Graham Pawelec ¹, Ed Remarque ², Yvonne Barnett ³, Rafael Solana ⁴

1 University of Tübingen, Tübingen, FRG ², University of Leiden, Leiden, Holland ³, University of Ulster, Coleraine, Northern Ireland ⁴, University of Córdoba, Córdoba, Spain

Received 12/29/97 Accepted 1/5/97

7. DOES TELOMERIC END LOSS CONTRIBUTE TO THE REPLICATIVE SENESCENCE OF NORMAL T CELLS?

Loss of telomeric DNA, and gradual shortening of telomeres, has been proposed to result, after a certain number of cell divisions, in the inability of cells to divide again (180). In human monoclonal fibroblast cultures, telomere length was found to be reduced with culture age and was directly proportional to the remaining replicative capacity of the clone (181). Telomere shortening might therefore act as a mechanism counting the number of cell divisions that a cell population has experienced. Loss of telomeric DNA has also been demonstrated in blood cells in vivo and shown to be related to donor age (182). It occurs more rapidly in premature aging syndromes, eg. Hutchinson-Gilford progeria (183) or trisomy-21 (184). In culture, lymphocytes from normal donors allow an estimated telomere DNA loss rate of 120 bp/cell doubling, comparable to that seen in other somatic cells. More recently, Weng et al . (185) reported that CD4⁺ memory phenotype cells showed consistently shorter telomeres than naive phenotype cells. Interestingly, this difference in telomere length between naive and memory cells was the same whether the cells were isolated from young or old donors. This may suggest that it is the T cell precursor rather than the mature T cell which has "aged", as defined by decrease of telomere length. Weng et al . also showed that telomere length decreased during autocrine expansion of both naive and memory cells, and that the latter completed less PD than the former. The authors concluded from this that the replicative potential of memory cells was less than that of naive cells and that this might be related to telomere shortening. However, what they actually measured in their experiments was autocrine proliferative capacity, not replicative potential. Autocrine proliferative capacity relies upon the stimulation of growth factor secretion, upregulation of the growth factor receptor and correct signal transduction. As we have shown for culture aged T cell clones (173), exogenous factor-dependent growth of the cells (ie. replicative potential) is retained for a period far greater than the capacity to secrete interleukin 2 (autocrine proliferative potential). It is therefore unlikely that the cessation of growth noted by Weng et al ., which was only 10 PD for memory cells and 20 PD for naive cells, actually reflects shortened telomere-triggered blockade of autocrine proliferative capacity, not replicative potential.

Early data showed that telomere length in sperm DNA does not decrease with increasing age of the donor, suggesting that a mechanism for maintaining telomere length may be active in germ cells but not somatic cells (183,186). Such a factor, telomerase, an enzyme responsible for maintaining telomere length in unicellular eukaryotes, was previously found in immortalized human cell lines and tumor cells, but not in normal somatic cells. However, data obtained using more sensitive assays indicate that the presence of telomerase may be more widespread than previously thought (187). Moreover, Hiyama et al . (188) have shown that telomerase activity is detectable at very low levels in normal human T and B cells and that it increases greatly after mitogenic stimulation. Because the increase in telomerase activity was transient, it did not prevent the decrease in telomere length during long-term culture (189). Furthermore, the number of donors with telomerase activity detectable in their lymphocytes decreased with age. In the age range 0 - 19 years, all donors were positive, whereas this fraction decreased in the next group (20 - 39 years) before plateauing in the groups 40 - 59, 60 - 79 and 80 years. Therefore, lymphocytes may be unusual among somatic non-transformed cells in expressing telomerase, which is upregulated when they are stimulated to divide. This might represent one prerequisite for enabling T cells to avoid the Hayflick limit. It would therefore be worth testing this hypothesis by measuring telomerase activity in T cell cultures of different ages and different proliferative lifespans. In this respect, Effros´s group has presented preliminary data on the induction of telomerase in cultured T cells. They indeed found that the degree to which either CD4+ or CD8+ cells upregulated telomerase after PHA stimulation was inversely related to the length of time that they had been in culture (R. Effros, personal communication, Decemner 1997). Previous studies demonstrating decreased telomere length in in vivo or in vitro aged lymphocytes have examined uncloned (heterogeneous) populations. The results of these studies could be reconciled with a unique T cell longevity by hypothesizing 1) that only a small proportion of T cell clones manifest full telomerase function and are effectively immortal (consistent with most studies in the literature) and 2) that the reductions measured had not yet resulted in a critical low repeat number that would activate full telomerase function, as in the study by Weng et al . (185) discussed above, in which replicative potential was confused with autocrine proliferative capacity. Further studies will undoubtedly continue to contribute to this area: for example, Monteiro et al . (190) reported that telomere lengths in the CD28-negative CD8+ population were shorter than in the CD28+ CD8+ population, and that in vitro clonal expansion of CD8 cells is associated with TL shortening.

Telomerase may not be the only factor determining telomere length and cell survival. Strahl & Blackburn reported (191) that inhibitors of retroviral reverse transcriptase (telomerase itself is a specialized cellular reverse transcriptase) could cause progressive telomere shortening of immortalized human lymphoid cell lines in vitro. Telomerase activity was present in these lines and its activity was blocked by the agents tested. Telomeres in the blocked lines eventually stabilized and remained short. It was, however, suggested that telomere lengths in lymphoid cells lines (which were unstable even in the absence of inhibitors) are determined both by telomerase and telomerase-independent mechanisms.