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. 2002 Aug 5;196(3):335-48.
doi: 10.1084/jem.20020307.

CD28-dependent activation of protein kinase B/Akt blocks Fas-mediated apoptosis by preventing death-inducing signaling complex assembly

Russell G Jones  1 , Alisha R Elford, Michael J Parsons, Linda Wu, Connie M Krawczyk, Wen-Chen Yeh, Razqallah Hakem, Robert Rottapel, James R Woodgett, Pamela S Ohashi
Affiliations

CD28-dependent activation of protein kinase B/Akt blocks Fas-mediated apoptosis by preventing death-inducing signaling complex assembly

Russell G Jones et al. J Exp Med. .

Abstract

The T cell costimulatory molecule CD28 is important for T cell survival, yet both the signaling pathways downstream of CD28 and the apoptotic pathways they antagonize remain poorly understood. Here we demonstrate that CD4(+) T cells from CD28-deficient mice show increased susceptibility to Fas-mediated apoptosis via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. Protein kinase B (PKBalpha/Akt1) is an important serine/threonine kinase that promotes survival downstream of PI3K signals. To understand how PI3K-mediated signals downstream of CD28 contribute to T cell survival, we examined Fas-mediated apoptosis in T cells expressing an active form of PKBalpha. Our data demonstrate that T cells expressing active PKB are resistant to Fas-mediated apoptosis in vivo and in vitro. PKB transgenic T cells show reduced activation of caspase-8, BID, and caspase-3 due to impaired recruitment of procaspase-8 to the death-inducing signaling complex (DISC). Similar alterations are seen in T cells from mice which are haploinsufficient for PTEN, a lipid phosphatase that regulates phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) and influences PKBalpha activity. These findings provide a novel link between CD28 and an important apoptosis pathway in vivo, and demonstrate that PI3K/PKB signaling prevents apoptosis by inhibiting DISC assembly.

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Figures

Figure 1.
Figure 1.
CD28-associated PI3K activity confers protection against Fas-mediated apoptosis. (a) Increased sensitivity of CD4+CD28−/− T cells to Fas-mediated apoptosis. Splenocytes were cultured with anti-CD3 and anti-CD28 antibodies and IL-2 for 4 d, and apoptosis induced by FasL. CD4+ cell death was measured 6 h after FasL treatment by Annexin V-FITC, CD4-PE, and 7AAD staining. The proportion of cells in each quadrant is indicated. Results are representative of four independent experiments. (b) Time course of Fas-mediated death for CD28−/− T cells. Activated, viable T cells were treated with 5 μg/ml hCD8-mFasL, and apoptosis measured as in panel a. C57BL/6 (B6), filled squares; CD28−/− (B6/CD28−/−), open squares; lpr (B6/lpr/lpr), filled triangles. (c) FasL preferentially kills CD4+ T cells. Activated, viable T cells were left untreated (−FasL) or treated with 5 μg/ml FasL (+FasL), and percent dead CD4+ and CD8+ cells were determined by staining with 7AAD after 6 h. Wild-type, black bars; CD28−/−, white bars. (d) CD28 ligation enhances T cell survival. Wild-type sorted T cells were cultured with anti-CD3 (black bars) or anti-CD3 and CD28 (white bars) antibodies and IL-2, and apoptosis induced by FasL. Cell death was measured 12 h after FasL treatment. (e) Enhanced Fas-mediated apoptosis in T cells with defective CD28-dependent P13K signals. Dose response of Fas-mediated death of activated, viable CD4+ T cells 6 h after the addition of FasL. Apoptosis was measured as in panel a. Wild-type, filled squares; CD28−/−, open squares; WT CD28 Tg, filled circles; Y170F Tg-1 (impaired PI3K binding), open circles.
Figure 1.
Figure 1.
CD28-associated PI3K activity confers protection against Fas-mediated apoptosis. (a) Increased sensitivity of CD4+CD28−/− T cells to Fas-mediated apoptosis. Splenocytes were cultured with anti-CD3 and anti-CD28 antibodies and IL-2 for 4 d, and apoptosis induced by FasL. CD4+ cell death was measured 6 h after FasL treatment by Annexin V-FITC, CD4-PE, and 7AAD staining. The proportion of cells in each quadrant is indicated. Results are representative of four independent experiments. (b) Time course of Fas-mediated death for CD28−/− T cells. Activated, viable T cells were treated with 5 μg/ml hCD8-mFasL, and apoptosis measured as in panel a. C57BL/6 (B6), filled squares; CD28−/− (B6/CD28−/−), open squares; lpr (B6/lpr/lpr), filled triangles. (c) FasL preferentially kills CD4+ T cells. Activated, viable T cells were left untreated (−FasL) or treated with 5 μg/ml FasL (+FasL), and percent dead CD4+ and CD8+ cells were determined by staining with 7AAD after 6 h. Wild-type, black bars; CD28−/−, white bars. (d) CD28 ligation enhances T cell survival. Wild-type sorted T cells were cultured with anti-CD3 (black bars) or anti-CD3 and CD28 (white bars) antibodies and IL-2, and apoptosis induced by FasL. Cell death was measured 12 h after FasL treatment. (e) Enhanced Fas-mediated apoptosis in T cells with defective CD28-dependent P13K signals. Dose response of Fas-mediated death of activated, viable CD4+ T cells 6 h after the addition of FasL. Apoptosis was measured as in panel a. Wild-type, filled squares; CD28−/−, open squares; WT CD28 Tg, filled circles; Y170F Tg-1 (impaired PI3K binding), open circles.
Figure 2.
Figure 2.
Impaired Fas-mediated apoptosis in PKB-transgenic mice. Splenocytes from C57BL/6 (B6), PKB-transgenic (B6/PKB), or lpr (B6/lpr/lpr) mice were activated with anti-CD3 and anti-CD28 antibodies and IL-2 for 4 d, followed by induction of Fas-specific apoptosis by addition of FasL. (a) Reduced FasL-induced apoptosis of activated PKB-transgenic T cells. Activated, viable T cells were left untreated (−FasL) or treated (+FasL) with various concentrations of FasL and cell death measured at 10 h by AnnexinV-FITC and PI staining. The proportion of cells in each quadrant is indicated. Results are representative of six independent experiments. (b) Time course of Fas-mediated death. Activated, viable T cells were treated with 5 μg/ml FasL, and apoptosis measured as in panel a. C57BL/6 (B6), closed circles; PKB transgenic (B6/PKB), open circles; lpr (B6/lpr/lpr), closed squares. (c) Elevated Fas surface expression on PKB transgenic T cells. Fas surface expression (open histogram) on activated T cells from C57BL/6 (B6) or PKB transgenic (B6/PKB) animals. IgG2a isotype control antibody staining is shown (shaded histogram).
Figure 3.
Figure 3.
Impaired Fas-dependent T cell deletion in PKB transgenic mice. (a and b) Impaired SEB-mediated deletion of PKB-transgenic T cells. Control C57BL/6 mice (B6, open circles), Fas-deficient lpr mice (B6/lpr, open squares), or animals expressing one or two gag-PKB alleles (B6/PKB+/−, filled circles, and B6/PKB+/+, filled squares, respectively) were injected with SEB (80 μg) and the proportion of Vβ8+CD4+ T cells from peripheral blood was measured by flow cytometry (a). The percentage of Vβ6+CD4+ T cells was measured as a negative control (b). (c) Normal peptide-mediated deletion of PKB-transgenic T cells. P14 TCR (open circles) or P14 TCR/PKB (filled circles) transgenic mice were injected with p33 peptide (three times with 5 μg over 6 d) and the proportion of Vα2+CD8+ T cells from peripheral blood was measured by flow cytometry. The percentage of CD4+ T cells was measured as a negative control (d).
Figure 4.
Figure 4.
Increased frequency of activated lymphocytes in MRL/PKB transgenic mice. Total numbers of activated T cells (a) and B cells (b) in Peyer's patches of 12-wk-old MRL mice (filled circles, n = 5), MRL/PKB transgenic mice (open circles, n = 8), and MRL/lpr (filled triangles, n = 5) mutant mice. Accumulation of activated cells in MRL/PKB mice relative to MRL/lpr mice is shown. Mean scores for each genotype are indicated.
Figure 5.
Figure 5.
PKB inhibits the activation of caspase-8 and multiple downstream targets of Fas. (a) Normal expression of components of the Fas death pathway. FADD, procaspase-8, procaspase-3, and Bid protein levels were determined in naive (−) or activated (+) T cells from wild-type (B6) or PKB transgenic (PKB) mice via Western blotting. (b) PKB prevents processing of procaspases-8 and -3, and degradation of PARP. Activated CD4+ T cells from C57BL/6 (B6) or PKB transgenic (PKB) animals cultured in the presence of IL-2 were left untreated (−FasL) or treated (+FasL) with 10 μg/ml FasL for various time points. Cell lysates were immunodetected for the presence of procaspase-8, procaspase-3, PARP, and PKB. Antibodies directed against GSK-3 were used to assess protein loading. (c) Decreased caspase activity in PKB transgenic T cells. Caspase-8- or caspase-3–specific activity was determined in cell lysates 4 h after addition of FasL. Fold increase in activity determined as ratio of activity relative to untreated cells. Mean values ± SEM are shown for one representative experiment performed in triplicate. (d) Active PKB prevents processing of Bid. Bid protein levels were detected in CD4+ T cell lysates after addition of FasL using antibodies directed against Bid. (e) Decreased mitochondrial permeability in PKB transgenic T cells. Specific loss of DiOC6+ staining was measured in T cells from C57BL/6 (closed circles), PKB transgenic (open circles), or lpr (filled squares) mice via flow cytometry after addition of FasL. Mean values ± SEM for samples performed in triplicate are shown.
Figure 6.
Figure 6.
PTEN+/− T cells show decreased Fas-induced apoptosis and caspase-8 activation. (a) Enhanced PKB activation in PTEN+/− T cells. T cells from C57BL/6 (B6), PTEN+/−, or PKB transgenic (B6/PKB) mice were stimulated with soluble anti-CD3 and anti-CD28 antibodies for the indicated times and the amount of phosphorylated PKB (P-PKB) and total PKB were analyzed via Western blot. Anti-GSK-3 antibodies were used to assess protein loading. (b) Activated PTEN+/− T cells show decreased susceptibility to Fas-mediated apoptosis. Activated, viable T cells were treated with FasL and cell death measured at 5 h by AnnexinV-FITC and PI staining. The proportion of cells in each quadrant is indicated. (c) Dose-dependent Fas-resistance in PTEN+/− T cells. Activated, viable T cells from C57BL/6 (B6, filled circles), gag-PKB transgenic (B6/PKB, open circles), and PTEN+/− (filled triangles) mice were treated with various concentrations of FasL and cell death measured at 8 h. Viability was determined as percent AnnexinV-PI- relative to untreated controls and performed in triplicate. Results are representative of two independent experiments. (d) Decreased PARP cleavage and procaspase-8 processing in PTEN+/− T cells. Cell lysates were analyzed for the presence of PARP (top panel) and procaspase-8 (middle panel) by Western blot. Antibodies directed against GSK-3 were used to assess protein loading (bottom panel).
Figure 7.
Figure 7.
(a) PKB inhibits the recruitment of procaspase-8 to the DISC. T cells from C57BL/6 (B6) and PKB transgenic (PKB) mice were activated with anti-CD3 and anti-CD28 antibodies for 24 h, followed by culture with IL-2 for 4 d. Fas was immunoprecipitated from activated T cells either left untreated (−) or treated (+) with FasL (10 μg/ml) for 1 h. T cells were maintained in IL-2 during stimulation with FasL. Immunoprecipitates were blotted with antibodies specific for procaspase-8, FADD, or Fas. Relative protein expression of DISC components in precleared lysates is shown. (b) Expression of IAPs and FLIP in PKB transgenic T cells. Protein levels of XIAP, cIAP-1, cIAP-2, and cFLIP in naive (N, no stimulation for 24 h) or activated (A, anti-CD3 and anti-CD28 treatment for 24 h) sorted CD4+ T cells from wild-type (B6) and PKB transgenic (PKB) mice were assessed by Western blot. Levels of p85 PI3K were measured to assess protein loading.
Figure 8.
Figure 8.
Activation of PKB by TCR/CD28 inhibits Fas-mediated death. In T cells, PKB is activated downstream of the TCR and CD28 by PI3K, a process antagonized by the lipid phosphatase PTEN. Triggering of Fas by FasL leads to recruitment of procaspase-8 to the DISC via binding to a FADD-Fas complex. Processing and activation of procaspase-8 leads to activation of downstream effectors (caspase-3, Bid) and subsequently to apoptosis. FLIP antagonizes this process through direct binding and inhibition of procaspase-8. PKB inhibits processing of procaspase-8 by preventing its translocation to the DISC, independent of FLIP expression.
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