Deletion of PKBalpha/Akt1 affects thymic development

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.

Figure 1. The deletion of PKBα leads to a reduced thymic size in mouse neonates.

A: The weight of freshly dissected thymi was measured in PKBα ^+/+ and PKBα ^−/− neonates (top panel) and expressed as ratio to body weight (bottom panel). The kidney was used as a control. Error bars represent standard error of the mean; n≥13. B: Western-blot analysis of 50 µg protein extracts from wild-type neonatal thymus using PKB isoform specific antibodies (top panel). Western-blot analysis of 50 µg protein extracts from PKBβ ^−/−, PKBβ ^+/+, PKBγ ^−/−, and PKBγ ^+/+ neonatal thymi using PKB isoform specific antibodies (bottom panel). Actin was used as a loading control. C: The weight of freshly dissected thymi was measured in PKBβ ^+/+, PKBβ ^−/−, PKBγ ^+/+, and PKBγ ^−/− neonates (top panels) and expressed as ratio to body weight (bottom panels). The kidney was used as a control. Error bars represent standard error of the mean. n≥7 (n = number of mice analyzed per genotype).

Figure 2. The deletion of PKBα leads to a reduced number of thymocytes in neonatal mice.

A: Thymocytes were isolated from neonatal PKBα ^+/+ and PKBα ^−/− littermates and their size compared by flow cytometry using the forward scatter (FSC) parameter. The histogram is representative of 3 litters. B: (left panel) Thymocytes were isolated and counted from PKBα ^+/+ and PKBα ^−/− neonatal mice. (right panel) Lymphocytes isolated from the spleen of PKBα ^+/+ and PKBα ^−/− neonates were stained with anti-CD19 and anti-CD3 antibodies. The number of T cells (CD3⁺CD19⁻) is shown. n≥3. Error bars represent standard error of the mean. C: TUNEL assay on neonatal thymus sections from PKBα ^+/+ and PKBα ^−/− littermates. The graph represents the quantification of TUNEL-positive cells from 5 fields on 3 sections. The result shown is representative of 3 independent experiments. The bar shown on the pictures represents 200 µm. Error bars represent standard error of the mean. D: Thymocytes were isolated from PKBα ^+/+ and PKBα ^−/− neonates and stained with annexin V and propidium iodide (PI). Histograms show results that are representative of 2 independent experiments; n≥3 (n = number of mice per genotype within the same experiment).

Figure 3. The lack of PKBα leads to an accumulation of DN3 and ISP8 early thymocyte subsets.

Flow cytometric analysis of early thymocytes at the transition from DN to DP. A: Density plots show thymocytes from PKBα ^+/+ and PKBα ^−/− mice that were stained with cell surface markers for identification of lineage-negative thymocytes DN1 (CD25⁻CD44⁺), DN2 (CD25⁺CD44⁺), DN3 (CD25⁺CD44⁻), and DN4 (CD25⁻CD44⁻). B: Histograms show the intracellular protein expression of TCRβ (iTCRβ) in DN3 thymocytes from PKBα ^+/+ and PKBα ^−/− mice. C: Histograms show the surface expression of CD25 on lineage-negative PKBα ^+/+ and PKBα ^−/− thymocytes. MFI: mean fluorescence intensity. D: Density plots and histograms show thymocytes from PKBα ^+/+ and PKBα ^−/− mice that were labeled with cell surface markers for identification of ISP8 (CD4⁻CD8⁺CD3⁻) thymocytes. E: Histograms show the intracellular protein expression of TCRβ (iTCRβ) in ISP8 thymocytes from PKBα ^+/+ and PKBα ^−/− mice. The results shown are representative of 3 independent experiments on 4 to 6 week-old mice. n≥4 (n = number of mice per genotype within the same experiment).

Figure 4. 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).

Figure 5. PKBα is the main isoform in DN3 and ISP8 thymocyte subsets.

A: mRNA levels of PKBα, PKBβ, and PKBγ isoforms in DN3 and ISP8 thymocyte subsets. The expression data obtained following microarray analysis were corrected for GC-bias within oligos, allowing gene expression signals to be expressed on the same scale; this permits a semi-quantitative comparison of the expression of different genes. B: Proposed model of PKBα mediating PI3K signaling at the transition from DN to DP thymocyte subsets. iTCRβ and TCR refer to intracellular and surface expression of TCRβ, respectively.