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CTLA-4 activation of phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death.

Schneider H, Valk E, Leung R, Rudd CE - PLoS ONE (2008)

Bottom Line: The level of activation was similar to that observed with CD28.CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL.This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Section, Department of Pathology, University of Cambridge, Cambridge, UK.

ABSTRACT
The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.

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Panel A: CTLA-4 mediated phosphorylation of GSK-3. Upper left panel: DC27.10-CTLA-4 and pre-activated peripheral T-cells were stimulated for 30 min as described for Figure 1A. Cell lysates were immunoblotted with anti-phospho-GSK-3 α/β antibody (lanes 1–7). Upper right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Lower left panel: Cells treated as described above were lysed and immunoblotted with an antibody against total GSK-3 α/β (lanes 1–7). Similar results were obtained from at least three other experiments. Lower right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Panel B: Ligation of CTLA-4 by natural ligand induces phosphorylation of GSK-3. DC27.10 cells transfected with mock (lanes 1–3) or CTLA-4 (lanes 4–6) were either left untreated (lanes 1, 4) or stimulated for 30 min with anti-CD3 (lanes 2, 5) or anti-CD3/CD80Ig (lanes 3, 6) and assesssed for phosphorylation of GSK-3 by immunoblotting with anti-phospho-GSK-3 α/β antibody (lanes 1–6). Lower panel: Equal amounts of cell lysates were immunoblotted for total GSK-3 α/β (lanes 1–6). Right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Results are representative of at least two experiments.
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pone-0003842-g002: Panel A: CTLA-4 mediated phosphorylation of GSK-3. Upper left panel: DC27.10-CTLA-4 and pre-activated peripheral T-cells were stimulated for 30 min as described for Figure 1A. Cell lysates were immunoblotted with anti-phospho-GSK-3 α/β antibody (lanes 1–7). Upper right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Lower left panel: Cells treated as described above were lysed and immunoblotted with an antibody against total GSK-3 α/β (lanes 1–7). Similar results were obtained from at least three other experiments. Lower right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Panel B: Ligation of CTLA-4 by natural ligand induces phosphorylation of GSK-3. DC27.10 cells transfected with mock (lanes 1–3) or CTLA-4 (lanes 4–6) were either left untreated (lanes 1, 4) or stimulated for 30 min with anti-CD3 (lanes 2, 5) or anti-CD3/CD80Ig (lanes 3, 6) and assesssed for phosphorylation of GSK-3 by immunoblotting with anti-phospho-GSK-3 α/β antibody (lanes 1–6). Lower panel: Equal amounts of cell lysates were immunoblotted for total GSK-3 α/β (lanes 1–6). Right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Results are representative of at least two experiments.

Mentions: As a read-out for PKB/AKT activity, we next assessed the phosphorylation of the PKB/AKT target GSK-3 α and β on inhibitory serine 21 and 9, respectively [14]. Anti-CTLA-4 readily induced GSK-3 α phosphorylation in DC27.10-CTLA-4 and activated peripheral T-cells (Fig. 2A, left and right panel, lane 4. histograms). The level of phosphorylation was comparable to that induced by anti-CD3 and anti-CD28 (lanes 2 and 3, respectively). Co-ligation of CTLA-4 with CD3 or CD28 often led to increased GSK-3 α phosphorylation (lanes 6, 7). No phosphorylation of the β isoform of GSK-3 could be detected despite equal expression levels of GSK-3 α and β in these cells (lower panel). CTLA-4 mediated phosphorylation of GSK3 α was also observed with natural ligand such as plate-bound CD80Ig together with anti-CD3 (Fig. 2B). Since both CD28 and CTLA-4 are expressed on activated peripheral T-cells (i.e. both bind to CD80), this assay was conducted using the DC27.10 T-cell hybridoma transfected with mock (i.e. express CD28 alone), or with CTLA-4 (i.e. express CD28 and CTLA-4). While anti-CD3 induced comparable GSK-3α phosphorylation in both transfectants (lane 2 vs. 5), CD80Ig/anti-CD3 revealed greater GSK-3 α phosphorylation when CTLA-4 was present (lane 6 vs. 3 and histogram). These observations indicated that CTLA-4 ligation by antibody or natural ligand can activate PKB/AKT and induce GSK-3 α phosphorylation.


CTLA-4 activation of phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death.

Schneider H, Valk E, Leung R, Rudd CE - PLoS ONE (2008)

Panel A: CTLA-4 mediated phosphorylation of GSK-3. Upper left panel: DC27.10-CTLA-4 and pre-activated peripheral T-cells were stimulated for 30 min as described for Figure 1A. Cell lysates were immunoblotted with anti-phospho-GSK-3 α/β antibody (lanes 1–7). Upper right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Lower left panel: Cells treated as described above were lysed and immunoblotted with an antibody against total GSK-3 α/β (lanes 1–7). Similar results were obtained from at least three other experiments. Lower right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Panel B: Ligation of CTLA-4 by natural ligand induces phosphorylation of GSK-3. DC27.10 cells transfected with mock (lanes 1–3) or CTLA-4 (lanes 4–6) were either left untreated (lanes 1, 4) or stimulated for 30 min with anti-CD3 (lanes 2, 5) or anti-CD3/CD80Ig (lanes 3, 6) and assesssed for phosphorylation of GSK-3 by immunoblotting with anti-phospho-GSK-3 α/β antibody (lanes 1–6). Lower panel: Equal amounts of cell lysates were immunoblotted for total GSK-3 α/β (lanes 1–6). Right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Results are representative of at least two experiments.
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pone-0003842-g002: Panel A: CTLA-4 mediated phosphorylation of GSK-3. Upper left panel: DC27.10-CTLA-4 and pre-activated peripheral T-cells were stimulated for 30 min as described for Figure 1A. Cell lysates were immunoblotted with anti-phospho-GSK-3 α/β antibody (lanes 1–7). Upper right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Lower left panel: Cells treated as described above were lysed and immunoblotted with an antibody against total GSK-3 α/β (lanes 1–7). Similar results were obtained from at least three other experiments. Lower right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Panel B: Ligation of CTLA-4 by natural ligand induces phosphorylation of GSK-3. DC27.10 cells transfected with mock (lanes 1–3) or CTLA-4 (lanes 4–6) were either left untreated (lanes 1, 4) or stimulated for 30 min with anti-CD3 (lanes 2, 5) or anti-CD3/CD80Ig (lanes 3, 6) and assesssed for phosphorylation of GSK-3 by immunoblotting with anti-phospho-GSK-3 α/β antibody (lanes 1–6). Lower panel: Equal amounts of cell lysates were immunoblotted for total GSK-3 α/β (lanes 1–6). Right panel: Histogram depiction of phosphorylated GSK-3 as detected by densitometric reading. Results are representative of at least two experiments.
Mentions: As a read-out for PKB/AKT activity, we next assessed the phosphorylation of the PKB/AKT target GSK-3 α and β on inhibitory serine 21 and 9, respectively [14]. Anti-CTLA-4 readily induced GSK-3 α phosphorylation in DC27.10-CTLA-4 and activated peripheral T-cells (Fig. 2A, left and right panel, lane 4. histograms). The level of phosphorylation was comparable to that induced by anti-CD3 and anti-CD28 (lanes 2 and 3, respectively). Co-ligation of CTLA-4 with CD3 or CD28 often led to increased GSK-3 α phosphorylation (lanes 6, 7). No phosphorylation of the β isoform of GSK-3 could be detected despite equal expression levels of GSK-3 α and β in these cells (lower panel). CTLA-4 mediated phosphorylation of GSK3 α was also observed with natural ligand such as plate-bound CD80Ig together with anti-CD3 (Fig. 2B). Since both CD28 and CTLA-4 are expressed on activated peripheral T-cells (i.e. both bind to CD80), this assay was conducted using the DC27.10 T-cell hybridoma transfected with mock (i.e. express CD28 alone), or with CTLA-4 (i.e. express CD28 and CTLA-4). While anti-CD3 induced comparable GSK-3α phosphorylation in both transfectants (lane 2 vs. 5), CD80Ig/anti-CD3 revealed greater GSK-3 α phosphorylation when CTLA-4 was present (lane 6 vs. 3 and histogram). These observations indicated that CTLA-4 ligation by antibody or natural ligand can activate PKB/AKT and induce GSK-3 α phosphorylation.

Bottom Line: The level of activation was similar to that observed with CD28.CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL.This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Section, Department of Pathology, University of Cambridge, Cambridge, UK.

ABSTRACT
The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.

Show MeSH
Related in: MedlinePlus