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Telomerase activation and rejuvenation of telomere length in stimulated T cells derived from serially transplanted hematopoietic stem cells.

Allsopp RC, Cheshier S, Weissman IL - J. Exp. Med. (2002)

Bottom Line: Telomeres shorten in hematopoietic cells, including hematopoietic stem cells (HSCs), during aging and after transplantation, despite the presence of readily detectable levels of telomerase in these cells.Southern analysis of telomere length in resting and anti-CD3/CD28 stimulated donor-derived splenic T cells revealed an increase in telomere size by approximately 7 kb for the population as a whole.Together, these results imply that one role for telomerase in T cells may be to renew or extend replicative potential via the rejuvenation of telomere length.

View Article: PubMed Central - PubMed

Affiliation: Beckman Center, Pathology Department, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA. rallsopp@stanford.edu

ABSTRACT
Telomeres shorten in hematopoietic cells, including hematopoietic stem cells (HSCs), during aging and after transplantation, despite the presence of readily detectable levels of telomerase in these cells. In T cells, antigenic stimulation has been shown to result in a marked increase in the level of telomerase activity. We now show that stimulation of T cells derived from serially transplanted HSC results in a telomerase-dependent elongation of telomere length to a size similar to that observed in T cells isolated directly from young mice. Southern analysis of telomere length in resting and anti-CD3/CD28 stimulated donor-derived splenic T cells revealed an increase in telomere size by approximately 7 kb for the population as a whole. Stimulation of donor-derived T cells from recipients of HSCs from telomerase-deficient mice did not result in regeneration of telomere length, demonstrating a dependence on telomerase. Furthermore, clonal anti-CD3/CD28 stimulation of donor-derived T cells followed by fluorescent in situ hybridization (FISH) analysis of telomeric signal intensity showed that telomeres had increased in size by approximately 50% for all clonal expansions. Together, these results imply that one role for telomerase in T cells may be to renew or extend replicative potential via the rejuvenation of telomere length.

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Analysis of TRF length of resting and stimulated donor-derived T cells. (A) Splenic T cells (5 × 104) from young adult mice and secondary HSC recipients were collected via FACS® and either transferred to growth media for stimulation or used for isolation of high molecular weight DNA. Anti-CD3/CD28 stimulated T cells were collected for isolation of DNA after 1–2 wk of growth. The extraction and restriction enzyme digestion of DNA was performed as described previously (reference 27). 1 μg of each DNA sample was resolved in a 0.75% agarose gel by field inversion gel electrophoresis (pulse conditions: 180 V forward; 120 V reverse for 16 h). The gel was dried and the DNA hybridized to a 32P-end labeled telomeric oligomer overnight followed by 3 × 5 min washes at 37°C in 0.35× SSC buffer. Images were collected using a Phosphor-Imager screen. Size of molecular weight standards (kilobases) are shown on the left. REST, resting; STIM, anti-CD3/CD28 stimulated. (B) The mean TRF length was calculated and averaged for all resting and stimulated T cell samples taken from a total of five adult mice and seven secondary recipients. Error bars (standard deviation) and P values (Student's t test) are shown.
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fig1: Analysis of TRF length of resting and stimulated donor-derived T cells. (A) Splenic T cells (5 × 104) from young adult mice and secondary HSC recipients were collected via FACS® and either transferred to growth media for stimulation or used for isolation of high molecular weight DNA. Anti-CD3/CD28 stimulated T cells were collected for isolation of DNA after 1–2 wk of growth. The extraction and restriction enzyme digestion of DNA was performed as described previously (reference 27). 1 μg of each DNA sample was resolved in a 0.75% agarose gel by field inversion gel electrophoresis (pulse conditions: 180 V forward; 120 V reverse for 16 h). The gel was dried and the DNA hybridized to a 32P-end labeled telomeric oligomer overnight followed by 3 × 5 min washes at 37°C in 0.35× SSC buffer. Images were collected using a Phosphor-Imager screen. Size of molecular weight standards (kilobases) are shown on the left. REST, resting; STIM, anti-CD3/CD28 stimulated. (B) The mean TRF length was calculated and averaged for all resting and stimulated T cell samples taken from a total of five adult mice and seven secondary recipients. Error bars (standard deviation) and P values (Student's t test) are shown.

Mentions: To assess the effect on telomere length of antigenic stimulation of T cells of donor type from adult mice and HSC transplant recipients, we collected donor-derived splenic T cells by FACS® for anti-CD3/CD28 stimulation in vitro and performed southern analysis of TRF length on the resting and stimulated T cells (Fig. 1) . The TRF length for resting splenic T cells isolated from secondary HSC recipients (mean ∼16 kb) was significantly shorter than that observed for resting T cells from young adult mice (mean ∼23 kb; P = 0.005). 1 wk after anti-CD3/CD28 stimulation, no change in the TRF length was detected for splenic T cells isolated from young adult mice. However, the TRF length of the stimulated splenic T cells isolated from HSC transplant recipients had increased significantly (Fig. 1; P = 0.002), to an average size approximately equal to that observed for splenic T cells from young adult mice.


Telomerase activation and rejuvenation of telomere length in stimulated T cells derived from serially transplanted hematopoietic stem cells.

Allsopp RC, Cheshier S, Weissman IL - J. Exp. Med. (2002)

Analysis of TRF length of resting and stimulated donor-derived T cells. (A) Splenic T cells (5 × 104) from young adult mice and secondary HSC recipients were collected via FACS® and either transferred to growth media for stimulation or used for isolation of high molecular weight DNA. Anti-CD3/CD28 stimulated T cells were collected for isolation of DNA after 1–2 wk of growth. The extraction and restriction enzyme digestion of DNA was performed as described previously (reference 27). 1 μg of each DNA sample was resolved in a 0.75% agarose gel by field inversion gel electrophoresis (pulse conditions: 180 V forward; 120 V reverse for 16 h). The gel was dried and the DNA hybridized to a 32P-end labeled telomeric oligomer overnight followed by 3 × 5 min washes at 37°C in 0.35× SSC buffer. Images were collected using a Phosphor-Imager screen. Size of molecular weight standards (kilobases) are shown on the left. REST, resting; STIM, anti-CD3/CD28 stimulated. (B) The mean TRF length was calculated and averaged for all resting and stimulated T cell samples taken from a total of five adult mice and seven secondary recipients. Error bars (standard deviation) and P values (Student's t test) are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194261&req=5

fig1: Analysis of TRF length of resting and stimulated donor-derived T cells. (A) Splenic T cells (5 × 104) from young adult mice and secondary HSC recipients were collected via FACS® and either transferred to growth media for stimulation or used for isolation of high molecular weight DNA. Anti-CD3/CD28 stimulated T cells were collected for isolation of DNA after 1–2 wk of growth. The extraction and restriction enzyme digestion of DNA was performed as described previously (reference 27). 1 μg of each DNA sample was resolved in a 0.75% agarose gel by field inversion gel electrophoresis (pulse conditions: 180 V forward; 120 V reverse for 16 h). The gel was dried and the DNA hybridized to a 32P-end labeled telomeric oligomer overnight followed by 3 × 5 min washes at 37°C in 0.35× SSC buffer. Images were collected using a Phosphor-Imager screen. Size of molecular weight standards (kilobases) are shown on the left. REST, resting; STIM, anti-CD3/CD28 stimulated. (B) The mean TRF length was calculated and averaged for all resting and stimulated T cell samples taken from a total of five adult mice and seven secondary recipients. Error bars (standard deviation) and P values (Student's t test) are shown.
Mentions: To assess the effect on telomere length of antigenic stimulation of T cells of donor type from adult mice and HSC transplant recipients, we collected donor-derived splenic T cells by FACS® for anti-CD3/CD28 stimulation in vitro and performed southern analysis of TRF length on the resting and stimulated T cells (Fig. 1) . The TRF length for resting splenic T cells isolated from secondary HSC recipients (mean ∼16 kb) was significantly shorter than that observed for resting T cells from young adult mice (mean ∼23 kb; P = 0.005). 1 wk after anti-CD3/CD28 stimulation, no change in the TRF length was detected for splenic T cells isolated from young adult mice. However, the TRF length of the stimulated splenic T cells isolated from HSC transplant recipients had increased significantly (Fig. 1; P = 0.002), to an average size approximately equal to that observed for splenic T cells from young adult mice.

Bottom Line: Telomeres shorten in hematopoietic cells, including hematopoietic stem cells (HSCs), during aging and after transplantation, despite the presence of readily detectable levels of telomerase in these cells.Southern analysis of telomere length in resting and anti-CD3/CD28 stimulated donor-derived splenic T cells revealed an increase in telomere size by approximately 7 kb for the population as a whole.Together, these results imply that one role for telomerase in T cells may be to renew or extend replicative potential via the rejuvenation of telomere length.

View Article: PubMed Central - PubMed

Affiliation: Beckman Center, Pathology Department, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA. rallsopp@stanford.edu

ABSTRACT
Telomeres shorten in hematopoietic cells, including hematopoietic stem cells (HSCs), during aging and after transplantation, despite the presence of readily detectable levels of telomerase in these cells. In T cells, antigenic stimulation has been shown to result in a marked increase in the level of telomerase activity. We now show that stimulation of T cells derived from serially transplanted HSC results in a telomerase-dependent elongation of telomere length to a size similar to that observed in T cells isolated directly from young mice. Southern analysis of telomere length in resting and anti-CD3/CD28 stimulated donor-derived splenic T cells revealed an increase in telomere size by approximately 7 kb for the population as a whole. Stimulation of donor-derived T cells from recipients of HSCs from telomerase-deficient mice did not result in regeneration of telomere length, demonstrating a dependence on telomerase. Furthermore, clonal anti-CD3/CD28 stimulation of donor-derived T cells followed by fluorescent in situ hybridization (FISH) analysis of telomeric signal intensity showed that telomeres had increased in size by approximately 50% for all clonal expansions. Together, these results imply that one role for telomerase in T cells may be to renew or extend replicative potential via the rejuvenation of telomere length.

Show MeSH
Related in: MedlinePlus