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Essential role of survivin, an inhibitor of apoptosis protein, in T cell development, maturation, and homeostasis.

Xing Z, Conway EM, Kang C, Winoto A - J. Exp. Med. (2003)

Bottom Line: Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number.In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis.These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Division of Immunology and Cancer Research Laboratory, University of California at Berkeley, 469 LSA, Berkeley, CA 94720, USA.

ABSTRACT
Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell-specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre-T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.

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BrdU incorporation study of CD4-survivin–deficient mice. (A) BrdU labeling of SP thymocytes and peripheral T cells from CD4-survivin mice and their littermate (+/−) controls. Mice were injected intraperitoneally with BrdU and 3 h later flow cytometric analyses were conducted for the respective T cell populations: CD4 or CD8 SP thymocytes and spleen CD8+ cells. (B) Side and forward scatter analysis of CD4 or CD8 SP thymocytes from CD4-survivin and littermate (+/−) controls. The percentages of the large (L) cells are indicated. (C) Mice were injected with BrdU every 6 h in a 25-h period before their thymocytes were harvested and stained with either anti-CD4 or anti-CD8 antibodies followed by anti-BrdU antibodies and 7-AAD. The BrdU versus 7-AAD profiles of CD4− gated thymocytes, which include CD8+ CD4− SP and DN cells, are shown. Similar findings were also observed for CD8− gated thymocytes.
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fig7: BrdU incorporation study of CD4-survivin–deficient mice. (A) BrdU labeling of SP thymocytes and peripheral T cells from CD4-survivin mice and their littermate (+/−) controls. Mice were injected intraperitoneally with BrdU and 3 h later flow cytometric analyses were conducted for the respective T cell populations: CD4 or CD8 SP thymocytes and spleen CD8+ cells. (B) Side and forward scatter analysis of CD4 or CD8 SP thymocytes from CD4-survivin and littermate (+/−) controls. The percentages of the large (L) cells are indicated. (C) Mice were injected with BrdU every 6 h in a 25-h period before their thymocytes were harvested and stained with either anti-CD4 or anti-CD8 antibodies followed by anti-BrdU antibodies and 7-AAD. The BrdU versus 7-AAD profiles of CD4− gated thymocytes, which include CD8+ CD4− SP and DN cells, are shown. Similar findings were also observed for CD8− gated thymocytes.

Mentions: To investigate further the mechanism underlying the low T cell number in the periphery despite a normal thymocyte profile in survivin-deficient mice, we conducted BrdU labeling experiments in newborn and adult mice to measure the rate of DNA synthesis. BrdU was injected intraperitoneally into newborn and adult mice and the extent of BrdU incorporation into recently replicating thymocytes and peripheral T cells was assessed 3 h later. Although adult thymocytes did not proliferate and hence contained few BrdU+ cells, thymocytes from newborn mice exhibited a significant number of BrdU+ cells (Fig. 7 A). This is consistent with the notion that SP thymocytes undergo homeostatic proliferation in newborn animals before export to the periphery (3). As the peripheral compartment is filled up, the rate of proliferation drops. In survivin-deficient mice, however, BrdU incorporation was not diminished. If any, a reproducible increase in BrdU labeling was detected instead (Fig. 7 A). This effect was more pronounced in CD8+ cells than CD4+ cells (percent BrdU incorporation in CD8+ SP cells: CD4-survivin: 27.42 ± 1.78%, wild-type littermates: 18.2 ± 2.86%; percent BrdU incorporation in CD4+ cells: CD4-survivin: 7.53 ± 3.94%, wild-type littermates: 5.495 ± 1.03%; n = 4). These observations were substantiated by FACS® analysis of the side and forward scatter plots of these survivin-deficient SP thymocytes, which revealed a similar increase in the proportion of larger than normal CD8+ and CD4+ SP thymocytes (Fig. 7 B). Interestingly, splenic CD8+ cells, which usually are not cycling, incorporated BrdU when survivin was deleted (Fig. 7 A, right). These data suggest that survivin-deficient T cells could synthesize DNA and indeed replicated at a higher rate than wild-type T cells, but CFSE and propidium iodide analysis suggests that they could not complete the cell cycle.


Essential role of survivin, an inhibitor of apoptosis protein, in T cell development, maturation, and homeostasis.

Xing Z, Conway EM, Kang C, Winoto A - J. Exp. Med. (2003)

BrdU incorporation study of CD4-survivin–deficient mice. (A) BrdU labeling of SP thymocytes and peripheral T cells from CD4-survivin mice and their littermate (+/−) controls. Mice were injected intraperitoneally with BrdU and 3 h later flow cytometric analyses were conducted for the respective T cell populations: CD4 or CD8 SP thymocytes and spleen CD8+ cells. (B) Side and forward scatter analysis of CD4 or CD8 SP thymocytes from CD4-survivin and littermate (+/−) controls. The percentages of the large (L) cells are indicated. (C) Mice were injected with BrdU every 6 h in a 25-h period before their thymocytes were harvested and stained with either anti-CD4 or anti-CD8 antibodies followed by anti-BrdU antibodies and 7-AAD. The BrdU versus 7-AAD profiles of CD4− gated thymocytes, which include CD8+ CD4− SP and DN cells, are shown. Similar findings were also observed for CD8− gated thymocytes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1887718&req=5

fig7: BrdU incorporation study of CD4-survivin–deficient mice. (A) BrdU labeling of SP thymocytes and peripheral T cells from CD4-survivin mice and their littermate (+/−) controls. Mice were injected intraperitoneally with BrdU and 3 h later flow cytometric analyses were conducted for the respective T cell populations: CD4 or CD8 SP thymocytes and spleen CD8+ cells. (B) Side and forward scatter analysis of CD4 or CD8 SP thymocytes from CD4-survivin and littermate (+/−) controls. The percentages of the large (L) cells are indicated. (C) Mice were injected with BrdU every 6 h in a 25-h period before their thymocytes were harvested and stained with either anti-CD4 or anti-CD8 antibodies followed by anti-BrdU antibodies and 7-AAD. The BrdU versus 7-AAD profiles of CD4− gated thymocytes, which include CD8+ CD4− SP and DN cells, are shown. Similar findings were also observed for CD8− gated thymocytes.
Mentions: To investigate further the mechanism underlying the low T cell number in the periphery despite a normal thymocyte profile in survivin-deficient mice, we conducted BrdU labeling experiments in newborn and adult mice to measure the rate of DNA synthesis. BrdU was injected intraperitoneally into newborn and adult mice and the extent of BrdU incorporation into recently replicating thymocytes and peripheral T cells was assessed 3 h later. Although adult thymocytes did not proliferate and hence contained few BrdU+ cells, thymocytes from newborn mice exhibited a significant number of BrdU+ cells (Fig. 7 A). This is consistent with the notion that SP thymocytes undergo homeostatic proliferation in newborn animals before export to the periphery (3). As the peripheral compartment is filled up, the rate of proliferation drops. In survivin-deficient mice, however, BrdU incorporation was not diminished. If any, a reproducible increase in BrdU labeling was detected instead (Fig. 7 A). This effect was more pronounced in CD8+ cells than CD4+ cells (percent BrdU incorporation in CD8+ SP cells: CD4-survivin: 27.42 ± 1.78%, wild-type littermates: 18.2 ± 2.86%; percent BrdU incorporation in CD4+ cells: CD4-survivin: 7.53 ± 3.94%, wild-type littermates: 5.495 ± 1.03%; n = 4). These observations were substantiated by FACS® analysis of the side and forward scatter plots of these survivin-deficient SP thymocytes, which revealed a similar increase in the proportion of larger than normal CD8+ and CD4+ SP thymocytes (Fig. 7 B). Interestingly, splenic CD8+ cells, which usually are not cycling, incorporated BrdU when survivin was deleted (Fig. 7 A, right). These data suggest that survivin-deficient T cells could synthesize DNA and indeed replicated at a higher rate than wild-type T cells, but CFSE and propidium iodide analysis suggests that they could not complete the cell cycle.

Bottom Line: Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number.In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis.These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Division of Immunology and Cancer Research Laboratory, University of California at Berkeley, 469 LSA, Berkeley, CA 94720, USA.

ABSTRACT
Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell-specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre-T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.

Show MeSH
Related in: MedlinePlus