103 170 45 39

a

Longevity is expressed as a percentage of established clones (i.e. those counted as positive in calculating the CE) which survive to 20, 30 or 40 PD. b CD34+, positively selected stem cells; CD3ÿCD7+, T cell progenitors; CD3+, normal peripheral T cells; young and old, apparently healthy donors under 30 year and SENIEUR-selected donors over 95 year, respectively; HS, human serum; X-V, X-Vivo 10 medium. c CE, cloning eciency (calculated from percentage of wells positive in cloning plates). d nyt=not yet tested.

longevities of mixed populations can only be done by examining each component clone separately. If this is not done, only the longevity of the last remaining surviving clone from the original polyclonal population can be measured. This is indeed one of the confounding factors even in investigations of a much more extensively studied cell type, the ®broblast, where almost all available data actually apply only to the longest-lived clone present in the study population, and not to the average longevity of the entire population. 2. The in vitro model of T cell senescence: longevities of individual cultured T cell clones derived from healthy young donors Human T cell clones can be generated and maintained with relative ease [6] but it remains extremely labour-intensive to culture every single clone from each separate experiment right up to the end of their lifespans. Perhaps for this reason, there are very few reports in the literature on the average longevities of whole T cell populations in vitro. In contrast, there are several reports on longevities of polyclonal populations, and on maximum lifespans of small numbers of selected clones. To redress this imbalance, we have (over the years) attempted to culture all derived clones from at least some experiments to the end of their ®nite lifespans. Moreover, we have attempted to compare average and maximum longevities of clones from di€erent sources: Mature CD3+ T cells from young donors or healthy old donors, as well as from patients with certain diseases (the latter not included in the present analysis), compared to extra-thymically di€erentiated T cells derived from either CD3-negative CD7+ progenitors or from CD34+ populations (referred to here as stem cells). Table 1 shows a summary of the cloning eciencies

(CE) and longevities of T cell clones (TCC) derived from these di€erent sources. CD3+ cells from normal healthy young adult donors commonly yield an average CE of around 50%. However, only about half of these clones reach a population size of one million (equivalent to 20 PD). TCC continue to be lost thereafter so that there are fewer and fewer survivors at 30 and 40 PD. The average longevity of the established clones (de®ned as those that reach 20 PD) in these experiments comes to approximately 33 PD, whereas the average lifespan of all cells in the starting population is considerably less, being estimated at only around 17 PD. This contrasts very markedly with the lifespan of the longest-lived clone ever seen in any of these experiments, which was about 170 PD. This would represent a truly astronomical number of cells, around 1051. Clearly, rare long-lived clones are the exception rather than the rule in this type of culture, but do occasionally arise. This implies that if similar phenomena occur in vivo, these longest-lived clones would remain in the repertoire long after other speci®c clones had senesced. TCC die by apoptosis in vitro at the end of their lifespans, and if this also occurs in vivo the T cells would thus be subject to peripheral clonal deletion from the repertoire. There is evidence from studies of human T cells ex vivo in the elderly that both repertoire restriction and increased clonal deletion by apoptosis do indeed occur. 3. Cloning of T cells from the very elderly Accumulated evidence suggests that T cell function in the elderly is compromised, and that certain biomarkers of ageing may be associated with this event (e.g. decreased expression of CD28, decreased telomere lengths, decreased secretion of IL-2). However, in strictly selected perfectly healthy elderly donors, many but not all of the age-associated T cell alterations

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observed in the overall elderly population are no longer seen. We were therefore interested in investigating whether the characteristics of TCC derived from such strictly selected healthy elderly donors were di€erent from those previously established using young donors (e.g. because elderly donors' T cells may have proliferated extensively already in vivo before cloning). First experiments supported this possibility, because cloning T cells from old (>95 year) donors selected according to the SENIEUR protocol using the same culture conditions as used successfully for young donors' T cells (RPMI 1640+10% human serum and IL-2) failed to result in the generation of any clones at all (Table 1). This ®nding was consistent with the hypothesis that T cell ageing may have occurred in vivo such that the replicative lifespan of the clones in vivo was already close to exhaustion. However, we have recently discovered that this cannot be true, because cloning and culture in a serum-free medium (X-Vivo 10, BioWhittaker) supported equally high cloning eciencies even in near-centenarians as in the young. As far as these experiments have been pursued up to now, the proportion of clones achieving 20 PD seems to be similar in old and young donors, and the maximum longevity of that fraction of clones which has already been cultured up to the end of their lifespans also seems comparable. These results suggest that even very old perfectly healthy donors do possess T cells with an intact replicative capacity compared to young donors and imply (1) that senescent cells have in fact not been accumulating in these (albeit rare, highly selected) donors and (2) that, unlike T cells from young donors, the growth of T cells from old donors is not supported by pooled human serum (derived predominantly from young or middle-aged males). Thus, in this extreme case, T cells from old donors behave comparably to those from young donors (at least regarding longevity). We were, therefore, interested to see whether T cells at the other extreme (as young as possible) behaved di€erently. Here, the hypothesis was that they would manifest increased longevities as a result of not having divided previously in vivo. For these experiments we could have used blood from new-borns, but we felt that a better way of making absolutely certain that T cells had not divided previously was to generate them in situ. To this end we ®rst invested much e€ort in establishing extra-thymic T cell di€erentiation systems using T cell progenitors [7,8]. Most recently, extrathymic T cell di€erentiation even of CD34+ stem cells has been accomplished [9]. The results so far on TCC longevities of such clones are also summarized in Table 1. In contrast to our hypothesis (and earlier data which had suggested that T cells from the ®rst of these sources did experience longer average lifespans in vitro), we now see that if anything, the life expectancies of these T cells are

lower than those of TCC derived from mature T cells. This suggests that it is the process of in vitro replication itself which in¯uences culture lifespan, regardless of the replicative history of the cells in vivo before isolation and culture. Alternatively, only those cells ex vivo which had not proliferated extensively in vivo are capable of establishing clones in this system. If the latter explanation is true, it suggests (1) that young and old donors possess similar fractions of cells which have or have not proliferated and (2) that previous proliferative activity of CD34+ stem cells also ``counts'' towards reducing the proliferative lifespan in vitro. Our feeling is that neither of these possibilities is likely. These results could therefore be interpreted as suggesting that culture ageing is a phenomenon resulting from the turning on of a longevity clock as soon as the cells are put into culture in vitro, and thus may not be relevant in vivo. We were therefore anxious to monitor structural and functional alterations to these T cells during culture (rather than simply measuring the time at which they stopped growing and died), because we felt that if biomarkers of ageing in vitro were identi®ed which could be validated in vivo, it would be unlikely that the ``ageing'' process in vitro was a culture artefact (something still argued about even in the ®broblast ®eld). 4. The search for biomarkers of ageing: antibody-de®ned surface structures ``Biomarkers of ageing'' must be able to distinguish between changes associated with growth and activation status of cells and their age. Moreover, in the case of sets of clones with markedly di€erent life expectancies, there are two possible ways of viewing such markers either in terms of absolute numbers of PD undergone by the cells or in terms of the fraction of each clone's lifespan completed. Our most extensive analysis so far has been con®ned to the former, i.e. by comparing ``young'' cells and ``old'' cells from the same clones. However, it should be pointed out that the ``young'' cells in these experiments were still older than the average lifespan even of established clones. Clearly, we are only dealing with a subset of clones which tend to have greater than average longevity; however, we believe that these are the ones relevant for memory maintenance in vivo, as discussed above. Using large panels of monoclonal antibodies (mAb) to CD antigens, we have screened cells from TCC at earlier and later times in their lives [10]. Most surface markers tested did not change with culture age. For example, the density of expression of the antigen receptor remained the same, CD45R family molecules did not change and several adhesion and regular MHC molecules did not change. Given the heterogeneity of the

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clones, however, these ®ndings do not exclude that age-associated alterations in the expression of these molecules might be observed on certain individual clones, or under certain di€erent culture conditions. However, some changes in the majority of clones in the expression of certain cell surface structures were in fact observed. One potentially important set of changes was reduction in the density of expression of CD28, CD134 and CD154 [10], all three of which have been implicated in accessory signalling to the T cell (``costimulation''). Prior to senescence, the level of CD28 expression was commonly so low that it was no longer detectable by FACS, and a functional consequence of this was that the degree of autocrine proliferation of the cells decreased in parallel [11]. We also observed decreased expression of IL-2, IL-4 and IL-7 receptors on the majority of clones, possibly as a result of the decreases in costimulation [10]. Moreover, not only were decreases in the level of expression of certain cell surface molecules recorded, but also increases in expression of others. Thus, the density of expression of the negative costimulatory receptor CD152 (CTLA-4) and of the ``killer-inhibitory receptor'' CD94 was increased on certain clones as they aged (Pawelec et al., unpublished results). We therefore searched the literature for evidence that similar changes occurred in vivo in an age-associated manner. For CD28 expression there are numerous publications and increasing evidence that this is indeed the case, both on CD4 and CD8 cells. Moreover, in many di€erent pathological conditions sharing only the characteristics of chronic antigenic stimulation in vivo, the number of cells expressing CD28 is reported to be decreased (reviewed in [12]). Regarding CD94 and CD134, no reports on age-associated change in vivo have yet appeared, as far as we are aware. However, age-associated decreased expression of CD154 on human T cells was recently reported in two separate publications [13,14], and in mouse there is one report of increased CD152 expression [15]. 5. Cytokine secretion T cell ageing is characterized by a progressive loss of ability to undergo autocrine proliferation, often many PD before replicative senescence occurs. At this time, TCC can still be maintained with exogenous IL-2 but no longer secrete it in large amounts themselves. Nonetheless, old cells from TCC retain antigen-speci®c TCR function, as shown by their retention of ability to secrete cytokines such as IFN-g, and even an enhanced ability to secrete IL-10 [10]. Decreased IL-2 secretion coupled with increased IL-10 and more or less constant IFN-g production quite accurately re¯ects a consensus of studies published over the years

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on cytokine secretion from freshly isolated cells in the elderly (reviewed in [12]). 6. Apoptosis induction Old CD4+ TCC manifest an aged-associated increased susceptibility to activation-induced cell death (AICD) prior to their eventual ®nal clonal deletion by apoptosis [16]. This may be related to decreased levels of CD28 expression (and possibly additional costimulators) and consequent decreased IL-2 secretion. However, even in the presence of exogenous IL-2, they still show increased susceptibility to AICD, which is mediated by fas/fas-ligand interactions. The clones, however, do not generally show an age-associated increase in the density of CD95 (fas) expression (but note that young clones already express a very large amount of fas); however, they do show an age-related increase in fas-ligand mRNA which may contribute to enhanced susceptibility to AICD (M. Adibzadeh, unpublished results). The situation in vivo is not yet completely clear. While all studies published report increased susceptibility to apoptosis of CD4+ cells; there is disagreement on CD8 with some claiming increased, others decreased, apoptosis. There is clearly a tendency towards increased apoptosis with age [17± 21]. These results are more fully discussed in Ref. [22]. 7. What is the e€ector mechanism of T cell growth arrest? Telomere attrition as a possible causative mechanism of replicative senescence in ®broblasts and some other cell types has received a great deal of attention over the past few years. However, relatively little is known of telomere length dynamics in T cells and even less on telomerase regulation. It is clear that, unlike ®broblasts, stimulated T cells can upregulate telomerase and possibly maintain telomere lengths for at least a limited period during which they proliferate. Telomerase activity is regulated in the G1 phase of the cell cycle in normal human T cells, as indicated by the ®nding that rapamycin (which blocks TCR-signal transduction and cdk2 activation) but not hydroxyurea (an S-phase inhibitor) prevents telomerase induction [23]. Telomerase is upregulated by T cells within 24 h, increases up to 72 h and then decreases again after 96 h if the cells are not restimulated [24]. Telomere lengths do not decrease during this period, but possibly because telomerase is downregulated again, decreases in telomere length are not prevented during long-term culture, although they may be prevented initially [25]. In CD8 cells, Monteiro et al. [26] reported that telomere lengths in the CD28-negative population were

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shorter than in the CD28+CD8+ population, and that in vitro clonal expansion of CD8 cells is associated with telomere shortening. Optimal telomerase induction requires optimal stimulation via CD3 and CD28 [27], so age-associated defects in CD28 may also contribute to suboptimal telomerase induction. Repetitive restimulations may result in ever-decreasing telomerase induction, failure to maintain telomere lengths and proliferative cessation [28,29]. However, these data

were generated in only a few experiments on uncloned T cells and are thus dicult to interpret because telomerase-negative clones may have been generated as the culture aged. In the TCC studied here, we have shown that telomerase reinduction occurs in parallel with autocrine proliferation. Stimulation results in the rapid upregulation of telomerase in young cells, and this is further enhanced, and the kinetic extended, in the presence of exogenous IL-2 (e.g. see Fig. 1, upper panel).

Fig. 1. IL-2 dependent telomerase-activity of young (23 PD, upper panel) and old (29 PD lower panel) cells from the same short-lived alloreactive TCC 549-9 as in Fig. 2. The telomerase-activity was determined at the beginning of the test (d0, 7 days after the last stimulation in clonal propagation culture, =``resting'') and 1, 2 and 3 days after restimulation. The test was performed with the TRAP-Telomerase ELISA kit (Appligene Oncor) with 1  105 cells/150 ml lysis-bu€er. The nature of the stimulators is indicated on the bottom of the graph: CHO cells co-transfected with human CD58 and CD80 genes, together with HLA-DR alpha and beta chains, were ®xed with glutaraldehyde and used in the presence or

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Here, T cells from a CD4+ clone 549-9 at 23 PD were stimulated with PHA in the presence of triple transfectant CHO cells expressing human CD58, CD80 and HLA-DR and telomerase activity was measured after di€erent periods of time using the Oncor±Appligene TRAP assay. Duplicate sets of experiments were carried out in the presence or absence of 40 U/ml of IL-2. Before stimulation (day 0; cells approximating to a resting state) low telomerase activity was detected in either group. One day after stimulation, enhanced telomerase activity was detected. This increased after 2 days but decreased again after 3 days in the absence of

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exogenous IL-2. In the presence of IL-2, telomerase activity continued to increase at 3 days. The ability of these cells to proliferate was measured at the same time, as shown in Fig. 2. Without stimulation (autologous), there was no proliferation measured, but upon stimulation, thymidine incorporation was measurable in the young cells (Fig. 2, upper panel). In contrast, later passage cells from the same clone no longer upregulate telomerase even in the presence of IL-2 (Fig. 1, lower panel). Here, the TCC 549-9 cells have only reached 29 PD, but for this short-lived clone, this represents the end of their lifespan. It is at this time point

Fig. 2. Proliferation-kinetic of young (23 PD, upper panel) and senescent (29 PD, lower panel) cells from the short-lived alloreactive TCC 549-9. The proliferation was measured by [3H]-thymidine incorporation at day 1/2, 2/3 and 3/4 using the same stimulators as in Fig. 1.

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that stimulation no longer results in proliferation (Fig. 2, lower panel), and telomerase activity can no longer be detected. Thus, as illustrated in Figs. 1 and 2, the parallel loss of both autocrine proliferative capacity and telomerase induction also occurs in clones which senesce at an early passage number, as well as those senescing at later passages (data not shown). These ®ndings may suggest that telomerase is a marker for T cell activation rather than being instrumental in triggering growth arrest of the cells, but this hypothesis still requires testing. Further studies are required to clarify the role of telomerase and its mechanism of induction, and whether telomere attrition in lymphocytes is really an e€ector mechanism of cellular senescence. Nonetheless, in vivo, an age-associated reduction in telomere lengths is observed [30] and telomere attrition occurs more rapidly in premature aging syndromes [31,32]. Weng et al. [33] reported that CD4+ memoryphenotype cells showed consistently shorter telomeres than naive-phenotype cells. Interestingly, this di€erence in telomere length between naive- and memory-phenotype cells was the same whether the cells were isolated from young or old donors. These data suggest a role for telomere attrition in T cell ageing and once again indicate that the in vitro and in vivo data are more closely in agreement than disagreement. 8. Expression of mitotic inhibitors Telomere attrition may trigger growth arrest by activating DNA damage limitation programs. Growth arrest mediated by mitotic inhibitors allows time for DNA repair mechanisms to attempt to make good the damage. In the case of telomere attrition they obviously cannnot repair this type of ``damage'' and so apoptosis is triggered to delete the potentially dangerous, neoplastic, cell. Preliminary results of experiments carried out as part of a collaborative workshop by a European Union consortium (EUCAMBIS; see www.medizin.uni-tuebingen.de/eucambis/) examined the expression of mRNA for p16-INK4a, p21sdi and p27kip in cells from young and old TCC [12]. Cells from old TCC were found to contain more message for all three mitotic inhibitors than young cells. In the case of p21, no message at all was detectable in the young cells. Although it was previously thought that p16 was not expressed in T cells, more recent work by Erickson et al. showed that both p16 and p15 proteins accumulate, whereas p21 levels were only slightly elevated, as PHA-stimulated T cells age in culture, and that there was increased binding of p16 to its target Cdk6 kinase [34]. Therefore, p16 may play an important role in growth control of lymphocytes as well and could be a target for manipulation in immunosenescence. The situation ex vivo in T cells from the young

and old has not yet been properly studied. Preliminary, unpublished, data from the same EUCAMBIS study mentioned before so far provides little evidence for any consistent di€erence in the level of expression of mRNA for p21 or p27 in resting or stimulated T cells from SENIEUR donors compared with JUNIEUR donors. Thus, p27 mRNA appeared after an average of 32212 PCR cycles in unstimulated SENIEUR cells and after 29 2 4 cycles in stimulated SENIEUR cells compared with 29 2 2 and 28 2 1 respectively in JUNIEUR cells. For p21 SENIEUR and JUNIEUR, these values were 27.5, 27.5, 27.0 and 26.0 respectively. 9. Conclusions The in vitro behaviour of cultured T cells at the clonal level is an accurate model for age-associated T cell alterations in vivo (Table 2). It may be possible to employ clonal T cell cultures to search for age-associated characteristics representing risk factors for immunosenescence in vivo. Furthermore, screening approaches to manipulate immunosenescence in vivo may be developed using this in vitro model. At the simplest level, this could be the testing of hormones, vitamin and mineral supplements, anti-oxidants etc. An understanding of and ability to in¯uence the upand down-regulation of CD28 and CD152, as well as CD134 and CD154, might also result in interventions to enhance immunity in the elderly. In addition, attempts to increase functional longevity of the T cells by more invasive manipulations, such as enforcing expression of telomerase or blockade of mitotic inhibitors at the same time as enhancing DNA repair capacity, might be an option [35]. Acknowledgements Support for the work from the author's laboratory, Table 2 Possible similarities between in vivo and in vitro T cell ageing Decreased

Increased

CD28 expression CD154 expression IL-2R expression IL-2 production bcl-2 telomere lengths telomerase induction CD134 DNA repair stress resistance and HSP expression TCR signalling

p27 p16 mutation frequency CD152 expression IL-10 production DNA damage apoptosis

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and the EUCAMBIS project, discussed here came from the Deutsche Forschungsgemeinschaft (Pa 361-2; Pa 361/5-1), the VERUM Foundation, the Mildred Scheel Foundation (10-1173-Pa3), the Sandoz (Novartis) Foundation for Gerontological Research, and the European Commission (BMHI-CT94-1209; project EUCAMBIS; BMH4-CT98-3058; project EUCAPS). We thank especially Dr. Yvonne Barnett (University of Ulster) and Dr. Ed Remarque (University of Leiden) for their input into the EUCAMBIS project related to the experiments on levels of expression of mitotic inhibitors in TCC and SENIEUR donors, and Dr. Erminia Mariani (University of Bologna), Prof. Rafael Solana (University of CordoÂba) and Dr. Ed Remarque for contributions of cells from JUNIEURand SENIEUR-selected donors collected in the EUCAMBIS/Messer-Griesheim Cell Bank, TuÈbingen. References [1] Pawelec G, Solana R, Remarque E, Mariani E. Impact of aging on innate immunity. J Leukocyte Biol 1998;64:703±12. [2] Steger MM, Maczek C, Grubeck-Loebenstein B. Morphologically and functionally intact dendritic cells can be derived from the peripheral blood of aged individuals. Clin Exp Immunol 1996;105:544±50. [3] Maini MK, Soares MVD, Zilch CF, Akbar AN, Beverley PCL. Virus-induced CD8(+) T cell clonal expansion is associated with telomerase up-regulation and telomere length preservation: a mechanism for rescue from replicative senescence. J Immunol 1999;162:4521±6. [4] Bruno L, Kirberg J, Von Boehmer H. On the cellular basis of immunological T cell memory. Immunity 1995;2:37±43. [5] E€ros RB, Pawelec G. Replicative senescence of T lymphocytes: does the Hay¯ick Limit lead to immune exhaustion? Immunol Today 1997;18:450±4. [6] Pawelec G. Cloning and propagation of human T lymphocytes. In: Gallagher G, Rees RC, Reynolds CW, editors. Tumour immunobiology. A practical approach. Oxford: IRL Press, 1993. p. 131±41. [7] Pohla H, Adibzadeh M, Buhring HJ, Siegelshubenthal P, Deikeler T, Owsianowsky M, Schenk A, Rehbein A, Schlotz E, Schaudt K, Pawelec G. Evolution of a CD3+CD4+ alpha/beta T-cell receptor plus mature T-cell clone from CD3ÿCD7+ sorted human bone marrow cells. Develop Immunol 1993;3:197±210. [8] Adibzadeh M, Buhring HJ, Daikeler T, Siegelshubenthal P, Owsianowsky M, Schenk A, Rehbein A, Schaudt K, Schlotz E, Pohla H, Pawelec G. Extrathymic development and function of human T-lymphocytes from bone marrow cells in vitro. Cell Immunol 1994;154:25±42. [9] Pawelec G, MuÈller R, Rehbein A, HaÈhnel K, Ziegler BL. Extrathymic T cell di€erentiation in vitro from CD34+ stem cells. J Leukocyte Biol 1998;64:733±9. [10] Pawelec G, Rehbein A, Haehnel K, Merl A, Adibzadeh M. Human T cell clones as a model for immunosenescence. Immunol Rev 1997;160:31±43. [11] Adibzadeh M, Pohla H, Rehbein A, Pawelec G. Long-term culture of monoclonal human T lymphocytes: models for immunosenescence? Mech Ageing Dev 1995;83:171±83. [12] Pawelec G, Wagner W, Adibzadeh M, Engel A, T cell immunosenescence in vitro and in vivo. Exp Gerontol 1999;34:419±29.

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