Transfer of the human telomerase reverse transcriptase(TERT) gene into T lymphocytes results in extension of replicative potential

Abstract

IntroductionAfter an antigenic challenge, naive T lymphocytes undergo differentiation and proliferation to become effector and memory T cells. T effector cells are the progeny of a limited number of antigenspecific precursor cells, and dramatic expansions of CD8 ϩ T cells to achieve adequate immune responses have been described in several viral models. 1,2 Moreover, memory responses are characterized by repetitive expansion of antigen-specific T-cell clones. 3 Limitations in the proliferative life span of such clones could eventually lead to immune senescence 4 and contribute to the increased incidence of infectious diseases observed in elderly individuals.Telomeres are specialized structures at the end of eukaryotic chromosomes whose length decreases with cell divisions in vitro and in vivo. 5,6 Recently, experiments have shown that artificial telomere elongation by transfection of the human telomerase reverse transcriptase (hTERT) gene results in extension of telomeres and increased proliferative life span of fibroblasts and of retinal pigment epithelial cells. 7,8 Moreover, those cells appear indistinguishable from young normal cells and do not show signs of chromosomal aberrations or cellular transformations. 9,10 Thus, these results have provided evidence for a direct linkage between telomere shortening and cellular senescence.Despite transient telomerase activity upon mitogenic and antigen stimulation, 11-13 cells of the immune system also show telomere shortening with age and with cellular replication in vitro. 14-16 Indeed, lymphocytes maintained in culture have a finite life span and undergo replicative senescence after 30 to 50 population doublings (PDs; reviewed in reference 4). Moreover, the telomere length of memory CD4 and CD8 T lymphocytes is significantly shorter than that of naive T cells. 16,17 Relative short telomere lengths have been found in lymphocytes from individuals with Down syndrome, 15 in the memory CD4 and CD8 subsets of elderly individuals, 18 and in the CD8 ϩ CD28 Ϫ subset of normal individuals 19 and those infected with human immunodeficiency virus. 20 Taken together, most observations support the notion that telomere length reflects the replicative history of T cells and suggest that the proliferative life span of particular T-cell clones could be restricted by progressive telomere shortening.Here we show that introduction of hTERT in human CD8 ϩ T clones is able to maintain or elongate the telomere lengths during cell divisions and significantly extends their proliferative life span. Moreover, the hTERT-transduced T cells retained their cytotoxic properties and had a normal 46,XY karyotype, and 2 of the clones studied did not present any signs of apoptosis or senescence after more than 170 PDs. Materials and methods Isolation of naive and memory CD8 ؉ T cells from peripheral bloodPeripheral blood mononuclear cells (PBMCs) were obtained following density centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). The PBMCs (10 7 ) isolated from a male healthy individual wer...

Rhadinovirus vector-derived human telomerase reverse transcriptase expression in primary T cells

“…44 Although we observed early transgene transcription and functional activity in hTERT-transduced T cells (Figures 1 and 2) in our experiments, hTERT transcript levels seemed to be reduced at week 5 in Dorf1RT-transduced T cells, whereas transcript levels of the orf75 fragment adjacent to H-DNA were sustained. Moreover, WT f -and RT-transduced T cells had similar activity but different mRNA levels.…”

Télomérase, élixir de jouvence des cellules humaines ?

“…En revanche, la fluorescence des télomères est homogène et intense sur les chromosomes provenant de lymphocytes surexprimant hTERT (B). Ces expériences montrent que l'introduction de la télo-mérase dans des clones de lymphocytes T permet l'élongation des télomères les plus courts [19]. tant ainsi de maintenir la taille des télomères, voire de rallonger les séquences télomériques (Figure 2).…”

“…C. Dans les lymphocytes, la présence de la télomérase (+) pourrait apporter un mécanisme de compensation partiel en réponse à la perte des télomères [20]. Cependant, les télomères raccourcissent progressivement, même dans les lymphocytes, et leur sénescence peut être observée dans des cultures in vitro [19,20]. D'après ce modèle, la limite de Hayflick dans les lymphocytes (M1') surviendrait après un nombre de divisions cellulaires supérieur à celui des fibroblastes (M1).…”

“…Ces résultats expliquent que les lymphocytes perdent progressivement les télomères et atteignent la limite de Hayflick (Figure 2). Ainsi, comme les fibroblastes, les lymphocytes ont une durée de vie qui est limitée par un nombre défini de divisions cellulaires (revue dans [18]) et des clones T sénescents, avec des télomères extrême-ment courts, sont observés après quelques mois de culture cellulaire in vitro (Figure 1) [19]. L'activité de la télomérase permettrait la prolongation du potentiel réplicatif des lymphocytes T humains, mais non leur immortalisation.…”

“…Trois études indépendantes ont récemment décrit l'introduction de hTERT et sa surexpression dans des lymphocytes T humains par l'emploi de vecteurs rétroviraux [19][20][21]. Deux types de cellules T ont été utilisées: des populations polyclonales de lymphocytes T CD8 + et des clones de cellules T, dérivés d'une seule cellule à partir de populations polyclonales.…”