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. 2000 May 15;191(10):1721-34.
doi: 10.1084/jem.191.10.1721.

Protein kinase B regulates T lymphocyte survival, nuclear factor kappaB activation, and Bcl-X(L) levels in vivo

R G Jones  1 , M Parsons, M Bonnard, V S Chan, W C Yeh, J R Woodgett, P S Ohashi
Affiliations

Protein kinase B regulates T lymphocyte survival, nuclear factor kappaB activation, and Bcl-X(L) levels in vivo

R G Jones et al. J Exp Med. .

Abstract

The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. We have generated transgenic mice expressing a constitutively active form of PKB (gag-PKB) to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB displayed increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolonged the survival of CD4(+)CD8(+) double positive (DP) thymocytes in fetal thymic organ culture, but was unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I-restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen-specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB displayed elevated levels of the antiapoptotic molecule Bcl-X(L). In addition, the activation of peripheral T cells led to enhanced nuclear factor (NF)-kappaB activation via accelerated degradation of the NF-kappaB inhibitory protein IkappaBalpha. Our data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules (PKB, Bcl-X(L), and NF-kappaB) in vivo in T lymphocytes.

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Figures

Figure 1
Figure 1
T cells from transgenic mice express activated PKB. (A) Illustration of the gag-PKB transgene. A 2.3-kb cDNA fragment containing the coding sequence of gag-PKB was inserted into the EcoRI and SmaI sites of the human CD2 minigene. The CD2 promoter, polyadenylation signal (Poly A), and enhancer/locus control region (LCR) are indicated. The construct was linearized at the KpnI and XbaI sites before microinjection. (B) gag-PKB T cells express transgenic gag-PKB mRNA. DNase-treated total RNA isolated from gag-PKB transgenic (B6/PKB) and nontransgenic (B6) LN T cells was subjected to RT-PCR using gag-PKB–specific primers. PCR was performed on the RNA to control for DNA contamination. In the right lane, gag-PKB cDNA was amplified as a positive control for the PCR reaction. RT-PCR products were analyzed on a 1% agarose gel stained with ethidium bromide. (C) Transgenic gag-PKB T cells display elevated levels of PKB protein and increased levels of PKB phosphorylation. Expression of PKB was assessed in thymus, LN, and purified CD4+ and CD8+ T cells from wild-type mice (B6) and gag-PKB transgenic mice from two independent founder lines (B6/PKB5-4 and B6/PKB4-1) by Western blot analysis with a polyclonal anti-PKB antibody. Phosphorylated PKB was detected for the same samples using a polyclonal antibody specific for PKB phosphorylated at Ser-473. Lysates from 2 × 106 cells per tissue sample were loaded per lane after normalization for protein content via the Bradford assay.
Figure 1
Figure 1
T cells from transgenic mice express activated PKB. (A) Illustration of the gag-PKB transgene. A 2.3-kb cDNA fragment containing the coding sequence of gag-PKB was inserted into the EcoRI and SmaI sites of the human CD2 minigene. The CD2 promoter, polyadenylation signal (Poly A), and enhancer/locus control region (LCR) are indicated. The construct was linearized at the KpnI and XbaI sites before microinjection. (B) gag-PKB T cells express transgenic gag-PKB mRNA. DNase-treated total RNA isolated from gag-PKB transgenic (B6/PKB) and nontransgenic (B6) LN T cells was subjected to RT-PCR using gag-PKB–specific primers. PCR was performed on the RNA to control for DNA contamination. In the right lane, gag-PKB cDNA was amplified as a positive control for the PCR reaction. RT-PCR products were analyzed on a 1% agarose gel stained with ethidium bromide. (C) Transgenic gag-PKB T cells display elevated levels of PKB protein and increased levels of PKB phosphorylation. Expression of PKB was assessed in thymus, LN, and purified CD4+ and CD8+ T cells from wild-type mice (B6) and gag-PKB transgenic mice from two independent founder lines (B6/PKB5-4 and B6/PKB4-1) by Western blot analysis with a polyclonal anti-PKB antibody. Phosphorylated PKB was detected for the same samples using a polyclonal antibody specific for PKB phosphorylated at Ser-473. Lysates from 2 × 106 cells per tissue sample were loaded per lane after normalization for protein content via the Bradford assay.
Figure 1
Figure 1
T cells from transgenic mice express activated PKB. (A) Illustration of the gag-PKB transgene. A 2.3-kb cDNA fragment containing the coding sequence of gag-PKB was inserted into the EcoRI and SmaI sites of the human CD2 minigene. The CD2 promoter, polyadenylation signal (Poly A), and enhancer/locus control region (LCR) are indicated. The construct was linearized at the KpnI and XbaI sites before microinjection. (B) gag-PKB T cells express transgenic gag-PKB mRNA. DNase-treated total RNA isolated from gag-PKB transgenic (B6/PKB) and nontransgenic (B6) LN T cells was subjected to RT-PCR using gag-PKB–specific primers. PCR was performed on the RNA to control for DNA contamination. In the right lane, gag-PKB cDNA was amplified as a positive control for the PCR reaction. RT-PCR products were analyzed on a 1% agarose gel stained with ethidium bromide. (C) Transgenic gag-PKB T cells display elevated levels of PKB protein and increased levels of PKB phosphorylation. Expression of PKB was assessed in thymus, LN, and purified CD4+ and CD8+ T cells from wild-type mice (B6) and gag-PKB transgenic mice from two independent founder lines (B6/PKB5-4 and B6/PKB4-1) by Western blot analysis with a polyclonal anti-PKB antibody. Phosphorylated PKB was detected for the same samples using a polyclonal antibody specific for PKB phosphorylated at Ser-473. Lysates from 2 × 106 cells per tissue sample were loaded per lane after normalization for protein content via the Bradford assay.
Figure 2
Figure 2
PKB kinase activity protects thymocytes from various apoptosis-inducing stimuli and spontaneous apoptosis in vitro. (A) Transgenic gag-PKB thymocytes are resistant to γ irradiation, dexamethasone, and anti-Fas–induced apoptosis. Thymocytes from control mice (B6, ▪) or mice expressing the gag-PKB transgene (B6/PKB5-4, □; or B6/PKB4-1, ○) were cultured at 106 cells/ml after exposure to γ radiation or incubation in the presence of dexamethasone or recombinant CD8-FasL fusion protein. Thymocyte viability was determined by trypan blue exclusion at various time points after apoptotic treatment. Viability is expressed normalized to untreated thymocytes and in triplicate (±SEM). The data are representative of four independent experiments. (B) PKB activity promotes survival in thymocytes. Thymocytes from transgenic mice (B6/PKB5-4, □; or B6/PKB4-1, ○) or nontransgenic littermate controls (B6, ▪) were cultured in 24-well tissue culture plates at 106 cells/ml in IMDM supplemented with 5% heat-inactivated FCS, 2 mM l-glutamine, and 50 μM β-mercaptoethanol. Cell viability was determined in triplicate by trypan blue exclusion over 8 d of culture at 37°C. The error bars represent the SEM expressed as cell numbers for each triplicate value. These data are representative of six independent experiments. (C) PKB activity selectively protects CD4+CD8+ DP thymocytes from spontaneous apoptosis. Thymocytes from B6/PKB and B6 mice were cultured as in B and harvested after 0, 4, or 8 d of culture. Cells were stained with FITC-conjugated anti-CD8α, PE-conjugated anti-CD4, and 7AAD, and analyzed via flow cytometry. Viable thymocytes were determined by negative staining with 7AAD. The mean percentage of DP thymocytes at each time point is indicated. The presented data are representative of three separate experiments with two mice per experiment.
Figure 2
Figure 2
PKB kinase activity protects thymocytes from various apoptosis-inducing stimuli and spontaneous apoptosis in vitro. (A) Transgenic gag-PKB thymocytes are resistant to γ irradiation, dexamethasone, and anti-Fas–induced apoptosis. Thymocytes from control mice (B6, ▪) or mice expressing the gag-PKB transgene (B6/PKB5-4, □; or B6/PKB4-1, ○) were cultured at 106 cells/ml after exposure to γ radiation or incubation in the presence of dexamethasone or recombinant CD8-FasL fusion protein. Thymocyte viability was determined by trypan blue exclusion at various time points after apoptotic treatment. Viability is expressed normalized to untreated thymocytes and in triplicate (±SEM). The data are representative of four independent experiments. (B) PKB activity promotes survival in thymocytes. Thymocytes from transgenic mice (B6/PKB5-4, □; or B6/PKB4-1, ○) or nontransgenic littermate controls (B6, ▪) were cultured in 24-well tissue culture plates at 106 cells/ml in IMDM supplemented with 5% heat-inactivated FCS, 2 mM l-glutamine, and 50 μM β-mercaptoethanol. Cell viability was determined in triplicate by trypan blue exclusion over 8 d of culture at 37°C. The error bars represent the SEM expressed as cell numbers for each triplicate value. These data are representative of six independent experiments. (C) PKB activity selectively protects CD4+CD8+ DP thymocytes from spontaneous apoptosis. Thymocytes from B6/PKB and B6 mice were cultured as in B and harvested after 0, 4, or 8 d of culture. Cells were stained with FITC-conjugated anti-CD8α, PE-conjugated anti-CD4, and 7AAD, and analyzed via flow cytometry. Viable thymocytes were determined by negative staining with 7AAD. The mean percentage of DP thymocytes at each time point is indicated. The presented data are representative of three separate experiments with two mice per experiment.
Figure 2
Figure 2
PKB kinase activity protects thymocytes from various apoptosis-inducing stimuli and spontaneous apoptosis in vitro. (A) Transgenic gag-PKB thymocytes are resistant to γ irradiation, dexamethasone, and anti-Fas–induced apoptosis. Thymocytes from control mice (B6, ▪) or mice expressing the gag-PKB transgene (B6/PKB5-4, □; or B6/PKB4-1, ○) were cultured at 106 cells/ml after exposure to γ radiation or incubation in the presence of dexamethasone or recombinant CD8-FasL fusion protein. Thymocyte viability was determined by trypan blue exclusion at various time points after apoptotic treatment. Viability is expressed normalized to untreated thymocytes and in triplicate (±SEM). The data are representative of four independent experiments. (B) PKB activity promotes survival in thymocytes. Thymocytes from transgenic mice (B6/PKB5-4, □; or B6/PKB4-1, ○) or nontransgenic littermate controls (B6, ▪) were cultured in 24-well tissue culture plates at 106 cells/ml in IMDM supplemented with 5% heat-inactivated FCS, 2 mM l-glutamine, and 50 μM β-mercaptoethanol. Cell viability was determined in triplicate by trypan blue exclusion over 8 d of culture at 37°C. The error bars represent the SEM expressed as cell numbers for each triplicate value. These data are representative of six independent experiments. (C) PKB activity selectively protects CD4+CD8+ DP thymocytes from spontaneous apoptosis. Thymocytes from B6/PKB and B6 mice were cultured as in B and harvested after 0, 4, or 8 d of culture. Cells were stained with FITC-conjugated anti-CD8α, PE-conjugated anti-CD4, and 7AAD, and analyzed via flow cytometry. Viable thymocytes were determined by negative staining with 7AAD. The mean percentage of DP thymocytes at each time point is indicated. The presented data are representative of three separate experiments with two mice per experiment.
Figure 3
Figure 3
Active PKB enhances CD4+CD8+ DP thymocyte survival in FTOC. FTOCs from P14 TCR transgenic mice with (P14/PKB, hatched bars) or without (P14, black bars) the gag-PKB transgene were cultured and stained with FITC-conjugated anti-CD8, PE-conjugated anti-CD4, and biotinylated anti-Vα2 or HSA antibodies. (A) Dot plots show CD4 and CD8 expression of thymocytes isolated from P14 and P14/PKB fetal thymic lobes after 6 d in culture. The percentage of cells from each thymocyte subpopulation is indicated. Histograms display the Vα2 expression levels of mature (HSAlo) CD8+ thymocytes. The data shown are representative of three independent experiments. (B) Total cell numbers from P14 (n = 4) and P14/PKB (n = 6) fetal thymic lobes are described in A.
Figure 3
Figure 3
Active PKB enhances CD4+CD8+ DP thymocyte survival in FTOC. FTOCs from P14 TCR transgenic mice with (P14/PKB, hatched bars) or without (P14, black bars) the gag-PKB transgene were cultured and stained with FITC-conjugated anti-CD8, PE-conjugated anti-CD4, and biotinylated anti-Vα2 or HSA antibodies. (A) Dot plots show CD4 and CD8 expression of thymocytes isolated from P14 and P14/PKB fetal thymic lobes after 6 d in culture. The percentage of cells from each thymocyte subpopulation is indicated. Histograms display the Vα2 expression levels of mature (HSAlo) CD8+ thymocytes. The data shown are representative of three independent experiments. (B) Total cell numbers from P14 (n = 4) and P14/PKB (n = 6) fetal thymic lobes are described in A.
Figure 4
Figure 4
Overexpression of gag-PKB does not prevent thymocyte negative selection in FTOC. FTOCs from P14 TCR transgenic mice with (P14/PKB) or without (P14) the gag-PKB transgene were cultured in the presence or absence of 10−6 M p33 peptide and stained with FITC-conjugated anti-CD8, PE-conjugated anti-CD4, and biotinylated anti-Vα2 or HSA antibodies. (A) The percentage of cells from each thymocyte subpopulation is indicated. Histograms display the Vα2 expression levels on immature (HSAhi) CD4+CD8+ DP thymocytes and mature (HSAlo) CD8+ thymocytes. (B) Total thymocyte and CD4+CD8+ DP thymocyte cell numbers from P14 (black bars) and P14/PKB lobes (hatched bars) after incubation in the presence or absence of p33 peptide. Values shown represent the mean ± SEM for four (P14) and six (P14/PKB) thymic lobes. These data are representative of two independent experiments.
Figure 4
Figure 4
Overexpression of gag-PKB does not prevent thymocyte negative selection in FTOC. FTOCs from P14 TCR transgenic mice with (P14/PKB) or without (P14) the gag-PKB transgene were cultured in the presence or absence of 10−6 M p33 peptide and stained with FITC-conjugated anti-CD8, PE-conjugated anti-CD4, and biotinylated anti-Vα2 or HSA antibodies. (A) The percentage of cells from each thymocyte subpopulation is indicated. Histograms display the Vα2 expression levels on immature (HSAhi) CD4+CD8+ DP thymocytes and mature (HSAlo) CD8+ thymocytes. (B) Total thymocyte and CD4+CD8+ DP thymocyte cell numbers from P14 (black bars) and P14/PKB lobes (hatched bars) after incubation in the presence or absence of p33 peptide. Values shown represent the mean ± SEM for four (P14) and six (P14/PKB) thymic lobes. These data are representative of two independent experiments.
Figure 6
Figure 6
PKB kinase activity protects mature T cells from apoptosis-inducing stimuli and spontaneous cell death in vitro. (A) Transgenic gag-PKB T cells are resistant to γ irradiation and dexamethasone-induced apoptosis. Purified CD4+ and CD8+ splenic T cells from mice expressing the gag-PKB transgene (B6/PKB5-4, □; or B6/PKB4-1, ○) or littermate controls (B6, ▪) were cultured at 106 cells/ml after exposure to γ radiation or dexamethasone. Viability was determined by trypan blue exclusion up to 48 h after treatment. Viability is expressed normalized to untreated T cells and in triplicate (±SEM). The data are representative of four independent experiments. (B) PKB kinase activity enhances T cell survival. Purified CD4+ and CD8+ splenic T cells were cultured at 106 cells/ml for 8 d at 37°C. Cell viability was determined in triplicate by trypan blue exclusion. The error bars represent the SEM expressed as cell numbers for each triplicate value. These data are representative of four independent experiments.
Figure 6
Figure 6
PKB kinase activity protects mature T cells from apoptosis-inducing stimuli and spontaneous cell death in vitro. (A) Transgenic gag-PKB T cells are resistant to γ irradiation and dexamethasone-induced apoptosis. Purified CD4+ and CD8+ splenic T cells from mice expressing the gag-PKB transgene (B6/PKB5-4, □; or B6/PKB4-1, ○) or littermate controls (B6, ▪) were cultured at 106 cells/ml after exposure to γ radiation or dexamethasone. Viability was determined by trypan blue exclusion up to 48 h after treatment. Viability is expressed normalized to untreated T cells and in triplicate (±SEM). The data are representative of four independent experiments. (B) PKB kinase activity enhances T cell survival. Purified CD4+ and CD8+ splenic T cells were cultured at 106 cells/ml for 8 d at 37°C. Cell viability was determined in triplicate by trypan blue exclusion. The error bars represent the SEM expressed as cell numbers for each triplicate value. These data are representative of four independent experiments.
Figure 5
Figure 5
Elevated Bcl-XL protein levels are detected in gag-PKB transgenic thymocytes. Cell lysates from 5 × 106 transgenic (B6/PKB) or nontransgenic (B6) thymocytes were resolved by 10% SDS-PAGE and analyzed via Western blotting. Blots were probed with antibodies specific for Bcl-XL (top) and Bcl-2 (bottom).
Figure 7
Figure 7
PKB is activated after TCR ligation and enhances antigen-specific proliferation. (A) PKB is activated in T cells in a TCR- and PI3K-dependent fashion. Purified T cells (5 × 106) from C57BL/6J animals were left untreated (NT) or stimulated with 10 μg/ml anti-CD3 antibody (αCD3), or anti-CD3 plus 2 μg/ml anti-CD28 antibody for 2 h at 37°C. W, pretreatment of cells with wortmannin (100 nM) for 30 min before stimulation. PKB activity was assayed by Western blot with antibodies specific for PKB phosphorylated on Ser-473. Endogenous PKB levels were measured using anti-PKB antibodies. (B) P14 TCR transgenic thymocytes expressing gag-PKB display enhanced antigen-specific proliferation over time. Splenocytes from P14 TCR transgenic (P14, •) or P14 TCR/gag-PKB double transgenic (P14/PKB, ○) animals were cultured for up to 5 d in the presence of APCs pre-pulsed with 10−5 M p33 peptide, and pulsed with [3H]thymidine at the indicated time points. To standardize for different experiments, scintillation counts obtained were normalized and expressed as a percentage of maximal proliferation (where maximal proliferation equals 100%). Mean proliferative values for each time point are indicated.
Figure 7
Figure 7
PKB is activated after TCR ligation and enhances antigen-specific proliferation. (A) PKB is activated in T cells in a TCR- and PI3K-dependent fashion. Purified T cells (5 × 106) from C57BL/6J animals were left untreated (NT) or stimulated with 10 μg/ml anti-CD3 antibody (αCD3), or anti-CD3 plus 2 μg/ml anti-CD28 antibody for 2 h at 37°C. W, pretreatment of cells with wortmannin (100 nM) for 30 min before stimulation. PKB activity was assayed by Western blot with antibodies specific for PKB phosphorylated on Ser-473. Endogenous PKB levels were measured using anti-PKB antibodies. (B) P14 TCR transgenic thymocytes expressing gag-PKB display enhanced antigen-specific proliferation over time. Splenocytes from P14 TCR transgenic (P14, •) or P14 TCR/gag-PKB double transgenic (P14/PKB, ○) animals were cultured for up to 5 d in the presence of APCs pre-pulsed with 10−5 M p33 peptide, and pulsed with [3H]thymidine at the indicated time points. To standardize for different experiments, scintillation counts obtained were normalized and expressed as a percentage of maximal proliferation (where maximal proliferation equals 100%). Mean proliferative values for each time point are indicated.
Figure 8
Figure 8
Mediators of survival in gag-PKB transgenic T cells. (A) Increased Bcl-XL levels, but no phosphorylated BAD, can be detected in gag-PKB transgenic T cells. Cell lysates from transgenic (B6/PKB) or nontransgenic (B6) T cells were resolved by 10% SDS-PAGE and analyzed by Western blot. Cell lysates from 5 × 106 thymocytes were probed with antibodies specific for Bcl-XL (top) and Bcl-2 (middle). Lysates from 2 × 107 thymocytes were probed with antibodies against BAD (bottom). (B) PKB activity enhances NF-κB activation in T cells. Primary T cells isolated from LNs and spleens of gag-PKB transgenic (B6/PKB) and control (B6) mice were left untreated (NT) or treated with the indicated stimuli for 4 h. Nuclear extracts were incubated with a radiolabeled probe containing NF-κB binding sites, and NF-κB activation was determined using a gel mobility shift assay as described in Materials and Methods. Bands corresponding to specific NF-κB–DNA complexes and nonspecific binding are indicated. (C) PKB activity enhances IκBα degradation in response to TCR stimulation. T cells purified from gag-PKB transgenic (B6/PKB) or control (B6) mice were pretreated with 50 μg/ml cyclohexamide for 15 min, followed by stimulation with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 antibodies for the times indicated. Whole cell lysates were analyzed for IκBα protein levels by Western blot. The blots were stripped and probed with an antibody to β-actin to assess for equal protein loading. This figure is representative of two independent experiments.
Figure 8
Figure 8
Mediators of survival in gag-PKB transgenic T cells. (A) Increased Bcl-XL levels, but no phosphorylated BAD, can be detected in gag-PKB transgenic T cells. Cell lysates from transgenic (B6/PKB) or nontransgenic (B6) T cells were resolved by 10% SDS-PAGE and analyzed by Western blot. Cell lysates from 5 × 106 thymocytes were probed with antibodies specific for Bcl-XL (top) and Bcl-2 (middle). Lysates from 2 × 107 thymocytes were probed with antibodies against BAD (bottom). (B) PKB activity enhances NF-κB activation in T cells. Primary T cells isolated from LNs and spleens of gag-PKB transgenic (B6/PKB) and control (B6) mice were left untreated (NT) or treated with the indicated stimuli for 4 h. Nuclear extracts were incubated with a radiolabeled probe containing NF-κB binding sites, and NF-κB activation was determined using a gel mobility shift assay as described in Materials and Methods. Bands corresponding to specific NF-κB–DNA complexes and nonspecific binding are indicated. (C) PKB activity enhances IκBα degradation in response to TCR stimulation. T cells purified from gag-PKB transgenic (B6/PKB) or control (B6) mice were pretreated with 50 μg/ml cyclohexamide for 15 min, followed by stimulation with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 antibodies for the times indicated. Whole cell lysates were analyzed for IκBα protein levels by Western blot. The blots were stripped and probed with an antibody to β-actin to assess for equal protein loading. This figure is representative of two independent experiments.
Figure 8
Figure 8
Mediators of survival in gag-PKB transgenic T cells. (A) Increased Bcl-XL levels, but no phosphorylated BAD, can be detected in gag-PKB transgenic T cells. Cell lysates from transgenic (B6/PKB) or nontransgenic (B6) T cells were resolved by 10% SDS-PAGE and analyzed by Western blot. Cell lysates from 5 × 106 thymocytes were probed with antibodies specific for Bcl-XL (top) and Bcl-2 (middle). Lysates from 2 × 107 thymocytes were probed with antibodies against BAD (bottom). (B) PKB activity enhances NF-κB activation in T cells. Primary T cells isolated from LNs and spleens of gag-PKB transgenic (B6/PKB) and control (B6) mice were left untreated (NT) or treated with the indicated stimuli for 4 h. Nuclear extracts were incubated with a radiolabeled probe containing NF-κB binding sites, and NF-κB activation was determined using a gel mobility shift assay as described in Materials and Methods. Bands corresponding to specific NF-κB–DNA complexes and nonspecific binding are indicated. (C) PKB activity enhances IκBα degradation in response to TCR stimulation. T cells purified from gag-PKB transgenic (B6/PKB) or control (B6) mice were pretreated with 50 μg/ml cyclohexamide for 15 min, followed by stimulation with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 antibodies for the times indicated. Whole cell lysates were analyzed for IκBα protein levels by Western blot. The blots were stripped and probed with an antibody to β-actin to assess for equal protein loading. This figure is representative of two independent experiments.
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