Akt1 and Akt2 are required for αβ thymocyte survival and differentiation

Akt1, Akt2, and Akt3 are Differentially Expressed During Thymocyte Development.

To understand the relative contributions of different Akt isoforms for T cell development, expression levels of Akt1, Akt2, and Akt3 were examined by real-time PCR analysis on sorted lymphocytes from the thymus and spleen of WT mice (Fig. 1A). Akt1 is the most highly expressed isoform in the DN and DP subsets, with more equal representation of Akt1, Akt2, and Akt3 in CD4 and CD8 single positive (SP) cells (Fig. 1A). All three Akt isoforms are also detectable by Western blot analysis of whole thymus and peripheral T cells (Fig. 1B). In addition to transcriptional regulation, Akt function depends on its phosphorylation by mTOR/Rictor and PDK1 (4). We found that direct stimulation of the TCR in thymocytes results in the rapid phosphorylation of Akt (Fig. 1C).

Akt1^−/−Akt2^−/− Thymocytes Display Defective Development Past the DN3 Stage.

To ascertain whether the loss of individual Akt isoforms would affect thymic development, we analyzed T cell subsets from mice with targeted deletions of either Akt1 or Akt2 [supporting information (SI) Fig. 7]. The overall thymic cellularity of adult Akt1 single knockout mice was consistently decreased compared with heterozygous littermates, but this did not affect the distribution of immature thymocytes or translate to altered peripheral T cell subsets (SI Fig. 7). Although there were fewer thymocytes present in the setting of Akt1 deficiency, loss of either Akt1 or Akt2 did not markedly affect thymocyte subsets or activation of mature T cells (data not shown).

To address the possibility that the lack of a developmental phenotype in the Akt-deficient mice was due to compensation by other isoforms, we next studied the T cell compartment when both Akt1 and Akt2 were eliminated. These experiments were challenging because Akt1^−/−Akt2^−/− mice die within a few hours of birth (12). Therefore, we made use of three complementary approaches: fetal thymic analysis to investigate the earliest stages of development, the OP9-DL1 coculture system to model further development ex vivo, and fetal liver bone marrow chimeras to assess full thymic development and the appearance of peripheral T cells. We first analyzed thymi from embryos and newborn pups. Consistent with our findings from adult single knockout mice, Akt1^−/− embryos had consistently lower thymic cellularity. This lower cellularity did not result in significantly altered thymic subsets (Fig. 2). In contrast, the loss of Akt1 and Akt2 caused both a reduction in thymocyte cellularity and altered T cell subsets (Fig. 2).

Decreased numbers of thymic progenitors may have affected later development in the absence of Akt1 and Akt2. Analysis of the early thymocyte progenitor (1) and DN2 populations showed no significant difference between Akt1^−/−Akt2^−/− embryos and littermate controls (data not shown). In contrast, the total number of DN3 cells in the Akt1^−/−Akt2^−/− thymus was decreased 2-fold compared with controls (SI Fig. 8A). Furthermore, the number of DN4 cells in the Akt1^−/−Akt2^−/− fetal thymus was reduced ≈4- to 5-fold compared with heterozygous controls (Fig. S2A). The increased DN3:DN4 ratio is consistent with a partial block at the DN3 stage (SI Fig. 8B).

Akt1^−/−Akt2^−/− T Cells Do Not Efficiently Reach the DP Stage in Vitro.

It remained unclear whether the altered thymic development found in the Akt1^−/−Akt2^−/− fetal thymus was due to delayed transition time from the DN3 to DP stage, decreased survival within or past the DN3 stage, or a secondary effect from Akt loss in the thymic stroma. To address the first possibility, we used the OP9-DL1 coculture model (13) to ask whether Akt1^−/−Akt2^−/− DN3 cells would differentiate to the DP stage if allowed a lengthier period than allowed by the short fetal lifespan. Fetal liver-derived progenitor cells from knockout embryos were cultured on OP9-DL1 monolayers for 21 days. We did not observe a defect in development before the DN3 stage, measured at day 10, in cultures from mutant embryos (data not shown). However, after 13 days of culture, there was a 33-fold reduction in the frequency of Akt1^−/−Akt2^−/− cells that up-regulated CD4 or CD8 compared with the Akt1^+/−Akt2^+/+ control (Fig. 3). Next, we continued the OP9-DL1 cultures for 7 days to determine whether the developmental defect was due to a delayed transition time. By day 21, 60% of the cells in Akt1^+/−Akt2^+/+ control cultures were positive for either CD4 or CD8, but less than 1% of Akt1^−/−Akt2^−/− cells became CD4 or CD8 positive (data not shown). Cells that had only one WT allele of either Akt1 or Akt2 displayed an intermediate phenotype. Collectively, the in vivo fetal thymic data and the in vitro OP9-DL1 coculture data demonstrate a partial defect at the β-selection checkpoint that is not caused by altered thymic stroma.

Akt1^−/−Akt2^−/− Fetal Liver Cells Fail to Reconstitute the Mature T Cell Population of an Irradiated Host.

Although the OP9-DL1 coculture system provides an excellent model for thymic development, this approach can only recapitulate a subset of the environmental cues that thymocytes normally receive in vivo. To measure T cell development in a more physiological setting, fetal liver cells were transferred to lethally irradiated congenic hosts. Analysis of the thymi 7–8 weeks after reconstitution revealed that Akt1^−/−Akt2^−/− thymocytes could differentiate to the DP and SP stages, but, consistent with our results in the fetal thymus and in OP9-DL1 co-cultures, there was a lower percentage of cells in the DP stage and a higher percentage of cells at the DN3 stage (Fig. 4A). Overall thymic cellularity in reconstituted chimeras was decreased 2-fold with the single loss of either Akt1 or Akt2 but decreased 45-fold with deficiency of both Akt1 and Akt2 (Fig. 4B). Analysis of thymic subsets revealed that the number of DN3 cells was 2-fold lower in the Akt1^−/−Akt2^−/− chimeras compared with the controls. The absence of a single isoform of Akt resulted in a 2-fold reduction in the DP population (Fig. 4B). In contrast, the total number of Akt1^−/−Akt2^−/− DP cells was decreased 70-fold compared with the control (Fig. 4B). The CD8 SP cells in the Akt1^−/−Akt2^−/− chimeras were mostly immature SPs, a step before the DP stage marked by low TCRβ levels and high CD24 expression (data not shown).

We next analyzed the peripheral T cell compartment to determine whether the few remaining Akt1^−/−Akt2^−/− SP thymocytes could exit the thymus as mature T cells. Overall splenic cellularity was decreased 2-fold in the Akt1^−/−Akt2^−/− chimeras with decreases in both the B220⁺ population and the Thy1.2⁺ populations (Fig. 4C and SI Fig. 9). However, T cells were more affected than B cells, and the ratio of Thy1.2⁺ cells to B220⁺ cells was decreased in the Akt1^−/−Akt2^−/− chimeras (SI Fig. 9), possibly because of the higher expression of Akt3 in B cells than in T cells (Fig. 1A). CD4⁺ and CD8⁺ cells respond differently to altered signals in the thymus, and the current model suggests that stronger or longer TCR signals favor the CD4⁺ lineage and weaker or shorter TCR signals direct cells to the CD8⁺ lineage (14). Therefore, as might be expected, we found that the CD4⁺ lineage was more affected than the CD8⁺ lineage in the Akt1^−/−Akt2^−/− chimeras, as manifested by a decreased ratio of CD4⁺ to CD8⁺ cells (SI Fig. 9). The decreased cellularity in the periphery did not affect all hematopoietic lineages equally, as no significant decrease in γδT cells, Mac-1⁺Gr-1⁺ neutrophils, or NK1.1⁺ NK cells was observed (SI Fig. 9). The normal development of Akt1^−/−Akt2^−/− γδT cells is further evidence that the DN stages are not as affected by the loss of Akt1 and Akt2, because the γδT lineage is thought to diverge from the αβT lineage before the DP stage (15).

Akt1^−/−Akt2^−/− Thymocytes have Decreased Glucose Uptake.

The reduction in DP cells in the Akt1^−/−Akt2^−/− chimeras could be due to either an increase in cell death or a decrease in the generation of DP cells. Because the 2-fold decrease in DN3 cells was not sufficient to explain the 70-fold decrease in DP cells, we first examined the metabolic capacity of DN3 cells by measuring the transport of glucose into the cell in vitro. Glucose uptake is a necessary step to sustain cell survival and metabolism for subsequent growth and division (16), and Akt has been shown to require glucose metabolism to prevent cell death (13). Akt1^−/−Akt2^−/− DN3 cells had reduced glucose uptake in vitro compared with controls (Fig. 5A). We consistently found that the glucose uptake relative to the total Glut1 receptor expression (data not shown) was reduced in Akt1^−/−Akt2^−/− cells, suggesting a requirement for these isoforms for efficient glucose transport in developing thymocytes.

Cellular metabolism is linked to cell size and overall protein content. Further, the activation of the Akt/mTOR pathway is critical for cell size determination (4). Consistent with this role for Akt, transgenic expression of myr-Akt in T cells results in an approximate 10% increase in cell size (17). Conversely and in support of our finding that glucose uptake was altered, we also found that Akt1^−/−Akt2^−/− DN3 cells were reduced in size by 10% compared with heterozygous controls (SI Fig. 10A). Further, Akt1^−/−Akt2^−/− DN3 cells lost a greater volume of their cell size with growth factor deprivation compared with controls (Fig. 10B).

Akt has a positive effect on proliferation in many model systems including peripheral T cells (4). During normal thymocyte development, CD25 is diluted from the surface as cells undergo multiple divisions to the DP stage, and thymocytes that have proliferative defects abnormally retain CD25 at the DP stage (18). We found that Akt1^−/−Akt2^−/− DP thymocytes retain surface CD25 (data not shown). To investigate more directly whether proliferation is altered in Akt1^−/−Akt2^−/− thymocytes, bone marrow chimeras were pulsed with BrdU in vivo (Fig. 5 B and C). We found no difference in the overall percentage of thymocytes in S phase in Akt1^−/−Akt2^−/− chimeras compared with controls (Fig. 5B). Because the formation of a pre-TCR regulates entry into the cell cycle, we also measured whether BrdU⁺ cells were also TCRβ⁺. The combined loss of Akt1 and Akt2 does not alter the percentage of total thymocytes that are intracellular (ic)TCRβ⁺ and BrdU⁺ compared with WT controls (Fig. 5C), although, a careful subset analysis suggested a modest defect in proliferation within the CD4⁻CD8-TCRβ⁺ population. These data indicate that loss of Akt1 and Akt2 does not preclude the proliferative response to pre-TCR signaling.

Defective Development Beyond the DN3 Stage Is Due to Increased Apoptosis in Akt1^−/−Akt2^−/− Mice.

Previous work reported increased DP thymocyte survival in transgenic mice that express myr–Akt1 in T cells (19). Myr-Akt1 was found to increase expression of Bcl-xL (19); however, we did not detect any alteration in Bcl-xL expression level in Akt1^−/−Akt2^−/− thymocytes relative to controls (data not shown). To complement the gain-of-function experiment, we analyzed Akt1^−/−Akt2^−/− thymocytes from chimeras and newborn pups for the frequency of spontaneously apoptotic cells (Fig. 6 A and B). A higher proportion of Akt1^−/−Akt2^−/− thymocytes were apoptotic in the chimeras at all points beyond the DN stage (Fig. 6A). The single loss of either Akt1 or Akt2 also resulted in slightly more apoptosis at the CD8⁺/immature SP stage (Fig. 6A). We also found an increasing pattern of apoptosis in thymocytes from newborn pups that lacked two, three, or four alleles of Akt (Fig. 6B). The loss of three or four Akt alleles correlated with a higher percentage of apoptotic cells at the TCRβ^hi and TCRβ^int stages of development. Based on this pattern of apoptosis, we hypothesized that post-DN3 cells may require Akt for survival upon TCR stimulation.

To test whether the absence of Akt in immature thymocytes causes apoptosis with pre-TCR stimulation, cells from day 10 OP9-DL1 cultures were stimulated with αCD3 and αCD28 to mimic β-selection. In contrast to cells expressing even a single copy of Akt1 or Akt2, Akt1^−/−Akt2^−/− cells exhibited increased cell death with pre-TCR stimulation (Fig. 6C). Additionally, Akt1^−/−Akt2^−/− DN3 cells were more susceptible to death by neglect and lost a greater percentage of cell size before death compared with controls (Fig. 6D and SI Fig. 10B). In contrast, Akt1^−/−Akt2^−/− DN3 cells were equally sensitive to dexamethasone-induced apoptosis (data not shown). Taken together, these data indicate that the combined loss of Akt1 and Akt2 causes increased apoptosis in immature thymocytes after expression of the TCRβ chain but a comparably modest effect on proliferation during the DN3 to DP transition.

Mice.

Mice lacking Akt1 or Akt2 on the C57BL/6 background were described in refs. 34 and 35. SJL.B6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were performed according to the guidelines of the University of Pennsylvania and Duke University Institutional Animal Care and Use Committees.

Flow Cytometric Analysis.

Single cell suspensions from thymi, spleens, and OP9-DL1 cultures were stained in FACS buffer (PBS/2% FBS/azide) and analyzed on a FACSCalibur or an LSRII (BD Biosciences, San Jose, CA), using FlowJo software. Annexin V staining was completed by using the manufacturer's protocol (BD Biosciences) except for an additional wash before analysis. DAPI was used as a live/dead exclusion marker. Cells were sorted on a FACSVantage SE (BD Biosciences) based on the surface expression of Thy1.2, CD4, CD44, and CD25. The lineage mixture for DN subset analysis includes CD8, NK1.1, CD11b, GR-1, CD11c, TCRγδ, B220, and Ter119. SI Materials and Methods contains further antibody information.

Statistical Analysis.

Statistical analysis was performed with two-tailed, unpaired Student's t tests with Prism software (significance: *, P < 0.05; **, P < 0.01; ***, P < 0.0001).

Real-time PCR.

TRIzol (Invitrogen, Carlsbad, CA)-isolated RNA was reverse transcribed with SuperScript II (Invitrogen). Quantitative RT-PCR was done by using SYBR Green Supermix (Bio-Rad, Hercules, CA) and was analyzed on an iCycler. All data were calculated relative to Akt1 levels in thymic CD4 SP cells and based on β2-microglobulin as the internal control. Primer sequences are available in SI Materials and Methods.

Biochemistry.

Thymocytes were resuspended at 10⁶ cells per 100 μl in PBS, rested for 20 min at 37°C, and stimulated with 10 μg/ml αCD3 (500A2-BD Biosciences). Cell lysates [RIPA buffer (36)] were run on SDS/PAGE. Membranes were incubated with p-Akt S473 (BD Biosciences) or total Akt (BD Biosciences), were developed with ECL (Amersham, Little Chalfont, U.K.) or AlexaFluor-680 anti-rabbit IgG (Invitrogen) and IR Dye 800 anti-mouse IgG (Li-Cor Biosciences, Lincoln, NE), and were detected with a Li-Cor Odyssey system for the Akt1, Akt2, or Akt3 (Cell Signaling, Danvers, MA) Western blot. T cells were purified by using StemSep magnetic purification kits (StemCell Technologies, Vancouver, BC, Canada).

OP9-DL1 Cultures.

Cultures were treated as described in ref. 37, except ≈10⁶ HSA-depleted (Miltenyi Biotec, Bergisch Gladbach, Germany) fetal liver cells (14.5–16.5 days post-coitum) were plated per well in a 24-well plate, and media were supplemented with 5 ng/ml each murine IL-7 and human Flt-3L (R&D Systems, Minneapolis, MN).

TCR Stimulation and Apoptosis Assays in Vitro.

Fetal liver cells were cultured with OP9-DL1 cells for 10 days and passed into 96-well plates containing OP9-DL1 cells. Cultures were stimulated with 20 μg/ml each αCD3 and αCD28 (BD Biosciences) in the presence of IL-7 and Flt-3L (described above). For neglect conditions, cells were plated without OP9-DL1 cells or cytokines. After 24–30 h, cell viability was defined by PI exclusion and cell size (FACScan).

Bone Marrow Chimeras.

Fetal livers were harvested from embryos 14.5–16.5 days postcoitum and were cultured overnight in αMEM supplemented with 20% FBS, 2.2 g/liter sodium bicarbonate, 2 mM glutamine, antibiotics, 10 ng/ml IL-6 (Peprotech), 20 ng/ml IL-3 (Peprotech), and 100 μg/ml stem cell factor (Peprotech, Rocky Hill, NJ). Embryos were genotyped by PCR amplification, and 0.25 × 10⁶ cells were injected into irradiated SJL.B6 mice (550 rads, two doses, 4 h apart).

Glucose Uptake.

Fetal liver cells cultured with OP9-DL1 cells for 10 days were resuspended in 0.5% BSA in Kreb's Ringers buffer. Uptake of ³H-2-deoxy-d-glucose (Amersham) was measured as described in ref. 8, using 2 μCi per reaction with a 10 min incubation.

In Vivo BrdU Labeling.

Mice were injected i.p. with 2 mg/200 μl of BrdU (BD Biosciences), and thymi were harvested 5 h later. The protocol for the BrdU kit (BD Biosciences) was followed except for the use of DAPI as marker for DNA content.