Negative regulation of T cell proliferation and interleukin 2 production by the serine threonine kinase GSK-3

T Ohteki, M Parsons, A Zakarian, R G Jones, L T Nguyen, J R Woodgett, P S Ohashi, T Ohteki, M Parsons, A Zakarian, R G Jones, L T Nguyen, J R Woodgett, P S Ohashi

Abstract

Glycogen synthase kinase (GSK)-3 is a protein serine/threonine kinase that regulates differentiation and cell fate in a variety of organisms. This study examined the role of GSK-3 in antigen-specific T cell responses. Using resting T cells from P14 T cell receptor (TCR)-transgenic mice (specific for the lymphocytic choriomeningitis virus and H-2D(b)), we demonstrated that GSK-3beta was inactivated by serine phosphorylation after viral peptide-specific stimulation in vitro. To further investigate the role of GSK-3, we have generated a retroviral vector that expresses a constitutively active form of GSK-3beta that has an alanine substitution at the regulatory amino acid, serine 9 (GSK-3betaA9). Retroviral transduction of P14 TCR-transgenic bone marrow stem cells, followed by reconstitution, led to the expression of GSK-3betaA9 in bone marrow chimeric mice. T cells from chimeric mice demonstrate a reduction in proliferation and interleukin (IL)-2 production. In contrast, in vitro assays done in the presence of the GSK-3 inhibitor lithium led to dramatically prolonged T cell proliferation and increased IL-2 production. Furthermore, in the presence of lithium, we show that nuclear factor of activated T cells (NF-AT)c remains in the nucleus after antigen-specific stimulation of T cells. Together, these data demonstrate that GSK-3 negatively regulates the duration of T cell responses.

Figures

Figure 1
Figure 1
TCR-specific stimulation leads to inactivation of GSK-3. CD8-purified P14 TCR–transgenic T cells were incubated with macrophages pulsed with the antigenic ligand p33 (P) or nonstimulatory ligand AV (A). After the indicated time periods, cells were lysed and analyzed by Western blot using phospho-serine–specific GSK-3 antibodies. Numbers indicate the densitometry measurements as fold increase relative to AV-treated cells. Total GSK-3 was shown as a control.
Figure 2
Figure 2
Retroviral expression of GSK-3βA9. (A) A retroviral vector was generated using MSCV and a constitutively active form of GSK-3β (GSK-3βA9) tagged with HA. GSK-3βA9 is expressed from the retroviral LTR, using the splice donor and splice acceptor sites. The selectable neomycin (G418)-resistant marker is expressed from the phosphoglycerate kinase (pgk) promoter. (B) NIH 3T3 cells were transduced with the retrovirus MSCV/GSK-3βA9, and expression of GSK-3βA9 was examined by Western blot using HA-specific antibodies, compared with control (con) NIH 3T3 cells.
Figure 2
Figure 2
Retroviral expression of GSK-3βA9. (A) A retroviral vector was generated using MSCV and a constitutively active form of GSK-3β (GSK-3βA9) tagged with HA. GSK-3βA9 is expressed from the retroviral LTR, using the splice donor and splice acceptor sites. The selectable neomycin (G418)-resistant marker is expressed from the phosphoglycerate kinase (pgk) promoter. (B) NIH 3T3 cells were transduced with the retrovirus MSCV/GSK-3βA9, and expression of GSK-3βA9 was examined by Western blot using HA-specific antibodies, compared with control (con) NIH 3T3 cells.
Figure 3
Figure 3
Overexpression of constitutively active GSK-3β inhibits antigen-specific T cell proliferation and IL-2 production. (A) Similar reconstitution of P14 TCR–transgenic T cells in retroviral transduced bone marrow chimeric mice. The spleen cells from chimeric mice, reconstituted with P14 TCR–transgenic bone marrow transduced with MSCV/GSK-3βA9 (left panel) or MSCV/neo (right panel), were stained with antibodies specific for CD8 and Vα2. T cells from bone marrow chimeric mice expressing GSK-3βA9 show decreased proliferation and IL-2 production. (B) Splenocytes from bone marrow chimeric mice transduced with MSCV/GSK-3βA9 show reduced proliferation in response to the strong agonist peptides p33 and weaker agonist A4Y compared with control MSCV-transduced T cells. Proliferative responses using the nonstimulatory AV peptide for the P14 TCR was

Figure 3

Overexpression of constitutively active GSK-3β…

Figure 3

Overexpression of constitutively active GSK-3β inhibits antigen-specific T cell proliferation and IL-2 production.…

Figure 3
Overexpression of constitutively active GSK-3β inhibits antigen-specific T cell proliferation and IL-2 production. (A) Similar reconstitution of P14 TCR–transgenic T cells in retroviral transduced bone marrow chimeric mice. The spleen cells from chimeric mice, reconstituted with P14 TCR–transgenic bone marrow transduced with MSCV/GSK-3βA9 (left panel) or MSCV/neo (right panel), were stained with antibodies specific for CD8 and Vα2. T cells from bone marrow chimeric mice expressing GSK-3βA9 show decreased proliferation and IL-2 production. (B) Splenocytes from bone marrow chimeric mice transduced with MSCV/GSK-3βA9 show reduced proliferation in response to the strong agonist peptides p33 and weaker agonist A4Y compared with control MSCV-transduced T cells. Proliferative responses using the nonstimulatory AV peptide for the P14 TCR was

Figure 4

Inhibition of GSK-3 leads to…

Figure 4

Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes…

Figure 4
Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes from P14 TCR–transgenic mice were cocultivated with APCs pulsed with the strong agonist ligand p33 or control AV peptide, in the presence of LiCl or KCl. (A) Proliferation of T cells was measured by thymidine incorporation. Proliferation in response to AV was below 300 cpm. (B) IL-2 production was measured by proliferation of the IL-2–dependent cell line CTLL-2 by the addition of supernatant from cultures harvested from the indicated days. (C) GSK-3 regulates NF-ATc nuclear localization. Purified CD8+ T cells from P14 TCR–transgenic RAG2−/− mice were cocultured with APCs pulsed with p33 for 3 d in the presence of LiCl or KCl. Cells were fixed, permeabilized, and stained using an NF-ATc–specific antibody.

Figure 4

Inhibition of GSK-3 leads to…

Figure 4

Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes…

Figure 4
Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes from P14 TCR–transgenic mice were cocultivated with APCs pulsed with the strong agonist ligand p33 or control AV peptide, in the presence of LiCl or KCl. (A) Proliferation of T cells was measured by thymidine incorporation. Proliferation in response to AV was below 300 cpm. (B) IL-2 production was measured by proliferation of the IL-2–dependent cell line CTLL-2 by the addition of supernatant from cultures harvested from the indicated days. (C) GSK-3 regulates NF-ATc nuclear localization. Purified CD8+ T cells from P14 TCR–transgenic RAG2−/− mice were cocultured with APCs pulsed with p33 for 3 d in the presence of LiCl or KCl. Cells were fixed, permeabilized, and stained using an NF-ATc–specific antibody.
All figures (7)
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References
    1. Qian D., Weiss A. T cell antigen receptor signal transduction. Curr. Opin. Cell Biol. 1997;9:205–212. - PubMed
    1. Alberola-Ila J., Takaki S., Kerner J.D., Perlmutter R.M. Differential signalling by lymphocyte antigen receptors. Annu. Rev. Immunol. 1997;15:125–154. - PubMed
    1. Rao A., Luo C., Hogan P.G. Transcription factors of the NFAT familyregulation and function. Annu. Rev. Immunol. 1997;15:707–747. - PubMed
    1. Woodgett J.R. Regulation and functions of the glycogen synthase kinase-3 subfamily. Cancer Biol. 1994;5:269–275. - PubMed
    1. Welsh G.I., Wilson C., Proud C.G. GSK3a SHAGGY frog story. Cell Biol. 1996;6:274–279. - PubMed
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Figure 3
Figure 3
Overexpression of constitutively active GSK-3β inhibits antigen-specific T cell proliferation and IL-2 production. (A) Similar reconstitution of P14 TCR–transgenic T cells in retroviral transduced bone marrow chimeric mice. The spleen cells from chimeric mice, reconstituted with P14 TCR–transgenic bone marrow transduced with MSCV/GSK-3βA9 (left panel) or MSCV/neo (right panel), were stained with antibodies specific for CD8 and Vα2. T cells from bone marrow chimeric mice expressing GSK-3βA9 show decreased proliferation and IL-2 production. (B) Splenocytes from bone marrow chimeric mice transduced with MSCV/GSK-3βA9 show reduced proliferation in response to the strong agonist peptides p33 and weaker agonist A4Y compared with control MSCV-transduced T cells. Proliferative responses using the nonstimulatory AV peptide for the P14 TCR was

Figure 4

Inhibition of GSK-3 leads to…

Figure 4

Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes…

Figure 4
Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes from P14 TCR–transgenic mice were cocultivated with APCs pulsed with the strong agonist ligand p33 or control AV peptide, in the presence of LiCl or KCl. (A) Proliferation of T cells was measured by thymidine incorporation. Proliferation in response to AV was below 300 cpm. (B) IL-2 production was measured by proliferation of the IL-2–dependent cell line CTLL-2 by the addition of supernatant from cultures harvested from the indicated days. (C) GSK-3 regulates NF-ATc nuclear localization. Purified CD8+ T cells from P14 TCR–transgenic RAG2−/− mice were cocultured with APCs pulsed with p33 for 3 d in the presence of LiCl or KCl. Cells were fixed, permeabilized, and stained using an NF-ATc–specific antibody.

Figure 4

Inhibition of GSK-3 leads to…

Figure 4

Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes…

Figure 4
Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes from P14 TCR–transgenic mice were cocultivated with APCs pulsed with the strong agonist ligand p33 or control AV peptide, in the presence of LiCl or KCl. (A) Proliferation of T cells was measured by thymidine incorporation. Proliferation in response to AV was below 300 cpm. (B) IL-2 production was measured by proliferation of the IL-2–dependent cell line CTLL-2 by the addition of supernatant from cultures harvested from the indicated days. (C) GSK-3 regulates NF-ATc nuclear localization. Purified CD8+ T cells from P14 TCR–transgenic RAG2−/− mice were cocultured with APCs pulsed with p33 for 3 d in the presence of LiCl or KCl. Cells were fixed, permeabilized, and stained using an NF-ATc–specific antibody.
All figures (7)
Figure 4
Figure 4
Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes from P14 TCR–transgenic mice were cocultivated with APCs pulsed with the strong agonist ligand p33 or control AV peptide, in the presence of LiCl or KCl. (A) Proliferation of T cells was measured by thymidine incorporation. Proliferation in response to AV was below 300 cpm. (B) IL-2 production was measured by proliferation of the IL-2–dependent cell line CTLL-2 by the addition of supernatant from cultures harvested from the indicated days. (C) GSK-3 regulates NF-ATc nuclear localization. Purified CD8+ T cells from P14 TCR–transgenic RAG2−/− mice were cocultured with APCs pulsed with p33 for 3 d in the presence of LiCl or KCl. Cells were fixed, permeabilized, and stained using an NF-ATc–specific antibody.
Figure 4
Figure 4
Inhibition of GSK-3 leads to prolonged T cell proliferation and IL-2 production. Splenocytes from P14 TCR–transgenic mice were cocultivated with APCs pulsed with the strong agonist ligand p33 or control AV peptide, in the presence of LiCl or KCl. (A) Proliferation of T cells was measured by thymidine incorporation. Proliferation in response to AV was below 300 cpm. (B) IL-2 production was measured by proliferation of the IL-2–dependent cell line CTLL-2 by the addition of supernatant from cultures harvested from the indicated days. (C) GSK-3 regulates NF-ATc nuclear localization. Purified CD8+ T cells from P14 TCR–transgenic RAG2−/− mice were cocultured with APCs pulsed with p33 for 3 d in the presence of LiCl or KCl. Cells were fixed, permeabilized, and stained using an NF-ATc–specific antibody.

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