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

Jones RG, Elford AR, Parsons MJ, Wu L, Krawczyk CM, Yeh WC, Hakem R, Rottapel R, Woodgett JR, Ohashi PS - J. Exp. Med. (2002)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Canada.

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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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.
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fig1: 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.

Mentions: To determine whether CD28 plays a role in Fas-mediated apoptosis, T cells from C57Bl/6 and CD28−/− (C57Bl/6 background) mice were activated with plate-bound anti-CD3 and anti-CD28 antibodies, followed by culture in the presence of IL-2. Fas-dependent apoptosis of viable T cells was induced after 4 d of culture through the addition of FasL. As shown in Fig. 1 a, CD4+ T cells lacking CD28 displayed dramatic sensitivity to Fas-mediated cell death relative to wild-type controls at various concentrations of FasL. This susceptibility to Fas-mediated cell death was characterized by both a decreased number of viable cells (AnnexinV-7AAD−, bottom left quadrant) and an increased proportion of apoptotic cells (AnnexinV+ 7AAD+, top right quadrant) when compared with controls, particularly at low concentrations of FasL which have little impact on wild-type cells. The kinetics of cell death over time revealed that CD28−/−CD4+ T cells underwent apoptosis with faster kinetics than wild-type cells, whereas T cells from lpr mice which have defective Fas signaling displayed no detectable increase in apoptosis after the addition of FasL (Fig. 1 b). Further assessment revealed that the lack of CD28 resulted in increased Fas-mediated death in both T cell subsets, but CD4+ T cells were clearly more sensitive to FasL treatment than CD8+ cells (Fig. 1 c). T cells from CD28−/− mice displayed normal surface expression of Fas (unpublished data), suggesting that the survival defect was not due to enhanced expression of Fas. Thus, our results indicate that Fas-mediated apoptosis of peripheral T cells is enhanced in the absence of CD28 expression, indicating that CD28 is an important guardian against Fas-mediated apoptosis in T cells.


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

Jones RG, Elford AR, Parsons MJ, Wu L, Krawczyk CM, Yeh WC, Hakem R, Rottapel R, Woodgett JR, Ohashi PS - J. Exp. Med. (2002)

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.
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fig1: 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.
Mentions: To determine whether CD28 plays a role in Fas-mediated apoptosis, T cells from C57Bl/6 and CD28−/− (C57Bl/6 background) mice were activated with plate-bound anti-CD3 and anti-CD28 antibodies, followed by culture in the presence of IL-2. Fas-dependent apoptosis of viable T cells was induced after 4 d of culture through the addition of FasL. As shown in Fig. 1 a, CD4+ T cells lacking CD28 displayed dramatic sensitivity to Fas-mediated cell death relative to wild-type controls at various concentrations of FasL. This susceptibility to Fas-mediated cell death was characterized by both a decreased number of viable cells (AnnexinV-7AAD−, bottom left quadrant) and an increased proportion of apoptotic cells (AnnexinV+ 7AAD+, top right quadrant) when compared with controls, particularly at low concentrations of FasL which have little impact on wild-type cells. The kinetics of cell death over time revealed that CD28−/−CD4+ T cells underwent apoptosis with faster kinetics than wild-type cells, whereas T cells from lpr mice which have defective Fas signaling displayed no detectable increase in apoptosis after the addition of FasL (Fig. 1 b). Further assessment revealed that the lack of CD28 resulted in increased Fas-mediated death in both T cell subsets, but CD4+ T cells were clearly more sensitive to FasL treatment than CD8+ cells (Fig. 1 c). T cells from CD28−/− mice displayed normal surface expression of Fas (unpublished data), suggesting that the survival defect was not due to enhanced expression of Fas. Thus, our results indicate that Fas-mediated apoptosis of peripheral T cells is enhanced in the absence of CD28 expression, indicating that CD28 is an important guardian against Fas-mediated apoptosis in T cells.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Canada.

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.

Show MeSH