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Deletion of PKBalpha/Akt1 affects thymic development.

Fayard E, Gill J, Paolino M, Hynx D, Holländer GA, Hemmings BA - PLoS ONE (2007)

Bottom Line: The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development.Protein kinase B (PKB) is the main effector of the PI3K pathway.Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT

Background: The thymus constitutes the primary lymphoid organ for the majority of T cells. The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development. Defects in single components of this pathway prevent thymocytes from progressing beyond early T cell developmental stages. Protein kinase B (PKB) is the main effector of the PI3K pathway.

Methodology/principal findings: To determine whether PKB mediates PI3K signaling in the thymus, we characterized PKB knockout thymi. Our results reveal a significant thymic hypocellularity in PKBalpha(-/-) neonates and an accumulation of early thymocyte subsets in PKBalpha(-/-) adult mice. Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus. Microarray analyses show that the absence of PKBalpha in early thymocyte subsets modifies the expression of genes known to be involved in pre-TCR signaling, in T cell activation, and in the transduction of interferon-mediated signals.

Conclusions/significance: This report highlights the specific requirements of PKBalpha for thymic development and opens up new prospects as to the mechanism downstream of PKBalpha in early thymocytes.

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Related in: MedlinePlus

The accumulation of early thymocytes is due to PKBα deficiency in the lymphoid compartment. A: Western-blot analysis of 50 µg protein extracts from PKBα+/+, PKBα+/−, and PKBα−/− isolated thymocytes using antibodies directed against either PKBα or phospho(Thr308)-PKB (PDK1 site). Actin was used as a loading control. B: Thymocytes were isolated from PKBα+/+ and PKBα−/− thymic grafts and counted 4 weeks post grafting (left panel). Lymphocytes were isolated from thymus and spleen of lethally irradiated congenic recipient mice injected with either PKBα+/+ or PKBα−/− fetal liver cells and counted 5 weeks post transplant (right panel). Error bars represent standard error of the mean; n≥5. C–D: Flow cytometric analysis of lymphocytes. C: Host-derived thymocytes developed in the PKBα+/+ or PKBα−/− fetal thymi grafted under the kidney capsule of wild-type mice were isolated 4 weeks post-grafting and stained with cell surface markers for identification of early thymocyte subsets. D: Thymocytes developed from PKBα+/+ or PKBα−/− fetal liver-derived HSC in lethally-irradiated wild-type congenic mice were isolated 5 weeks after reconstitution and stained with cell surface markers for identification of early thymocyte subsets. Representative density plots and histograms are shown. n≥5 (n = number of mice per genotype within the same experiment).
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pone-0000992-g004: The accumulation of early thymocytes is due to PKBα deficiency in the lymphoid compartment. A: Western-blot analysis of 50 µg protein extracts from PKBα+/+, PKBα+/−, and PKBα−/− isolated thymocytes using antibodies directed against either PKBα or phospho(Thr308)-PKB (PDK1 site). Actin was used as a loading control. B: Thymocytes were isolated from PKBα+/+ and PKBα−/− thymic grafts and counted 4 weeks post grafting (left panel). Lymphocytes were isolated from thymus and spleen of lethally irradiated congenic recipient mice injected with either PKBα+/+ or PKBα−/− fetal liver cells and counted 5 weeks post transplant (right panel). Error bars represent standard error of the mean; n≥5. C–D: Flow cytometric analysis of lymphocytes. C: Host-derived thymocytes developed in the PKBα+/+ or PKBα−/− fetal thymi grafted under the kidney capsule of wild-type mice were isolated 4 weeks post-grafting and stained with cell surface markers for identification of early thymocyte subsets. D: Thymocytes developed from PKBα+/+ or PKBα−/− fetal liver-derived HSC in lethally-irradiated wild-type congenic mice were isolated 5 weeks after reconstitution and stained with cell surface markers for identification of early thymocyte subsets. Representative density plots and histograms are shown. n≥5 (n = number of mice per genotype within the same experiment).

Mentions: The thymus is composed of a heterogeneous population of cells, including thymocytes at various developmental stages and different stromal cells that are either hematopoietic, mesenchymal, or epithelial in origin. In thymocytes, PKBα was the main isoform located downstream of PDK1 since PKBα−/− thymocytes showed only minimally phosphorylated PKB levels at the PDK1 dependent-Thr308 residue (Figure 4A). PKBα expression was also observed in thymic epithelial cells (JG and GAH, unpublished), which are the most abundant component of the stromal compartment. Therefore, ablation of PKBα expression in either of these compartments could potentially account for the impairment in the transition from DN to DP thymocytes. To determine whether the observed phenotype was due to a lack of PKBα in non-hematopoietic stromal and/or in blood-borne cells, we next performed thymic grafting and fetal liver cell transfer experiments, respectively. In the first instance, we assessed the ability of PKBα−/− thymic stroma to support T cell development. For this purpose, embryonic day E15.5 thymi were isolated from both PKBα−/− and wild-type embryos. The fetal lobes were treated in vitro with deoxyguanosine for 6 days to deplete lymphoid cells, and then grafted under the kidney capsule of wild-type recipient mice. Four weeks post transplantation, the number of wild-type host-derived thymocytes developing within the PKBα−/− grafted thymic stroma was significantly reduced when compared to control tissue but regular thymocyte development was not affected (Figure 4B and 4C). In a second series of experiments, we evaluated the capacity of fetal liver derived-hematopoietic stem cells (HSC) from wild-type and PKBα−/− embryonic day E15.5 donors (CD45.2) to recapitulate normal thymopoiesis in wild-type thymic stromal environment of lethally-irradiated congenic (CD45.1) mice. Five weeks after reconstitution, the bone marrow chimeras had similar overall numbers of thymocytes and peripheral lymphocytes, irrespective whether they were derived from PKBα−/− or wild-type fetal liver cells (Figure 4B). Flow cytometric analyses further showed that PKBα−/− HSC were able to give rise to all thymocyte subsets (DN, DP, SP CD4+, and SP CD8+), but again both DN3 and ISP8 cells accumulated to the same extent as what had been observed in unmanipulated PKBα−/− mice (Figure 4D). Taken together, these data indicate that the accumulation of thymocytes during early T cell development observed in PKBα-deficient mice is the specific consequence of a lack of PKBα in lymphoid cells.


Deletion of PKBalpha/Akt1 affects thymic development.

Fayard E, Gill J, Paolino M, Hynx D, Holländer GA, Hemmings BA - PLoS ONE (2007)

The accumulation of early thymocytes is due to PKBα deficiency in the lymphoid compartment. A: Western-blot analysis of 50 µg protein extracts from PKBα+/+, PKBα+/−, and PKBα−/− isolated thymocytes using antibodies directed against either PKBα or phospho(Thr308)-PKB (PDK1 site). Actin was used as a loading control. B: Thymocytes were isolated from PKBα+/+ and PKBα−/− thymic grafts and counted 4 weeks post grafting (left panel). Lymphocytes were isolated from thymus and spleen of lethally irradiated congenic recipient mice injected with either PKBα+/+ or PKBα−/− fetal liver cells and counted 5 weeks post transplant (right panel). Error bars represent standard error of the mean; n≥5. C–D: Flow cytometric analysis of lymphocytes. C: Host-derived thymocytes developed in the PKBα+/+ or PKBα−/− fetal thymi grafted under the kidney capsule of wild-type mice were isolated 4 weeks post-grafting and stained with cell surface markers for identification of early thymocyte subsets. D: Thymocytes developed from PKBα+/+ or PKBα−/− fetal liver-derived HSC in lethally-irradiated wild-type congenic mice were isolated 5 weeks after reconstitution and stained with cell surface markers for identification of early thymocyte subsets. Representative density plots and histograms are shown. n≥5 (n = number of mice per genotype within the same experiment).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1991598&req=5

pone-0000992-g004: The accumulation of early thymocytes is due to PKBα deficiency in the lymphoid compartment. A: Western-blot analysis of 50 µg protein extracts from PKBα+/+, PKBα+/−, and PKBα−/− isolated thymocytes using antibodies directed against either PKBα or phospho(Thr308)-PKB (PDK1 site). Actin was used as a loading control. B: Thymocytes were isolated from PKBα+/+ and PKBα−/− thymic grafts and counted 4 weeks post grafting (left panel). Lymphocytes were isolated from thymus and spleen of lethally irradiated congenic recipient mice injected with either PKBα+/+ or PKBα−/− fetal liver cells and counted 5 weeks post transplant (right panel). Error bars represent standard error of the mean; n≥5. C–D: Flow cytometric analysis of lymphocytes. C: Host-derived thymocytes developed in the PKBα+/+ or PKBα−/− fetal thymi grafted under the kidney capsule of wild-type mice were isolated 4 weeks post-grafting and stained with cell surface markers for identification of early thymocyte subsets. D: Thymocytes developed from PKBα+/+ or PKBα−/− fetal liver-derived HSC in lethally-irradiated wild-type congenic mice were isolated 5 weeks after reconstitution and stained with cell surface markers for identification of early thymocyte subsets. Representative density plots and histograms are shown. n≥5 (n = number of mice per genotype within the same experiment).
Mentions: The thymus is composed of a heterogeneous population of cells, including thymocytes at various developmental stages and different stromal cells that are either hematopoietic, mesenchymal, or epithelial in origin. In thymocytes, PKBα was the main isoform located downstream of PDK1 since PKBα−/− thymocytes showed only minimally phosphorylated PKB levels at the PDK1 dependent-Thr308 residue (Figure 4A). PKBα expression was also observed in thymic epithelial cells (JG and GAH, unpublished), which are the most abundant component of the stromal compartment. Therefore, ablation of PKBα expression in either of these compartments could potentially account for the impairment in the transition from DN to DP thymocytes. To determine whether the observed phenotype was due to a lack of PKBα in non-hematopoietic stromal and/or in blood-borne cells, we next performed thymic grafting and fetal liver cell transfer experiments, respectively. In the first instance, we assessed the ability of PKBα−/− thymic stroma to support T cell development. For this purpose, embryonic day E15.5 thymi were isolated from both PKBα−/− and wild-type embryos. The fetal lobes were treated in vitro with deoxyguanosine for 6 days to deplete lymphoid cells, and then grafted under the kidney capsule of wild-type recipient mice. Four weeks post transplantation, the number of wild-type host-derived thymocytes developing within the PKBα−/− grafted thymic stroma was significantly reduced when compared to control tissue but regular thymocyte development was not affected (Figure 4B and 4C). In a second series of experiments, we evaluated the capacity of fetal liver derived-hematopoietic stem cells (HSC) from wild-type and PKBα−/− embryonic day E15.5 donors (CD45.2) to recapitulate normal thymopoiesis in wild-type thymic stromal environment of lethally-irradiated congenic (CD45.1) mice. Five weeks after reconstitution, the bone marrow chimeras had similar overall numbers of thymocytes and peripheral lymphocytes, irrespective whether they were derived from PKBα−/− or wild-type fetal liver cells (Figure 4B). Flow cytometric analyses further showed that PKBα−/− HSC were able to give rise to all thymocyte subsets (DN, DP, SP CD4+, and SP CD8+), but again both DN3 and ISP8 cells accumulated to the same extent as what had been observed in unmanipulated PKBα−/− mice (Figure 4D). Taken together, these data indicate that the accumulation of thymocytes during early T cell development observed in PKBα-deficient mice is the specific consequence of a lack of PKBα in lymphoid cells.

Bottom Line: The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development.Protein kinase B (PKB) is the main effector of the PI3K pathway.Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT

Background: The thymus constitutes the primary lymphoid organ for the majority of T cells. The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development. Defects in single components of this pathway prevent thymocytes from progressing beyond early T cell developmental stages. Protein kinase B (PKB) is the main effector of the PI3K pathway.

Methodology/principal findings: To determine whether PKB mediates PI3K signaling in the thymus, we characterized PKB knockout thymi. Our results reveal a significant thymic hypocellularity in PKBalpha(-/-) neonates and an accumulation of early thymocyte subsets in PKBalpha(-/-) adult mice. Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus. Microarray analyses show that the absence of PKBalpha in early thymocyte subsets modifies the expression of genes known to be involved in pre-TCR signaling, in T cell activation, and in the transduction of interferon-mediated signals.

Conclusions/significance: This report highlights the specific requirements of PKBalpha for thymic development and opens up new prospects as to the mechanism downstream of PKBalpha in early thymocytes.

Show MeSH
Related in: MedlinePlus