Unequal Contribution of Akt Isoforms in the Double-Negative to Double-Positive Thymocyte Transition

Changchuin Mao, Esmerina G. Tili, Marei Dose, Mariëlle C. Haks, Susan E. Bear, Ioanna Maroulakou, Kyoji Horie, George A. Gaitanaris, Vincenzo Fidanza, Thomas Ludwig, David L. Wiest, Fotini Gounari and Philip N. Tsichlis
J Immunol May 1, 2007, 178 (9) 5443-5453; DOI: https://doi.org/10.4049/jimmunol.178.9.5443

Abstract

Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing β-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.

Differentiation of thymocytes is an ordered process with multiple steps that are defined by the sequential loss or gain of expression of specific cell surface markers (1). Multipotent T cell precursors that migrate into the thymus are not yet committed to the T cell lineage and retain the ability to differentiate into dendritic cells, NK cells, B cells, and αβ or γδ T cells (2). Differentiation of these precursors generates T cell lineage-committed thymocytes (3) that are double negative (DN)5 for CD4 and CD8, comprising ∼5% of the total cell number in the adult thymus. These cells are distributed into four distinct differentiation stages that are defined by ordered changes in the surface expression of CD44 and CD25: DN1 (CD44+CD25−), DN2 (CD44+CD25+), DN3 (CD44−CD25+) and DN4 (CD44−CD25−). Transition from DN1 to DN2 is associated with the first major wave of proliferative expansion (4). The next wave of proliferation takes place in DN3 cells after the assembly of pre-TCR and continues into the DN4 stage (5, 6).

The cells progressing through the DN3 stage can be subdivided into two subsets. One subset (85% of the DN3 compartment), known as DN3a, is comprised of small resting CD27− cells that either have not completed rearrangements of the TCR β-chain or have undergone out-of-frame rearrangements. The second subset (15% of the DN3 compartment), known as DN3b, is comprised of larger, actively dividing blastoid CD27+ cells (7) that carry a productively rearranged TCR β-chain locus. The TCR β-chain encoded by this locus interacts with the invariant pre-Tα (pT-α) chain and with signal-transducing CD3 proteins to form the pre-TCR complex (8). Signals originating in the pre-TCR instruct DN3b cells to commit to the αβ T cell lineage and to progress to the double-positive (DP) stage (7). They provide cues for cell maturation, survival, proliferative expansion, allelic exclusion at the Tcrb locus, and subsequent differentiation to the CD4+CD8+ DP stage (7, 8), in a process known as β-selection (9). Mutant mice lacking the recombinase proteins Rag1 (10) or Rag2 (11), TCR-β (12), or pT-α (13) cannot form a pre-TCR complex. As a result, they exhibit a T cell developmental arrest at the DN3 stage.

Genetic ablation and overexpression studies in mice identified a number of molecules that are required for the transition of DN thymocytes to the DP stage. Such molecules include members of the Notch family, which appear to play a critical role before the assembly of pre-TCR but are dispensable once pre-TCR is assembled (14). Other molecules such as Ras (15), Lck, and Fyn (16), Zap70 (17), Syk (17), and linker for activation of T cells (LAT) (18) may contribute to β-selection by transducing signals to the MAPK pathway, which is required for the transition of DN cells to the DP stage (19) and yet others, such as Pim1, may modulate the process by unknown mechanisms (20).

The PI3K/Akt pathway may also contribute to β-selection. Earlier studies indeed showed that ablation of PI3Kγ inhibits the transition of DN thymocytes to the DP stage (21, 22). Combined ablation of PI-3Kγ and PI3Kδ gives rise to an intrinsic thymocyte differentiation defect which results in the partial depletion of DP thymocytes. The latter depends, at least in part, on apoptosis in the DP thymocyte compartment (23). Ablation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) had the opposite effect promoting DN thymocyte differentiation to the DP stage in the absence of pre-TCR signals (24). Recent studies indeed showed that DN3 cells expressing constitutively active Akt do not require Notch signals to progress to the DP stage upon cocultivation with the stromal cell line OP-9. The partial substitution of Notch signals by Akt was interpreted to suggest that Akt functions downstream of Notch (25). In other studies, it was shown that β-catenin, a molecule that is regulated by Gsk-3β, a potential Akt target, is also involved in β-selection (26, 27). Finally, the adaptor molecule Shc was found to contribute to the transition of DN thymocytes to the DP stage, via a nonredundant MAPK-independent pathway. Because Shc is required for the activation of Akt by a variety of signals, these findings raise the question of whether this MAPK-independent pathway depends on Akt (28).

Akt encodes a serine/threonine protein kinase that is activated via PI3K-dependent mechanisms (29). Akt1 is the cellular homolog of the v-akt protooncogene, which was transduced by AKT-8, a transforming retrovirus isolated from an AKR mouse T cell lymphoma (30). Three major isoforms of Akt, Akt1, Akt2, and Akt3, encoded by three distinct loci, have been identified in mammals. All three isoforms are ubiquitously expressed, although, their levels of expression vary among tissues (29). Akt has been shown to regulate a diverse array of cellular functions including apoptosis, proliferation, differentiation, and intermediary metabolism (29). In T lymphocytes, Akt is activated by cytokines, chemokines, cell adhesion molecules, and immune recognition receptors. Activated Akt regulates targets that control cell proliferation and survival (31).

In this report, we present evidence that the PI3K/Akt pathway is regulated by pre-TCR signals and that its activity is required for the transition of DN thymocytes to the DP stage. In addition, we show that the same pathway also promotes the survival of DP thymocytes. Two complementary genetic approaches, genetic ablation of various Akt isoforms and thymic overexpression of constitutively active Akt1 transgenes (32) as well as pharmacological inhibition of Akt in fetal thymic organ cultures (FTOC), were used to address the requirement for Akt activity during β-selection. Our data indicate that Akt is activated by pre-TCR signals and that Akt activity (Akt1, Akt2, and to a lesser degree Akt3) is required for β-selection and for the transition of DN thymocytes to the DP stage and for the survival of DP thymocytes.

Materials and Methods

Mice

Akt1 knockout mice were generated by blastocyst injection of mouse embryonic stem cells carrying a replication-defective retroviral vector integrated 3 bp upstream of the Akt1 ATG codon. The mutant cells were isolated from a embryonic stem cell library generated by retroviral mutagenesis (33). These mice are Akt1null, in that they do not express Akt1 (Fig. 1⇓A). Conditional Akt1 knockout mice (Akt1fl/fl) were generated by inserting LoxP sites in the second and fourth intron of the Akt1 gene. The Cre transgene used in the experiments described in this study is under the transcriptional control of the proximal Lck promoter. Lck-cre mice (34) were crossed with Akt1fl/fl mice to generate Lck-cre+/Akt1fl/fl mice. Cre-induced recombination between the LoxP sites deleted exons 3 and 4. Splicing between exons 2 and 5 disrupts the open reading frame. Therefore, the mRNA transcribed from the Akt1 gene following LoxP recombination is expected to be translated into a protein that contains only part of the pleckstrin homology (PH) domain (aa 1–58) encoded by the first two exons (Fig. 1⇓, B–D). Western blotting of lysates derived from cells that underwent Cre-mediated recombination of the Akt1 gene failed to detect this truncated protein, suggesting that either the rearranged RNA or the truncated protein are unstable (data not shown). Akt2−/− mice (from M. Birnbaum, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA) and Akt3−/− mice have been described elsewhere (35, 36). Double and triple knockout mice and their wild-type controls were littermates derived by crossing mice heterozygous for the ablation of multiple Akt isoforms. The latter mice were generated by crossing single knockouts of different Akt isoforms. Of the double knockout mice the Akt2/3 double knockouts were viable and fertile. Akt1/2, Akt1/3 double knockout and Akt1/2/3 triple knockout mice were generated by crossing Akt2, Akt3, and Akt2/3 knockout mice with our conditional Akt1 knockouts.

FIGURE 1.
FIGURE 1.

Establishment and characterization of regular and conditional Akt1 knockout mice. A, Akt1−/− mice harboring a replication-defective retroviral vector 3 bp upstream of the Akt1 ATG codon do not express Akt1. A Western blot of mammary tissue lysates from a wild-type mouse and Akt1−/−, Akt2−/−, and Akt3−/− mice was probed with an Ab specific for Akt1. B, Upper panel, Schematic diagram of the intron/exon structure of the Akt1 gene. Middle panel, The conditional Akt1fl allele was constructed by inserting a loxP/NeoR cassette in intron 2 and an additional LoxP site in intron 4 as indicated. Lower panel, Cre-mediated recombination gives rise to a defective Akt1 allele lacking exons 3 and 4. Arrowheads indicate primer locations. C, PCR using genomic DNA from wild-type mice and Akt1fl/fl mice, as well as from DP thymocytes of Lck-Cre+/Akt1fl/+ and Lck-Cre+/Akt1fl/fl mice. D, Exons 2 and 5 are not in frame. The RNA encoded by the Cre-rearranged locus, therefore, has the potential to encode the peptide shown here.

Transgenic mice expressing a tetracycline-inducible TCR β-chain in the Rag1−/− genetic background (TetOβ-LTH-Rag1−/−) have been reported elsewhere (37). Transgenic mice expressing constitutively active Akt1 transgenes (myristoylated Akt1 Myr-Akt1 or Akt1E40K) from proximal Lck promoter constructs have been described elsewhere (32).

Rag1- and 2-deficient and TCR-α-deficient mice were obtained from The Jackson Laboratory. The studies have been reviewed and approved by the institutional animal care and use committee.

Western blotting

Cells were lysed in lysis buffer (50 mM Tris Cl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) and the lysates were analyzed by SDS-PAGE. Western blots were probed with various Akt Abs (Cell Signaling Technology) following standard procedures.

FTOCs

Development of early T cells was studied using FTOCs from day 14 embryos of C57BL/6 mice. Fetal thymuses were dissected on day 14 and they were cultured in FTOC medium (38). FTOCs were treated throughout the culture period with 10 μM SB202190 (Calbiochem), 10 μM LY294002 (Calbiochem), 10 μM UO126 (Calbiochem), or with the combination of LY294002 and UO126.

Abs and flow cytometry

Single-cell suspensions of thymocytes were incubated in staining buffer (1% normal rabbit sera, 0.01% NaN3 in PBS) with the following fluorochrome-conjugated Abs, purchased from BD Pharmingen, or eBioscience (CA 92121): FITC or allophycocyanin anti-CD4 (GK1.5), PE anti-CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), B220 (RA3-6B2), DX5, Gr-1 (RB6-8C5), TCRγδ (UC7-13D5), TER119, TCR-β (H57-597), allophycocyanin anti-CD25 (PC61.5), FITC anti-CD44 (1M7). Propidium iodide was used to identify and exclude dead cells. Flow cytometry was conducted using a CyAn LX High-Performance Flow Cytometer (DakoCytomation). Cell sorting was conducted in a MoFlo cell sorter (DakoCytomation). Data were analyzed using the software program Summit (DakoCytomation). Cell aggregates were excluded by gating, based on forward scatter and pulse width.

Cell cycle analysis

To determine the cell cycle distribution of subpopulations of thymocytes, surface-stained cells were resuspended in 0.1 ml of 0.15 M NaCl, 5 mM Na EDTA, 0.02% saponin, and 5 μg/ml 7-aminoactinomycin D (Sigma- Aldrich) for 20 min at room temperature (RT) in the dark. The stained cells were centrifuged and resuspended in staining buffer. They were analyzed for their cell cycle distribution by flow cytometry.

Intracellular TCR-β staining

Following surface staining, cells were washed twice in PBS, fixed by treatment with 0.5% paraformaldehyde for 15 min at RT and washed twice in PBS. Cells were permeabilized with 0.5% saponin in PBS for 10 min at RT and washed twice in PBS. Permeabilized cells were stained for 30 min at 4°C with PE-conjugated anti-TCRβ in 2% FBS and 0.5% saponin, and washed in PBS.

Anti-CD3ε injection

Anti-CD3ε (20 μg/mouse; BD Pharmingen) was diluted in 200 μl of PBS and was i.p. injected into 4-wk-old Rag1−/− mice. Rag1−/− mice injected with 200 μl of PBS were used as controls. Thymocytes were harvested at 18 h after the injection.

Polymerase chain reaction

The rearrangement of the floxed Akt1 locus in cells expressing Cre was detected by PCR. Rearrangements at the Tcrb locus were also detected by PCR, using purified thymic genomic DNA (39). Quantitative real-time RT-PCR was used to examine the expression of Akt1, Akt2, Akt3, p27, p21, c-myc, Gadd45a, Egr1, Egr2, Id2, Id3, and E47 in sorted DN thymocyte subpopulations. The list of primers used in all the PCR can be obtained by contacting us.

Cell culture

Sci/ET27F is a DN thymocyte cell line, derived from a spontaneously arising SCID thymoma. These cells express pre-Tα, but they do not express TCR-β. As a result, they are pre-TCR negative. Stable expression of TCR-β into Sci/ET27F cells gave rise to the pre-TCR-positive Sci/ET27F derivative line SCB.29 (40). All these cell lines were maintained in IMDM supplemented with 10% FBS, penicillin (10 U/ml), streptomycin (10 U/ml), and 2-ME (55 μM) (Invitrogen Life Technologies). OP9-GFP and OP9-DL1 cells, a gift from Dr. J. C. Zuniga-Pflucker (University of Toronto, Toronto, Ontario, Canada), were maintained according to the protocol described by Zuniga-Pflucker and colleague (41). Following cocultivation of Sci/ET27F or SCB.29 cells with the OP9-GFP and OP9-DL1 stromal cell lines, lymphoid cells were separated from the GFP-positive stromal cells by FACS sorting. Western blots of lysates of sorted cells were probed with total Akt and phospho-Akt Abs as shown in Fig. 2⇓D.

FIGURE 2.
FIGURE 2.

Akt is activated by pre-TCR signals in the thymus. A, Rag2−/− mice harboring a tetracycline-regulated Tcrb transgene (Tet-Off) were kept on tetracycline or off tetracycline. Alternatively, doxycycline was withdrawn from mice kept on the drug. Expression levels of both total Akt and Thr308-phosphorylated Akt in thymocytes derived from these mice were measured by Western blotting of cell lysates harvested from mice treated as indicated. The intensity of the Western blot bands was determined by densitometry. The bars show the ratio of phosphorylated to total Akt. B, Western blot of total thymocyte lysates from Rag1−/− mice inoculated with anti-CD3ε i.p. was probed with the phospho-Thr308 Akt Ab or with an Ab against total Akt. C, Anti-CD3ε treatment activates Akt only in the DN pre-TCR-positive cell line (SCB.29) but not in its parental pre-TCR-negative line (Sci/ET27F). Cells were serum-starved overnight and they were harvested 60 min after the start of the anti-CD3ε treatment. D, Whereas pre-TCR is required for Akt activation in the OP9 cocultivation system, Notch is not. Following cocultivation, the Sci/ET27F and the SCB.29 cells were sorted from the GFP-positive OP9-DL1 and OP9-GFP stromal cell lines, before lysis. The lysates in all panels were from the same experiment. However, they were electrophoresed in separate gels to make comparisons that specifically address the role of Notch and the role of pre-TCR in Akt activation during β-selection. See Results for details.

Results

Akt activation in DN3 thymocytes depends on pre-TCR

The rearrangement of the Tcrb gene, in the course of thymocyte differentiation, takes place within the CD25+CD44− DN3 compartment. The TCR-β protein encoded by a rearranged Tcrb gene associates with pT-α to form the pre-TCR, which is required for β-selection. To determine the effects of pre-TCR signaling on the activity of Akt, we withdrew tetracycline from TetOβ-LTH-Rag1−/− transgenic mice (37) expressing a rearranged Tcrb transgene from a tetracycline-regulated promoter. Withdrawal of tetracycline induces the expression of TCR-β, which is required for the assembly of pre-TCR. Monitoring the phosphorylation of Akt at Thr308 upon induction of TCR-β confirmed that Akt activation coincides with the assembly of pre-TCR (Fig. 2⇑A). This suggested that Akt may be activated by signals originating in the pre-TCR.

To address this hypothesis, we examined the phosphorylation of Akt in DN thymocytes of Rag1−/− mice, before and after i.p. injection of an anti-CD3ε Ab which is known to induce developmental events, mimicking β-selection in Rag1, Rag2, or pT-α-deficient mice (11). Injection of anti-CD3ε enhanced the phosphorylation of Akt at Thr308 (Fig. 2⇑B), indicating that Akt becomes phosphorylated upon engagement of the pre-TCR. This hypothesis was finally addressed using a pre-T cell line that expresses pT-α but lacks TCR-β (Sci/ET27F) and a derivative of this cell line, SCB.29, engineered to stably express TCR-β. Monitoring Akt phosphorylation at Thr308 before and after treatment with anti-CD3 confirmed that anti-CD3 treatment promotes Thr308 phosphorylation only in cells that express TCR-β (Fig. 2⇑C). The same was observed upon induction of TCR-β by tetracycline withdrawal in a Sci/ET27F-derivative line that expresses TCR-β inducibly from a tetracycline-regulated promoter (data not shown).

It is well-established that β-selection depends on both pre-TCR and Notch signals and that both signals can be partially replaced by an activated form of Akt (25). This suggests that the two signals may cooperate to activate Akt. To address the role of pre-TCR in Akt activation, Sci/ET27F and SCB.29 cells were cocultivated with a derivative of the bone marrow stromal cell line OP9 that stably expresses the Notch ligand Delta-like-1 and GFP (OP9-DL1) and they were harvested at 0, 1, 5, and 16 h from the start of the cocultivation. Western blots of these cells were probed with anti-Akt and anti-Thr308 phosphorylated Akt. Fig. 2⇑D (upper panel) shows that although Akt undergoes phosphorylation in both Sci/ET27F and SCB.29 cells, its phosphorylation is significantly weaker in the pre-TCR− Sci/ET27F cells, suggesting that Akt activation is pre-TCR dependent. To address the role of Notch in Akt activation, the pre-TCR-positive cell line SCB.29 was cocultivated with OP9-DL1 or OP9-GFP cells. Cell lysates harvested at the indicated time points were probed again for total and Thr308 phosphorylated Akt. The results (Fig. 2⇑D, middle panel) revealed that the extent of Akt phosphorylation in pre-TCR-positive cells is independent of Notch signaling. The low level of Akt phosphorylation observed in the pre-TCR-negative Sci/ET27F cell line cocultivated with OP9-DL1 cells (Fig. 2⇑D, upper panel) raised the question of whether Notch signals may induce phosphorylation of Akt in the absence of pre-TCR signals. To address this question, the pre-TCR-negative cell line Sci/ET27F was cocultivated with OP9-DL1 or OP9-GFP cells. Probing cell lysates harvested at the indicated time points for total or phosphorylated Akt revealed that the weak Akt activation in Sci/ET27F cells is also independent of Notch signaling. We conclude that Akt is activated by pre-TCR signals and that Notch is not involved in its activation. The weak activation of Akt in Sci/ET27F (pre-TCR−) cells following their cocultivation with either OP9-GFP or OP9-DL1 suggests that the OP9 stromal cells may be producing other factors that also contribute to the activation of Akt.

Treatment of FTOCs with the PI3K inhibitor LY294002 blocks the transition of DN thymocytes to the DP stage

To determine whether the activity of Akt is necessary for the transition of DN thymocytes to the DP stage, we treated FTOCs from wild-type C57BL/6 day 14 embryos with the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl chromone (LY294002). Phenotypic analysis of the cells harvested 4 days later revealed a drastic reduction of DP cells, suggesting that PI3K inhibition may block the transition of DN thymocytes to the DP stage (Fig. 3⇓). The MEK inhibitor UO126, which blocks ERK activation, also inhibited the DN to DP transition. The two drugs combined (LY294002 plus UO126) had a more severe effect than either drug alone. SB202190, which inhibits p38 MAPK, caused an overall decrease in the number of thymocytes but affected only slightly the percentage of different thymocyte subpopulations (Fig. 3⇓).

FIGURE 3.
FIGURE 3.

Akt is required for the transition of DN thymocytes to the DP stage. A, FTOCs of day 14 C57BL/6 embryos were cultured in FTOC medium in the presence or absence of SB202190 (10 μM), LY294002 (10 μM), UO126 (10 μM), or LY294002 plus UO126 (10 μM each). Cells were harvested, counted, and analyzed by flow cytometry on day 4. CD4/CD8 profiles (upper panel) and TCRγδ profiles (lower panel). B, Absolute numbers of DN and DP thymocytes in FTOCs treated with the indicated drugs. The experiment was repeated twice with similar results.

LY294002 induced an overall decrease in thymocyte number. However, the decrease in the number of DP cells was disproportionately large (Fig. 3⇑B), suggesting that LY294002 selectively inhibits the development and/or survival of DP thymocytes. The selectivity of LY294002 was further supported by findings showing that whereas the number of DP thymocytes in LY294002-treated FTOCs was significantly decreased, the number of γδ T cells was only slightly affected (Fig. 3⇑). Similar results were obtained in two independent experiments. These data indicate that the PI3K pathway is critical for the differentiation of thymocytes from the DN to the DP stage. However, because the generation of γδ T cells was also slightly impaired, general toxicity may contribute to the overall effects of LY294002. This conclusion is consistent with the established role of PI3K in the transduction of antiapoptotic signals in T cells and other cell types.

Genetic ablation of Akt inhibits the transition of thymocytes from the DN to the DP stage

To determine whether the inhibition of the PI3K pathway impairs thymocyte differentiation by blocking Akt, we compared thymocyte differentiation in wild-type and Akt knockout mice. Before this analysis, we used real-time RT-PCR to examine the expression of the three Akt isoforms in DN3 and DN4 thymocytes (Fig. 4⇓A). Akt3 is expressed at low levels relative to Akt1 and Akt2, which are expressed at similar levels.

FIGURE 4.
FIGURE 4.

The differentiation of DN thymocytes to the DP stage is impaired in the thymocytes of Lck-Cre+/Akt1fl/fl2−/−, Lck-Cre+/Akt1fl/fl2−/−3−/−, and, to a lesser extent, Lck-Cre/Akt1fl/fl3−/− mice. A, Expression of all three Akt isoforms in DN3 and DN4 thymocytes was measured by real-time RT-PCR. Histograms and error bars show the mean values ± the SD of data obtained from experiments conducted using samples from two mice (three measurements in each sample). B, Expression of Akt1 in DN3 and DN4 cells from wild-type (wt) and Lckcre+/Akt1fl/fl mice measured by real-time RT-PCR. Representative results from one of two separate measurements, each using one distinct group of samples that had one wt and one knockout (ko) mouse are shown. C, Thymocytes were stained with anti-CD4 and anti-CD8 and they were analyzed by flow cytometry. Akt1/2, and to a lesser extent Akt1/3 double knockout thymocytes were defective in β-selection and the progression to the DP stage. Thymocyte differentiation in single Akt knockouts and in Akt2/3 double knockout mice was normal. D, Lin− thymocytes were stained with anti-CD25 (x-axis) and anti-CD44 (y-axis). DN3 cells in Lck-Cre+/Akt1fl/fl2−/− and, to a lesser extent, Lck-Cre+/Akt1fl/fl3−/− mice merge with, and are not clearly separated from, the DN4 cells because of an increase in the relative abundance of a subpopulation of CD44− cells that express intermediate levels of CD25. E, Ablation of the floxed Akt1 allele in Akt1fl/fl/Akt2−/−3−/− mice by an Lck-Cre transgene has profound effects in thymocyte differentiation. Three of six Lck-Cre+/Akt1fl/fl2−/−3−/− mice had thymuses too small to be analyzed. Three of six Lck-Cre+/Akt1fl/fl2−/−3−/− mice also had very small thymuses but they yielded sufficient numbers of thymocytes for analysis. The analysis revealed an almost complete absence of DP thymocytes, as well as a defect in the differentiation of DN3 cells to the DN4 stage. The latter was again characterized by the loss of clear boundaries between DN3 and DN4 cells, which was reminiscent of a similar defect in Lck-Cre+/Akt1fl/fl2−/−mice. F, Weight comparison of the thymus of wild-type mice with the thymus of the Lck-Cre/Akt1fl/fl2−/−3−/− mice.

Thymocyte differentiation experiments were conducted using the following mice: constitutive and conditional Akt1−/− mice (Fig. 4⇑B), and constitutive Akt2−/− and Akt3−/−, Akt1−/−2−/−, Akt1−/−3−/−, Akt2−/−3−/−, and Akt1−/−2−/−3−/− mice and their wild-type controls that were generated by crossing mice heterozygous for the ablation of multiple Akt isoforms as described in Materials and Methods.

The size of the thymus of Akt1−/−, Akt2−/−, and Akt3−/− mice was similar to the size of the thymus of age-matched wild-type controls (data not shown). To address the role of each of the three isoforms in differentiation, thymocytes of wild-type mice (n = 7), Akt1−/− (n = 8), Akt2−/− (n = 5), and Akt3−/− (n = 9) mice were stained with anti-CD4 and anti-CD8 Abs and they were analyzed by flow cytometry. In addition, thymocytes from the same mice were stained with Abs against the lineage (lin) markers CD4, CD8, CD11b, B220, DX5, Gr-1, TCRγδ, and TER119, and against CD44 and CD25. Lin− cells were separated into DN1, 2, 3, and 4 subpopulations, based on the expression of CD44 and CD25. The results revealed no differences in the number of thymocyte subpopulations and their distribution between these mice (Fig. 4⇑, C and D, upper panels), suggesting that if Akt plays a role in thymocyte differentiation, there is significant compensation between Akt isoforms.

To overcome the problem of compensation between Akt isoforms, the same analyses were conducted in Akt2−/−3−/− double knockout mice (expressing only Akt1), Lck-cre+/Akt1fl/fl3−/− mice (expressing only Akt2) and Lck-cre+/Akt1fl/fl2−/− mice (expressing only Akt3). Expression of Lck-Cre during thymocyte differentiation starts at the DN2 stage (14). As a result, the expression of Akt1, measured via real-time RT-PCR in Lck-cre+/Akt1fl/fl mice, decreased at the DN3 stage and was not detectable at the DN4 stage (Fig. 4⇑B). This resulted in a decreased thymic cellularity (Table I⇓) and partial block in the transition of DN3 to DN4 and DN4 to DP stage in Lck-cre+/Akt1fl/fl2−/− mice (Fig. 4⇑, C and D, lower panel, and Table I⇓). A lesser block was observed in Lck-cre+/Akt1fl/fl3−/− mice (Fig. 4⇑, C and D, lower panel, and Table I⇓). Thymocyte differentiation was essentially normal in Akt2−/−3−/− double knockout mice.

View this table:
Table I.

Thymocytes from single, double, and triple Akt knockout mice

The low number of DP thymocytes in the thymus of the Akt1/Akt2 double knockout mice could be the result of a defect in differentiation of DN thymocytes and/or an increase in the rate apoptosis of DP thymocytes. The preceding data suggested a defect in differentiation but did not exclude an increase in the rate of apoptosis of DP thymocytes. To address this question, we first examined the percentage of propidium iodide (PI)-staining DP cells in five wild-type and six Lck-cre/Akt1fl/fl2−/− mice. The results showed that 5.0 ± 3.3% wild-type and 7.7 ± 3% Lck-cre/ Akt1fl/fl2−/− thymocytes were PI positive. The percentage of dead (PI+) DP thymocytes was therefore slightly higher in the double knockout mice. However, the difference was not statistically significant (p < 0.11). Annexin V staining detected also relatively small, although statistically significant differences between the DP thymocytes of five wild-type and three Lck-cre/Akt1fl/fl2−/− mice (2.6 ± 1.9% vs 15.9 ± 2.3%, p < 0.00081). Parallel experiments addressing the cellularity of the DP and DN thymic compartments revealed that the thymi of Akt1/2 double knockout mice contained similar numbers of DN thymocytes with the thymi of wild-type mice (4.1 ± 2.3 × 106 cells in the Akt1/2 double knockout and 3.9 ± 2.0 × 106 cells in the wild-type mice), but that the two differed by nearly 50-fold in the number of DP thymocytes (2.7 ± 2.8 × 106 cells in the Akt1/2 double knockout and 126 ± 57 × 106 cells in the wild-type mice). The small differences in the rate of apoptosis between the wild-type and Akt1/2 double knockout mice cannot explain the large differences in the number of DP thymocytes between the two. These data suggest that the very low numbers of DP thymocytes in the Akt1/2 double knockout mice are due both to a defect in the transition of DN thymocytes to the DP stage as well as to an increase in the rate of apoptosis in the DP compartment.

All the preceding experiments were conducted under conditions where at least one of the Akt isoforms is active. To determine the phenotypic effects of ablation of all Akt isoforms, we conducted the same analyses on Lck-cre+/Akt1fl/fl2−/−3−/− mice. The weight and cellularity of the thymus of the triple Akt knockout mice were greatly reduced compared with those of age-matched wild-type controls (Fig. 4⇑F and data not shown). Flow cytometric analysis of their thymocytes revealed that these mice had an almost complete block in the transition to the DP stage (0.05% of the wild type) (Fig. 4⇑E and Table I⇑). The CD25/CD44 profile of the DN cells of these mice revealed a β-selection defect, characterized by the loss of clear boundaries between the DN3 and DN4 compartment. Collectively, these data indicate that all three Akt isoforms contribute to β-selection and the transition of DN thymocytes to the DP stage, although Akt1 and Akt2 appear to play a more important role than Akt3.

In a separate experiment, DN3 cells (CD44−CD25+) were sorted from the thymus of wild-type and Lck-cre+/Akt1fl/fl2−/− mice and were cocultivated with OP9-GFP or OP9-DL1 cells. Four days later, thymocytes were harvested and analyzed for their expression of CD4 and CD8 (Fig. 5⇓). Although wild-type cells cocultivated with the OP9-DL1 stromal cell line differentiate to the CD4/CD8 DP and/or SP stage, Lck-cre+/Akt1fl/fl2−/− cells do not. These data demonstrate that the β-selection defect of Akt1/2 double knockout mice can be faithfully reproduced in the OP9-DL1 cocultivation system.

FIGURE 5.
FIGURE 5.

The combination of Akt1 and Akt2 is required for the differentiation of DN3 thymocytes, upon cocultivation with OP-9 DL1 cells. DN3 cells from wild-type (left panels) and Lck-Cre+/Akt1fl/fl2−/− mice (right panel) were cocultivated with OP9-GFP or OP9-DL1 cells as indicated. The CD4/CD8 phenotype of the cocultivated lymphoid cells was analyzed by flow cytometry. Representative results from one of three coculture experiments are shown.

Constitutively active Lck-MyrAkt1 transgenes promote the transition of DN3 cells to the DN4 stage

The preceding data suggested that Akt is activated by signals originating in pre-TCR and that the activated Akt is required for the differentiation of DN3 thymocytes and the transition of DN4 thymocytes to the DP stage. If this were the case, a constitutively active Akt1 transgene expressed in the thymus would be expected to promote the transition through these differentiation steps. Flow cytometry of anti-CD4 and anti-CD8 stained thymocytes of wild-type and Lck-MyrAkt1 transgenic mice showed that the Lck-MyrAkt1 transgene gives rise to a significant increase in the percentage of DN thymocytes and to a smaller but consistent increase in the percentage of CD4 SP and CD8 SP cells (Fig. 6⇓, A and B, upper panels). Thus, the Lck-MyrAkt1 transgene promotes the expansion of DN cells, perhaps by enhancing their survival and/or proliferation. Staining DN thymocytes with anti-CD44 and anti-CD25 revealed an unexpected dramatic increase of DN1 cells, with a parallel decrease of both DN3 and DN4 cells (Fig. 6⇓, A and B, lower panels). Because the expression of Lck-MyrAkt1 does not start until the DN2 stage, we speculated that the increase in the DN1 population results from the Akt1-mediated up-regulation of CD44 in DN4 thymocytes, making them appear as DN1 cells. To address this hypothesis, we stained DN cells of wild-type and transgenic mice for intracellular TCR-β and we analyzed them by flow cytometry. In support of our hypothesis, the DN1 cells of Lck-MyrAkt1 transgenic mice expressed intracellular TCR-β, suggesting that they had undergone β-selection (Fig. 6⇓C). To confirm this finding, we analyzed the DN1 cells of the MyrAkt1 transgenic mice for VDJ rearrangements. PCR analyses confirmed that the DN1 cells of the MyrAkt1 mice, but not of the wild-type mice, indeed carry VDJ rearrangements (data not shown). We conclude that the MyrAkt1 transgene promotes the transition of DN3 cells to the DN4 stage.

FIGURE 6.
FIGURE 6.

The Lck-MyrAkt1 transgene promotes β-selection. A, Upper panel, Flow cytometry of wild-type and Lck-MyrAkt1 transgenic thymocytes stained with CD4 and CD8. Lower panel, Flow cytometry of Lin−, DN thymocytes from Lck-MyrAkt1 transgenic and wild-type mice stained with anti-CD44 and anti-CD25 shows a dramatic increase of DN1 cells. B, Upper panel, Distribution of major thymocyte subpopulations in thymuses of five wild-type and seven Lck-MyrAkt1 transgenic mice (M1). Bars depict mean values ± SD. Lower panel, Thymocyte distribution among the four DN groups in the same five wild-type and seven MyrAkt1 mice (M1). C, Staining all populations of DN cells in the thymus of a representative wild-type and a representative Lck-MyrAkt1 (M1) transgenic mouse for intracellular TCR-β expression revealed that the DN1 cells of the Lck-MyrAkt1 transgenic mice but not the DN1 cells of the wild-type controls express intracellular TCR-β chains.

Constitutively active Akt1 rescues DP cell development in mice lacking a functionally rearranged Tcr-β gene

Rag1 and Rag2-deficient mice cannot rearrange their Tcr genes. As a result their thymus of these mice contains <2 million cells that are developmentally blocked at the DN3 stage (10, 11). To determine whether constitutively active Akt1 transgenes can rescue the DN to DP transition in these mice, we crossed the Lck-MyrAkt1 transgene onto the Rag2−/− genetic background. The MyrAkt1 transgene indeed promoted the transition of DN cells to the DP stage (Fig. 7⇓, upper panel) by allowing Rag2−/−MyrAkt1 transgenic thymocytes to bypass the DN3 block caused by the ablation of Rag2 (Fig. 7⇓, lower panel). Given the long latency of Lck-MyrAkt1-induced thymic lymphoma (32), it is unlikely that the DP thymocytes developing in the Rag2−/−MyrAkt1 transgenic mice are transformed. If these cells are indeed not transformed, the preceding data suggest that Akt may be not only necessary but also sufficient for the differentiation of DN3 cells and for the transition of DN cells to the DP stage.

FIGURE 7.
FIGURE 7.

Constitutively active Akt1 transgenes rescue DP cell development in mice lacking a functionally rearranged Tcrb gene. Thymocytes of wild-type Rag2−/− and Rag2−/−/Lck-MyrAkt1 (M1) mice were stained for CD4, CD8, CD44, and CD25 and they were analyzed by flow cytometry. Similar data were obtained when Lck-Akt1E40K rather than Lck-MyrAkt1 transgenic mice were analyzed (data not shown).

Similar data were obtained when the Lck-AktE40K transgene, rather than the Lck-MyrAkt1 transgene, was crossed to the Rag1−/− genetic background. This experiment showed that Akt1E40K, in addition to promoting the differentiation of DN cells to the DP stage, also induced a significant age-dependent increase in the total thymocyte number (data not shown). The increase in cell numbers suggests that Akt1 promotes the proliferative expansion and/or the survival of cells that transit into the DP stage.

Rag2−/−MyrAkt1 transgenic mice failed to produce CD4+ or CD8+ SP cells, suggesting that Akt1 promotes the transition from the DN to the DP stage but is not sufficient to promote the transition from the DP to the SP stage. To confirm this result Akt1 transgenic mice were crossed to TCR-α−/− mice. TCR-α−/− thymocytes arrest at the DP stage because they fail to undergo positive selection (42). CD4 and CD8 staining of thymocytes from TCR-α−/−/Lck-Akt1-E40K and TCR-α−/−/Lck-MyrAkt1 transgenic mice revealed that constitutively active Akt1 does not overcome this block (data not shown).

The combined ablation of Akt1 and Akt2 induces a selective proliferation block in DN4 cells, while the combined ablation of all three Akt isoforms also increases the death rate of all subpopulations of DN thymocytes

To explore the mechanism by which Akt regulates the transition of DN thymocytes to the DP stage, we first examined the effects of the genetic ablation of Akt1, Akt2, Akt3, and the combinations Akt1/2, Akt2/3, and Akt1/3 on the rate of proliferation of DN3 and DN4 cells. Thymocyte suspensions were surface stained with CD44 and CD25, followed by intracellular staining with the DNA dye 7-AAD and they were analyzed by flow cytometry. The ablation of combinations of Akt isoforms does not induce statistically significant changes in the percentage of DN3 cells in the S-G2-M phases of the cell cycle, suggesting that Akt ablation does not affect the proliferation of DN3 cells as a whole (Fig. 8⇓A, upper panel). Next, we gated the dividing cells and examined the percentage of such cells in the DN3 and DN4 compartments. Fig. 8⇓A shows that the ratio of dividing DN3/DN4 thymocytes is significantly higher in the Lck-Cre+/Akt1fl/fl2−/− (n = 4), and to a lesser degree in the Lck-Cre+/Akt1fl/fl3−/− mice (n = 6), suggesting that in these mice, the DN3 cells proliferate more rapidly, or the DN4 cells exhibit a proliferative defect, or both. No changes were found in Akt2−/−3−/− mice (n = 4). To determine whether the combined ablation of Akt1 and Akt2 induces a proliferation block in DN4 cells, as suggested by the preceding experiment, we examined the cell cycle distribution of the DN4 cells from mice of all Akt genotypes. The results (Fig. 8⇓B, upper panel) revealed that the combined ablation of Akt1 and Akt2 is associated with a significant reduction in the percentage of DN4 cells in the S-G2-M phases of the cell cycle, suggesting that these isoforms transduce signals that are required for the proliferation of DN4 cells. However, we could not demonstrate a reduction in the percentage of cycling DN4 cells in Lck-Cre+/Akt1fl/fl3−/− mice. Finally, a similar experiment using thymocytes from a single Lck-Cre+/Akt1fl/fl2−/−3−/− mouse revealed an even larger decrease in the percentage of cycling DN4 thymocytes. In agreement with these data, DN4 thymocytes of Lck-Cre+/Akt1fl/fl2−/− and Lck-Cre+/Akt1fl/fl2−/−3−/− mice are smaller than DN4 cells of wild-type mice (data not shown).

FIGURE 8.
FIGURE 8.

The combined ablation of Akt1 and Akt2, or all three Akt isoforms induces a selective DN4-cell proliferation block. A, Upper panel, Percentage of cycling DN3 thymocytes from wild-type (n = 11), Akt2−/−3−/− (four mice), Lck-Cre+/Akt1fl/fl3−/− (six mice), Lck-Cre+/Akt1fl/fl 2−/− (four mice). Middle panel, Distribution of cycling cells between the DN3 and the DN4 compartments in the indicated genetically defined strains of mice. Lower panel, Cumulative data of the experiment shown in the middle panel. B, Upper panel, Representative results of cell cycle distribution of DN4 thymocytes from the same mice used in A and Lck-Cre+/Akt1fl/fl2−/−3−/− (one mouse). The percentage of cells in S-G2-M in the Lck-Cre+/Akt1fl/fl2−/− and Lck-Cre+/Akt1fl/fl2−/−3−/− mice was significantly lower. Lower panel, Cumulative data of the experiment shown in the upper panel.

DN thymocytes of wild-type, Akt2−/−3−/−, Lck-Cre+/Akt1fl/fl3−/−, Lck-Cre+/Akt1fl/fl2−/−, and Lck-Cre+/Akt1fl/fl2−/− 3−/− mice were also stained with PI as well as with Abs against CD44 and CD25 and they were analyzed by flow cytometry. Fig. 9⇓ shows that the combined ablation of Akt1/2/3 is associated with an increase in the relative numbers of dead cells in all subpopulations of DN thymocytes. However, the fraction of dead DN thymocytes was not increased in the double Akt knockout mice, including those with the combined ablation of Akt1 and Akt2. These findings suggest that the phenotype of the Akt1/2 double knockout mice is due primarily to the effect of the ablation on the proliferation of DN4 thymocytes and that the more severe phenotype of the triple Akt knockout mice may be due to the additional effects of the combined ablation of all three isoforms on cell survival.

FIGURE 9.
FIGURE 9.

The combined ablation of Akt1, Akt2, and Akt3 promotes cell death in all subpopulations of DN thymocytes. Percentage of PI-positive cells in all four subpopulations of DN thymocytes in wild-type (n = 20), Akt2/3 (n = 5), Akt1/3 (n = 6), Akt1/2 (n = 4), and Akt1/2/3 (n = 3) knockout mice.

DN4 thymocytes of not only Akt1/2 but also Akt1/3 and Akt2/3 double knockout mice also express higher levels of CD25 (Fig. 4⇑, D and E, data not shown). This is reminiscent of up-regulation of CD25 in PDK1 (43), LAT (18), and Notch1 (14) knockout mice, which may define a DN3 to DN4 differentiation defect. However, this abnormality is not sufficient to give rise to the observed partial block in the transition of DN thymocytes to the DP stage, which correlates instead with a defect in the proliferation and survival of the DN4 cells caused by the combined ablation of Akt1, and 2, or Akt1, 2, and 3.

DN3 and DN4 cells from Lck-Cre+/Akt1fl/fl2−/− mice exhibit changes in gene expression consistent with their phenotype

DN3 cells undergo proliferative arrest before the rearrangement of the TCR genes and the differentiation to the DN4 stage (7). Fig. 10⇓ shows that DN3 thymocytes from Lck-Cre+/Akt1fl/fl2−/− mice express higher than normal levels of the proliferative gene c-myc and lower than normal levels of the cyclin-dependent kinase inhibitor p27 and the antiproliferative c-myc target gene Gadd45α. These changes would be expected to promote the proliferation of Akt1/2 double knockout DN3 cells. However, parallel changes in the expression of p21 and the transcription factors Egr1 and Egr2 and their target gene Id3 may counter the suggested proliferative phenotype. Following their induction by Egr family members, Id2 and Id3 bind to and inactivate the transcription factor E2A (44) which exerts an antiproliferative effect in DN3 cells, before the expression of TCR-β. Fig. 10⇓ shows that p21 is up-regulated and Egr1, Egr2, Id2, and Id3 are expressed at low levels in the DN3 cells of the Akt1/2 double knockout mice, which may inhibit cell proliferation.

FIGURE 10.
FIGURE 10.

Expression profile of selected genes in DN3 and DN4 cells in Lck-Cre+/Akt1fl/fl2−/− mice. The expression of selected genes was measured in sorted populations of DN3 and DN4 cells from wild-type and Lck-Cre+/Akt1fl/fl2−/− mice (two mice per group) by real-time RT-PCR.

Following differentiation to the DN4 stage, the Akt1/2 double knockout cells exhibit a proliferative defect, which correlates with the lower than normal levels of c-myc. These cells, however, also express higher than normal levels of Egr2, Id2, and Id3, which promote cellular proliferation by inhibiting E2A. Because E47, a component of E2A, is expressed at very low levels in DN4 cells however (Fig. 10⇑), the proliferative effects of the up-regulation of Id2 and Id3 may be neutralized by the deficiency of the antiproliferative target of these molecules.

Discussion

Signals triggered by pre-TCR and Notch1 are required for β-selection and the progression of DN thymocytes to the DP stage (25). In this report, we showed that pre-TCR signals activate Akt and that Akt activation is required for β-selection and the DN to DP transition of differentiating thymocytes. Interestingly, although Notch is required for these thymocyte differentiation steps, and constitutively active Akt1 may partially replace the requirement for Notch (25) our data demonstrated that Notch is not required for the activation of Akt by pre-TCR signals.

The activation of Akt by pre-TCR signals that are known to play an essential role in β-selection and the DN4 to DP transition suggested that Akt signals may also be required for these differentiation events. The role of the pre-TCR-activated Akt in these processes was confirmed by pharmacological inhibition of the PI3K/Akt pathway as well as by genetic ablation of all three Akt isoforms and combinations of them. Both pharmacological inhibition and the combined ablation of Akt1 and Akt2 reduced the number of DP cells. Moreover, the combined ablation of Akt1 and 2, or Akt1, 2, and 3 gave rise to a slight increase in the ratio of DN3 to DN4 cells and to a population of cells that express intermediate levels of CD25. The latter cells may identify an intermediate stage between DN3 and DN4. Finally, CD25 expression was up-regulated in all the double knockout combinations. These data combined suggest a partial block in the differentiation of DN3 cells to DN4 and ultimately to the DP stage. Expression of a constitutively active Akt1 transgene in the thymus had the opposite effect.

The fraction of actively cycling DN4 thymocytes was reproducibly lower in the thymus of Akt1/2 double knockout mice than in the thymus of normal mice. The rate of cell death was not affected in DN thymocytes of all the single and double Akt knockouts. However, in triple Akt knockouts, survival was impaired in all DN thymocyte subpopulations. The survival defect in all DN thymocytes correlates with the severe phenotype of these mice. These findings collectively suggest that signals originating in the pre-TCR complex activate Akt, which then selectively promote cell survival and proliferation. In addition, whereas proliferative expansion of DN4 cells depends primarily on Akt1 and Akt2, Akt3 appears to be required for cell survival.

The preceding data suggested that the decreased cellularity of the DP thymocyte compartment in Akt1/2 double knockout mice may be due to defects in the transition of DN thymocytes to the DP stage. The cellularity of this compartment however, would also be decreased if the ablation of Akt1 and Akt2 impaired the survival of the developing DP thymocytes. To address the role of Akt1 and Akt2 in cell survival in the DP compartment, we measured the percentage and absolute number of apoptotic DP thymocytes in Akt1/2 double knockout mice. The results showed that their numbers were increased relative to their numbers in wild-type mice. However, the increase in apoptosis was relatively small by comparison with the decreased cellularity of the DP compartment which was nearly 50-fold. We conclude that the decreased cellularity of the DP compartment may be due to a combination of defects in the transition of DN thymocytes to the DP stage and the survival of DP thymocytes.

The transduction of survival signals via Akt, in cells that have rearranged the Tcrb gene, has been highlighted in previous reports (45). However, a Bcl-2 transgene, which promotes T cell survival failed to rescue the development of DP thymocytes in CD3γ knockout or SCID mice (46), suggesting that survival signals alone may not be sufficient to promote the transition of DN cells to the DP stage. Therefore, the ability of Akt to promote this transition may depend on its ability to elicit both survival and proliferation signals. Moreover, because Akt ablation alters significantly the pattern of gene expression in DN3 cells, in addition to DN4, we conclude that Akt may also directly influence developmental events associated with the differentiation of DN3 thymocytes.

Several germline mutations and constitutively active transgenes promote the transition of DN cells to the DP stage in Rag−/− mice. The genes identified by these experiments as regulators of the DN to DP transition may be functioning in the same pathway either upstream or downstream of Akt. A number of signaling molecules involved in TCR and perhaps pre-TCR-signaling upstream of the PI3K and Akt1 including Lck, Fyn, Zap70, Syk, LAT, Ras, PI3Kγ, PI3Kδ, and PTEN have been shown to regulate this transition (21, 22, 23, 24, 47, 48). The products of these genes are likely to transmit developmental T cell signals that target Akt. Other genes that also regulate the transition of DN thymocytes to the DP stage, such as p53 (46), Ikaros (49), Egr-1 (38), NF-κB (50), c-Myb (51, 52), β-catenin (26), and TCF/LEF (53), may be regulated by Akt either at the level of gene expression or at the level of protein function and they are therefore likely to function downstream of PI3K/Akt pathway. Thus it has been reported that Akt phosphorylates Mdm2 and promotes degradation of p53 (54) whose inactivation promotes the DN to DP transition. Moreover, Akt activated by IL-2 in T cells (31) promotes c-Myb transcription (55) and Akt activated by TCR cross-linking in mature T cells promotes phosphorylation of Iκ-Bα and activation of NF-κB (56, 57). Pim1 also promotes the transition from the DN to the DP stage (20). Pim1 and its homolog Pim2 belong to a family of transcriptionally regulated serine-threonine kinases, which regulate cell size and mitochondrial membrane potential via an Akt-independent pathway (58).

To further explore the interplay of Akt and Notch during β-selection, we examined the effects of ablation of combinations of Akt isoforms on the differentiation of DN3 thymocytes cocultivated with OP9-DL1 cells. The results showed that DN3 thymocytes lacking both Akt1 and Akt2 fail to differentiate upon cocultivation, suggesting that in the absence of Akt, Notch signals are not sufficient to induce differentiation of DN3 cells. Notch may be required for events preceding the rearrangement of the TCR-β gene (14). Given that Akt is activated by pre-TCR signals, the inability of Notch signals to induce β-selection in cells that lack Akt may suggest that Notch is required for developmental events that lead to the assembly of pre-TCR and Akt activation (14, 39). This does not exclude the possibility that Akt may also undergo activation by signals that precede the rearrangement of pre-TCR and may contribute to the regulation of Notch. The possibility that Akt is required for the transduction of Notch signals is unlikely because of our data, which indicate that Notch is not needed for the activation of Akt.

Pre-TCR signals in differentiating thymocytes with productively rearranged TCR-β genes promote thymocyte commitment to the αβ lineage (59, 60) and promote the differentiation of DN thymocytes to the DP stage. Data presented in this report showed that the PI3K inhibitor LY294002 inhibits the development of DP cells and that genetic ablation of Akt1and 2 produced a similar, although less severe phenotype. Interestingly, however, LY294002 did not affect the total number of γδ T cells in FTOCs treated with the drug. We conclude that the PI3K pathway is obligatory for the development of αβ but not γδ T cells and that the PI3K signals responsible for these effects are transduced at least in part via Akt.

In summary, this study demonstrates that early thymocyte development is regulated by the PI3K/Akt pathway, which promotes both the transition of DN thymocytes to the DP stage and the survival of DP thymocytes. Of the three Akt isoforms, loss of Akt1 and Akt2 together suppresses proliferative expansion of DN4 cells and the survival of DP thymocytes. Loss of all three Akt isoforms inhibits survival of all the DN thymocyte subsets in addition to inhibition of proliferation of the DN4 cells. The fact that the combined ablation of Akt1 and Akt2 had significant effects on gene expression in DN3 cells suggests that Akt may also contribute to the regulation of developmental events associated with the differentiation of DN3 thymocytes.

Acknowledgments

We thank Dr. M. Birnbaum (University of Pennsylvania, Philadelphia, PA) for his gift of Akt2−/− mice and Dr. J. C. Zuniga-Pflucker for providing us with the OP9-GFP and OP9-DL1 cells. We also thank Drs. R. Van Etten and H. Wortis for reviewing the manuscript.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by National Institutes of Health Grants R01-CA057436 (to P.N.T.) and R01-AI059676 (to F.G.). C.M. was supported by National Institutes of Health Training Grant T32-CA009429.

  • ↵2 Current address: Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210.

  • ↵3 Current address: Department of Biology, Pine Manor College, Chestnut Hill, MA 02467.

  • ↵4 Address correspondence and reprint requests to Dr. Philip N. Tsichlis, Molecular Oncology Research Institute, Tufts-New England Medical Center, 750 Washington Street, No. 5609, Boston, MA 02111. E-mail address: ptsichlis{at}tufts-nemc.org

  • ↵5 Abbreviations used in this paper: DN, double negative; pT-α, pre-Tα; DP, double positive; LAT, linker for activation of T cells; FTOC, fetal thymic organ culture; PI, propidium iodide; RT, room temperature.

  • Received October 20, 2006.
  • Accepted February 13, 2007.

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