Studies have suggested that GSK-3 may be involved in lymphocyte activation 1112
. To determine whether antigen-specific stimulation of lymphocytes inactivates GSK-3, naive CD8+
splenocytes were sorted from P14 TCR–transgenic mice (specific for LCMV-gp [amino acid 33–41, p33] and H-2Db
) and stimulated with macrophages pulsed with the antigenic ligand p33 or a control peptide AV (). Western blot analysis using an antibody specific for inactive GSK-3β (GSK-3 serine phosphorylated at position 9) shows an increase in phosphorylated GSK-3β after peptide-specific stimulation. Densitometry analysis indicates that the amount of phosphorylated GSK-3β has increased one- to twofold. This demonstrates that GSK-3β becomes inactivated after antigen-specific T cell stimulation.
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 (more ...)
Previous studies have demonstrated that a substitution of alanine for serine at amino acid position 9 of GSK-3β leads to constitutive kinase activity in human cell lines and transgenic mice 718
. To understand the role of GSK-3 in T cell responses, an HA-tagged constitutively active form of GSK-3β (GSK-3βA9) was cloned into the retroviral vector murine stem cell virus (MSCV; A; reference 14). Expression was confirmed by transfecting NIH 3T3 cells with MSCV/GSK-3βA9 and examining expression by Western blot analysis using HA-specific mAbs ( B). A high-titer packaging cell line for MSCV/GSK-3βA9 and control MSCV was obtained (106
CFU/ml) and used to infect bone marrow stem cells from P14 TCR–transgenic mice. The frequency of bone marrow precursors that were transduced with the retrovirus was quantitated by selecting cells with neomycin. Routinely, 60–90% of stem cells carried the selectable marker. These cells were used to generate bone marrow chimeric mice with C57BL/6 irradiated host animals.
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.
Chimeric mice were compared from P14 TCR–transgenic bone marrow transduced with MSCV/GSK-3βA9 and control MSCV. After reconstitution, flow cytometry analysis using antibodies specific for CD4, CD8, and Vα2 (expressed by the P14 transgenic TCR) showed comparable numbers of P14-transgenic CD8+ T cells in the thymus (data not shown) and spleen ( A). This suggests that positive selection and survival of peripheral T lymphocytes was not dramatically affected by the presence of GSK-3βA9. Mature T cell LCMV peptide-specific responses were assessed using a strong agonist ligand p33, the weaker agonist A4Y, and nonstimulatory control peptide AV. Proliferative responses were consistently reduced in GSK-3βA9–reconstituted animals ( B). IL-2 production from these cultures was also assessed by measuring proliferation of an IL-2–dependent line, CTLL-2. C shows that IL-2 production was also reduced in T cells from MSCV/GSK-3βA9 chimeric mice. Therefore, inactivation of GSK-3 is required for maximal proliferation and IL-2 production.
T cell responses were also examined in the presence of LiCl, which selectively inhibits GSK-3 activity in a variety of cells, including COS cells, rat PC12 cells, Drosophila, Xenopus,
and Dictyostelium 91920
. Splenocytes from P14 TCR–transgenic mice were incubated with the strong antigenic peptide p33 and weaker agonist A4Y ligand, in the presence of 10 mM LiCl or 10 mM KCl, and pulsed with [3
H]thymidine at several time points. The initial proliferative responses after 24 h were similar (data not shown). However, in the presence of lithium, dramatically increased and prolonged proliferative responses to p33 and A4Y were seen after 3, 4, and 5 d ( A). In addition, IL-2 production was dramatically increased in the presence of lithium, as measured by proliferation of the IL-2–dependent line CTLL-2 ( B). Together, these data suggest that GSK-3 regulates the duration of T lymphocyte responses.
Previous studies have shown that GSK-3 phosphorylates NF-ATc, which promotes cytoplasmic localization and also export from the nucleus 11
. Therefore, the presence of LiCl should inactivate GSK-3β, resulting in prolonged dephosphorylation of NF-ATc and sustained nuclear localization. We examined the localization of NF-ATc 3 d after T cells were stimulated with the antigenic peptide p33 in the presence or absence of LiCl. Fluorescent microscopy showed that NF-ATc was found in the nucleus in the presence of inactive GSK-3 ( C). This demonstrates that GSK-3 plays an important role in antigen-specific T cell activation by regulating NF-ATc localization.
Our studies suggest that TCR signals inactivate GSK-3, which negatively regulates T cell proliferation and IL-2 production by altering the nuclear import/export of NF-AT. Evidence from other systems support this model. Studies using HeLa and BHK cells have shown that Crm1 and Ran are involved in NF-AT export from the nucleus and have suggested that GSK-3 contributes to this process 2122
. In addition, mutation of the serine residue in NF-AT that is phosphorylated by GSK-3 leads to constitutive nuclear localization 23
. Astoul et al. 24
have examined the role of GSK-3 in B cell receptor signaling using the B lymphoma cell line A20. Cross-linking the B cell receptor with F(ab′)2
fragments lead to the phosphorylation and inactivation of the GSK-3α isoform. Together, studies from a variety of models support a role for GSK-3 in receptor-mediated signals that shuttle NF-AT from the nucleus.
Surprisingly little is known about the negative regulation of T cell activation. Cell surface interactions through molecules such as CTLA-4 negatively regulate T cell responses by interacting with the costimulatory ligands B7-1 and B7-2 25
. Other molecules such as CD5 have been shown to negatively influence TCR-mediated signals 2627
. In addition, signaling molecules such as the tyrosine kinase Csk 28
, SHP-1 (motheaten; PTP-1C; references 29303132
), Cabin 1 33
, and the adaptor Cbl 3435
have also been demonstrated to play a role in negatively regulating T cell responses. In this study, we have shown that GSK-3, a molecule that is generally known to be active in resting cells, is inactivated upon TCR stimulation. We show that GSK-3 is involved in the regulation of TCR-mediated proliferation and that one role of GSK-3 is to negatively regulate NF-ATc activity and IL-2 production. However, GSK-3 has a multitude of substrates, and it will be interesting to understand how these different effectors interplay with other pathways to orchestrate resting T cell survival and activation.