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• W3163101880 abstract "•Conditional knockouts reveal a non-redundant role for GSK-3α and GSK-3β in T cells.•GSK-3α and GSK-3β act together to enhance PD-1 expression.•GSK-3β depletion alone enhances T cell mediated tumor rejection.•GSK-3β plays an important role in regulating T cell-mediated anti-tumor immunity. Glycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3β. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b−/− mice suppressed tumor growth to the same degree as Gsk3a/b−/− mice, whereas Gsk3a−/− mice behaved similarly to wild-type, revealing an important role for GSK-3β in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and β isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy. Glycogen synthase kinase-3 (GSK-3) is a positive regulator of PD-1 expression in CD8+ T cells and GSK-3 inhibition enhances T cell function and is effective in the control of tumor growth. GSK-3 has two co-expressed isoforms, GSK-3α and GSK-3β. Using conditional gene targeting, we demonstrate that both isoforms contribute to T cell function to different degrees. Gsk3b−/− mice suppressed tumor growth to the same degree as Gsk3a/b−/− mice, whereas Gsk3a−/− mice behaved similarly to wild-type, revealing an important role for GSK-3β in regulating T cell-mediated anti-tumor immunity. The individual GSK-3α and β isoforms have differential effects on PD-1, IFNγ, and granzyme B expression and operate in synergy to control PD-1 expression and the infiltration of tumors with CD4 and CD8 T cells. Our data reveal a complex interplay of the GSK-3 isoforms in the control of tumor immunity and highlight non-redundant activity of GSK-3 isoforms in T cells, with implications for immunotherapy. Glycogen synthase kinase-3 (GSK-3) was first discovered in 1980 as a regulatory kinase which phosphorylates and inhibits glycogen synthase (Embi et al., 1980Embi N. Rylatt D.B. Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase.Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (774) Google Scholar) and has since been implicated in several disease states and key cellular processes, including Wnt, insulin, and Hedgehog signaling (Matthews and Cantrell, 2009Matthews S.A. Cantrell D.A. New insights into the regulation and function of serine/threonine kinases in T lymphocytes.Immunol. Rev. 2009; 228: 241-252Crossref PubMed Scopus (23) Google Scholar). Two isoforms of GSK-3 have been reported in mammals; a 51 kDa GSK-3α and a 47 kDa GSK-3β isoform, encoded by the unlinked Gsk3a and Gsk3b genes, respectively. These two isoforms exhibit 98% homology in their kinase domains but only 36% identity in the last 76 C-terminal amino acid residues. Both isoforms are highly expressed in many tissues (Woodgett, 1990Woodgett J.R. Molecular cloning and expression of glycogen synthase kinase-3/factor A.EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1101) Google Scholar) and have been implicated in processes ranging from glycogen metabolism to gene transcription, apoptosis and microtubule stability (Eldar-Finkelman and Martinez, 2011Eldar-Finkelman H. Martinez A. GSK-3 inhibitors: preclinical and focus on CNS.Front. Mol. Neurosci. 2011; Crossref PubMed Google Scholar; Frame and Cohen, 2001Frame S. Cohen P. GSK3 takes centre stage more than 20 years after its discovery.Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1237) Google Scholar). GSK-3β is thought to be of prime importance in diabetes (McManus et al., 2005McManus E.J. Sakamoto K. Armit L.J. Ronaldson L. Shpiro N. Marquez R. Alessi D.R. Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis.EMBO J. 2005; 24: 1571-1583Crossref PubMed Scopus (467) Google Scholar), Alzheimer disease (Phiel et al., 2003Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides.Nature. 2003; 423: 435-439Crossref PubMed Scopus (1044) Google Scholar), and inflammation (Martin et al., 2005Martin M. Rehani K. Jope R.S. Michalek S.M. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3.Nat. Immunol. 2005; 6: 777-784Crossref PubMed Scopus (860) Google Scholar). An unusual aspect of GSK-3 is that it is constitutively active in resting cells (Embi et al., 1980Embi N. Rylatt D.B. Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase.Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (774) Google Scholar; Woodgett, 1990Woodgett J.R. Molecular cloning and expression of glycogen synthase kinase-3/factor A.EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1101) Google Scholar) and its inactivation occurs through phosphorylation of specific serine residues (Ser9 in GSK-3β, Ser21 in GSK-3α) (Hughes et al., 1993Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (500) Google Scholar; Rayasam et al., 2009Rayasam G.V. Tulasi V.K. Sodhi R. Davis J.A. Ray A. Glycogen synthase kinase 3: more than a namesake.Br. J. Pharmacol. 2009; 156: 885-898Crossref PubMed Scopus (365) Google Scholar). This phosphorylation allows the phosphoserine tail of GSK-3 to bind and block its own active site (Doble and Woodgett, 2003Doble B.W. Woodgett J.R. GSK-3: tricks of the trade for a multi-tasking kinase.J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1686) Google Scholar; Rayasam et al., 2009Rayasam G.V. Tulasi V.K. Sodhi R. Davis J.A. Ray A. Glycogen synthase kinase 3: more than a namesake.Br. J. Pharmacol. 2009; 156: 885-898Crossref PubMed Scopus (365) Google Scholar). In contrast to this, tyrosine phosphorylation of GSK-3 (Tyr216 in GSK-3β, Tyr279 in GSK-3α) enhances its ability to bind and phosphorylate substrates (Frame and Cohen, 2001Frame S. Cohen P. GSK3 takes centre stage more than 20 years after its discovery.Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1237) Google Scholar; Hughes et al., 1993Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (500) Google Scholar). Furthermore GSK-3 has a preference for substrates which have already been phosphorylated by a “priming kinase” (Picton et al., 1982Picton C. Woodgett J. Hemmings B. Cohen P. Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3.FEBS Lett. 1982; 150: 191-196Crossref PubMed Scopus (115) Google Scholar). For example, glycogen synthase is primed by casein kinase 2 (CK2) prior to its subsequent phosphorylation and inactivation by GSK-3 (Picton et al., 1982Picton C. Woodgett J. Hemmings B. Cohen P. Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3.FEBS Lett. 1982; 150: 191-196Crossref PubMed Scopus (115) Google Scholar). GSK-3 can phosphorylate more than one hundred substrates (Sutherland, 2011Sutherland C. What Are the bona fide GSK3 Substrates?.Int. J. Alzheimers Dis. 2011; 2011: 505607Crossref PubMed Scopus (221) Google Scholar) and plays a key role in T cell activation (Ohteki et al., 2000Ohteki T. Parsons M. Zakarian A. Jones R.G. Nguyen L.T. Woodgett J.R. Ohashi P.S. Negative regulation of T cell proliferation and interleukin 2 production by the serine threonine kinase GSK-3.J. Exp. Med. 2000; 192: 99-104Crossref PubMed Scopus (118) Google Scholar; Rudd et al., 2020Rudd C.E. Chanthong K. Taylor A. Small molecule inhibition of GSK-3 specifically inhibits the transcription of inhibitory Co-receptor LAG-3 for enhanced anti-tumor immunity.Cell Rep. 2020; 30: 2075-2082.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar; Taylor et al., 2016Taylor A. Harker J.A. Chanthong K. Stevenson P.G. Zuniga E.I. Rudd C.E. Glycogen synthase kinase 3 inactivation drives T-bet-mediated downregulation of co-receptor PD-1 to enhance CD8(+) cytolytic T cell responses.Immunity. 2016; 44: 274-286Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar; Taylor and Rudd, 2020Taylor A. Rudd C.E. Glycogen synthase kinase 3 (GSK-3) controls T-cell motility and interactions with antigen presenting cells.BMC Res. Notes. 2020; 13: 163Crossref PubMed Scopus (4) Google Scholar). Active GSK-3 blocks T cell activation and cytokine production (Ohteki et al., 2000Ohteki T. Parsons M. Zakarian A. Jones R.G. Nguyen L.T. Woodgett J.R. Ohashi P.S. Negative regulation of T cell proliferation and interleukin 2 production by the serine threonine kinase GSK-3.J. Exp. Med. 2000; 192: 99-104Crossref PubMed Scopus (118) Google Scholar), and we previously showed that the inhibition of GSK-3 downregulate PD-1 and LAG-3 gene expression (Taylor et al., 2016Taylor A. Harker J.A. Chanthong K. Stevenson P.G. Zuniga E.I. Rudd C.E. Glycogen synthase kinase 3 inactivation drives T-bet-mediated downregulation of co-receptor PD-1 to enhance CD8(+) cytolytic T cell responses.Immunity. 2016; 44: 274-286Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Other substrates include transcription factors such as cyclic AMP response element binding protein, the nuclear factor of activated T cells (NFATs), β-catenin, c-Jun, and NF-κB (Cohen and Frame, 2001Cohen P. Frame S. The renaissance of GSK3.Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1232) Google Scholar; Eldar-Finkelman and Martinez, 2011Eldar-Finkelman H. Martinez A. GSK-3 inhibitors: preclinical and focus on CNS.Front. Mol. Neurosci. 2011; Crossref PubMed Google Scholar; Grimes and Jope, 2001Grimes C.A. Jope R.S. CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium.J. Neurochem. 2001; 78: 1219-1232Crossref PubMed Scopus (323) Google Scholar). In the case of NFAT, GSK-3 inactivates the pathway by phosphorylating NFAT and facilitating its exit from the nucleus in T cells (Beals et al., 1997Beals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.Science. 1997; 275: 1930-1934Crossref PubMed Scopus (624) Google Scholar; Neal and Clipstone, 2001Neal J.W. Clipstone N.A. Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc.J. Biol. Chem. 2001; 276: 3666-3673Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Active GSK-3 inhibits T cell proliferation (Ohteki et al., 2000Ohteki T. Parsons M. Zakarian A. Jones R.G. Nguyen L.T. Woodgett J.R. Ohashi P.S. Negative regulation of T cell proliferation and interleukin 2 production by the serine threonine kinase GSK-3.J. Exp. Med. 2000; 192: 99-104Crossref PubMed Scopus (118) Google Scholar), whereas T cell receptor (TCR) and CD28 ligation induces GSK-3 phospho-inactivation (Appleman et al., 2002Appleman L.J. van Puijenbroek A.A. Shu K.M. Nadler L.M. Boussiotis V.A. CD28 costimulation mediates down-regulation of p27kip1 and cell cycle progression by activation of the PI3K/PKB signaling pathway in primary human T cells.J. Immunol. 2002; 168: 2729-2736Crossref PubMed Scopus (159) Google Scholar; Ohteki et al., 2000Ohteki T. Parsons M. Zakarian A. Jones R.G. Nguyen L.T. Woodgett J.R. Ohashi P.S. Negative regulation of T cell proliferation and interleukin 2 production by the serine threonine kinase GSK-3.J. Exp. Med. 2000; 192: 99-104Crossref PubMed Scopus (118) Google Scholar; Wood et al., 2006Wood J.E. Schneider H. Rudd C.E. TcR and TcR-CD28 engagement of protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3) operates independently of guanine nucleotide exchange factor VAV-1.J. Biol. Chem. 2006; 281: 32385-32394Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) dependent on phosphatidylinositol 3-kinase (PI3-K) (Taylor and Rudd, 2017Taylor A. Rudd C.E. Glycogen synthase kinase 3 inactivation compensates for the lack of CD28 in the priming of CD8(+) cytotoxic T-cells: implications for anti-PD-1 immunotherapy.Front. Immunol. 2017; 8: 1653Crossref PubMed Scopus (24) Google Scholar). As a regulator of PD-1 and LAG3 expression, we previously showed that small molecule inhibitors (SMIs) and siRNA down-regulation of GSK-3 are effective in promoting viral clearance (Taylor et al., 2016Taylor A. Harker J.A. Chanthong K. Stevenson P.G. Zuniga E.I. Rudd C.E. Glycogen synthase kinase 3 inactivation drives T-bet-mediated downregulation of co-receptor PD-1 to enhance CD8(+) cytolytic T cell responses.Immunity. 2016; 44: 274-286Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and suppressing tumor growth (Taylor et al., 2018Taylor A. Rothstein D. Rudd C.E. Small-molecule inhibition of PD-1 transcription Is an effective alternative to antibody blockade in cancer therapy.Cancer Res. 2018; 78: 706-717Crossref PubMed Scopus (43) Google Scholar). Mechanistically, this was found to operate by enhancing Tbet (Tbx21) transcription which, in turn, inhibits Pdcd1 gene expression by repressing the Pdcd1 promoter (Hui et al., 2017Hui E. Cheung J. Zhu J. Su X. Taylor M.J. Wallweber H.A. Sasmal D.K. Huang J. Kim J.M. Mellman I. Vale R.D. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.Science. 2017; 355: 1428-1433Crossref PubMed Scopus (662) Google Scholar; Rudd et al., 2020Rudd C.E. Chanthong K. Taylor A. Small molecule inhibition of GSK-3 specifically inhibits the transcription of inhibitory Co-receptor LAG-3 for enhanced anti-tumor immunity.Cell Rep. 2020; 30: 2075-2082.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar; Taylor et al., 2016Taylor A. Harker J.A. Chanthong K. Stevenson P.G. Zuniga E.I. Rudd C.E. Glycogen synthase kinase 3 inactivation drives T-bet-mediated downregulation of co-receptor PD-1 to enhance CD8(+) cytolytic T cell responses.Immunity. 2016; 44: 274-286Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar; Taylor and Rudd, 2017Taylor A. Rudd C.E. Glycogen synthase kinase 3 inactivation compensates for the lack of CD28 in the priming of CD8(+) cytotoxic T-cells: implications for anti-PD-1 immunotherapy.Front. Immunol. 2017; 8: 1653Crossref PubMed Scopus (24) Google Scholar, Taylor and Rudd, 2019Taylor A. Rudd C.E. Small molecule inhibition of glycogen synthase kinase-3 in cancer immunotherapy.Adv. Exp. Med. Biol. 2019; 1164: 225-233Crossref PubMed Scopus (4) Google Scholar). Tbet also regulates an array of other genes, including cytokines such as interleukin-2 and effector proteins such as granzyme B which are needed for optimal CD8 cytolytic function (Lazarevic and Glimcher, 2011Lazarevic V. Glimcher L.H. T-bet in disease.Nat. Immunol. 2011; 12: 597-606Crossref PubMed Scopus (172) Google Scholar; Sullivan et al., 2003Sullivan B.M. Juedes A. Szabo S.J. von Herrath M. Glimcher L.H. Antigen-driven effector CD8 T cell function regulated by T-bet.Proc. Natl. Acad. Sci. U S A. 2003; 100: 15818-15823Crossref PubMed Scopus (302) Google Scholar). An unanswered question concerns the relative roles of the two isoforms of GSK-3 in the modulation of PD-1 and protective immunity against cancer. Here, we show the alpha and beta isoforms differentially regulate PD-1, IFNγ and Granzyme B expression, whilst deletion of both isoforms synergizes to reduce PD-1 expression and promote the T cell infiltration into tumors. Our previous studies have demonstrated a clear role for GSK-3 in the regulation of tumor growth, with the inhibition of GSK-3 through SMIs potentiating T cell reactivity leading to diminished growth (Rudd et al., 2020Rudd C.E. Chanthong K. Taylor A. Small molecule inhibition of GSK-3 specifically inhibits the transcription of inhibitory Co-receptor LAG-3 for enhanced anti-tumor immunity.Cell Rep. 2020; 30: 2075-2082.e4Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar; Taylor et al., 2018Taylor A. Rothstein D. Rudd C.E. Small-molecule inhibition of PD-1 transcription Is an effective alternative to antibody blockade in cancer therapy.Cancer Res. 2018; 78: 706-717Crossref PubMed Scopus (43) Google Scholar). However, it is unclear if the different isoforms of GSK-3 work in a similar manner, if both isoforms are required for the function of GSK-3, or if the action of one is dominant over the other, particularly in the context of cancer. Several SMIs are available for GSK-3 and cited ki values suggest that it is possible for SMIs to preferentially target one isoform over the other; although in practice, particularly in vivo, evidence suggests that this is not the case and that inhibitors tend to act against both isoforms, albeit at varying levels (Lo Monte et al., 2012Lo Monte F. Kramer T. Gu J. Anumala U.R. Marinelli L. La Pietra V. Novellino E. Franco B. Demedts D. Van Leuven F. et al.Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3alpha.J. Med. Chem. 2012; 55: 4407-4424Crossref PubMed Scopus (39) Google Scholar; Martinez and Perez, 2008Martinez A. Perez D.I. GSK-3 inhibitors: a ray of hope for the treatment of Alzheimer's disease?.J. Alzheimers Dis. 2008; 15: 181-191Crossref PubMed Scopus (112) Google Scholar). To unequivocally answer this question, we have used Gsk3α-flox/flox-β−flox/flox mice crossed with an Lck-Cre line (Jackson labs) which uses the distal Lck promoter to delete both Gsk3a and Gsk3b in post-selection CD4+CD8+ DP cells without affecting the T cell repertoire (Chiang and Hodes, 2016Chiang Y.J. Hodes R.J. T-cell development is regulated by the coordinated function of proximal and distal Lck promoters active at different developmental stages.Eur. J. Immunol. 2016; 46: 2401-2408Crossref PubMed Scopus (12) Google Scholar) to generate Gsk3ab cKO mice. These mice were then used further to generate individual α and β isoform-specific cKO mice; Gsk3a-flox/flox-Lck-Cre+ (Gsk3a cKO) and Gsk3b-flox/flox-Lck-Cre+ (Gsk3b cKO), respectively, resulting in mice with T cells devoid of either GSK-3α or GSK-3β (Figure 1). This was apparent in both CD4 and CD8 T cell populations as shown in Figure 1A. Importantly, the loss of GSK-3 expression in T cells had no effect on the total number of CD8+ and CD4+ splenic T cells when compared to control mice (Figure 1B). Previously, we showed that using GSK-3 SMIs in in vivo mouse models of cancer led to suppression of tumor growth (Taylor et al., 2018Taylor A. Rothstein D. Rudd C.E. Small-molecule inhibition of PD-1 transcription Is an effective alternative to antibody blockade in cancer therapy.Cancer Res. 2018; 78: 706-717Crossref PubMed Scopus (43) Google Scholar). These studies focused on the CD8+ T cell population as the effector cells. However, SMIs act across all tissues and although CD8+ T cells were investigated ex vivo there is still the possibility that other cells might play a role within these models. We have now utilized the newly generated cKO mice to test if GSK-3 deletion in T cells alone is sufficient to suppress tumor growth and whether deletion of both isoforms is required. We first determined the tumor growth rate and survival kinetics in the three different strains of cKO mice bearing EL4 tumors in the flank (Figure 2A) The mean survival time of 13 days for wt littermate mice was similar to that seen in Gsk3a cKO mice, with the exception of 1 mouse surviving of 8. In contrast, depletion of Gsk3b alone or in combination with Gsk3a gave a clear increase in the overall survival rate, with 50% of Gsk3ab cKO mice and 60% of Gsk3b cKO still surviving at day 40. This survival was concurrent with diminished tumor growth in these animals and the eventual eradication of tumor which was apparent within 15–20 days post-tumor challenge (lower panel Figure 2A). In a repeat study, EL4 tumor bearing mice were sacrificed at day 10 (when tumors were present in all mice) to analyze splenic and tumor-infiltrating cells. In agreement with the results from tumor-free animals (Figure 1B), no difference was seen in the frequency of splenic CD4+ or CD8+ T cells between the different cKO mice bearing EL4 tumors (Figure 2B). Previous studies using GSK-3 SMIs have shown that control of tumor growth is associated with a decrease in PD-1 expression, correlated with an increase in the expression of the transcription factor T-bet (Taylor et al., 2016Taylor A. Harker J.A. Chanthong K. Stevenson P.G. Zuniga E.I. Rudd C.E. Glycogen synthase kinase 3 inactivation drives T-bet-mediated downregulation of co-receptor PD-1 to enhance CD8(+) cytolytic T cell responses.Immunity. 2016; 44: 274-286Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Here, using quantitative real-time polymerase chain reaction (qRT-PCR) of splenic CD8+ T cells, we found Pdcd1 gene expression to be significantly lower in the Gsk3ab cKO compared to wt littermates, and this was associated with increased Tbx21 expression (Figure 2C). A decrease in Pdcd1 expression was also seen in the Gsk3b cKO, whilst statistically significant, the reduction in expression of Pdcd1 in Gsk3b cKO T cells was not as great as that seen in the double cKO. There was no effect on Pdcd1 expression in Gsk3a cKO cells but all three cKO strains showed increased Tbx21 gene expression (Figure 2C). Analysis of the tumors revealed no difference in the infiltration of CD4+ or CD8+ T cells in the double cKO mice when compared to wt littermates, in agreement with results obtained using GSK-3 SMIs (Taylor et al., 2018Taylor A. Rothstein D. Rudd C.E. Small-molecule inhibition of PD-1 transcription Is an effective alternative to antibody blockade in cancer therapy.Cancer Res. 2018; 78: 706-717Crossref PubMed Scopus (43) Google Scholar). However, deletion of Gsk3b alone resulted in significantly increased infiltration of both CD4+ and CD8+ T cells (Figure 2D). Tumor-infiltrating CD8+ T cells from the double cKO mice had significantly higher levels of granzyme B expression and increased numbers of IFNγ expressing cells. Although the latter was true for all three strains of GSK-3 cKO, the levels of granzyme B were only increased when both GSK-3 isoforms were depleted (Figure 2D). These findings indicate that the regulatory effects of GSK-3 on these events are more nuanced that previously appreciated. It appears the required events that determine tumor infiltration are different from those needed for the differentiation of T cells leading to Granzyme B expression. Unlike these differences, cell surface PD-1 expression was measured on tumor-infiltrating lymphocytes (TILs) and showed a clear reduction in the double and Gsk3b cKO mouse cells (Figure 2E) and a lesser reduction in TILs from the Gsk3a cKO mice, consistent with the Pdcd1 gene expression data from the spleen (Figure 2C). We next sought to confirm the results obtained from cKO mice bearing EL4 tumors with those bearing a second tumor type, B16 melanoma, and to determine whether deletion of one GSK-3 isoform could lead to suppression of both flank tumors and pulmonary metastasis. B16 tumor cells tagged with luciferase were injected intradermally into the three different strains of cKO mice and control animals and the tumor growth rate and survival kinetics assessed. The wt littermate mice and Gsk3a cKO mice bearing B16 flank tumors demonstrated a mean survival of 15 and 18 days, respectively, with the exception of 1 surviving mouse of 8 in the Gsk3a cKO (Figure 3A). In contrast, the double GSK-3 cKO demonstrated a 50% survival rate at day 50. This was slightly lower in the Gsk3b cKO, with 40% of mice surviving at day 50; here increased survival was associated with diminished tumor growth and the eventual eradication of tumor was apparent within 15–20 days post-tumor challenge (lower panel Figure 3A). It should be noted that there was an overall prolonged survival of the Gsk3b cKO mice of 5–10 days compared to all other strains, with tumor growth being slower in these mice (Figure 3A). Splenic and tumor-infiltrating cells were analyzed from B16 flank tumor bearing mice which were sacrificed at day 12 (a time point at which tumors were present in the majority of animals to enable TIL isolation). As expected, no difference was seen in the number of splenic CD4+ or CD8+ T cells from the different cKO mice compared to wt littermates (Figure 3B). qRT-PCR of splenic CD8+ T cells demonstrated Pdcd1 transcription to be significantly lower in the double GSK-3 cKO compared to wt littermates, and this was associated with increased Tbx21 gene expression (Figure 3C). Furthermore, as seen in the EL4 lymphoma model, a significant decrease in Pdcd1 gene expression was also seen in the Gsk3b cKO but not in Gsk3a cKO cells (Figure 3C). All three cKO strains showed increased Tbx21 gene expression. The Gsk3b cKO had a significantly higher number of infiltrating CD4+ and CD8+ T cells compared to the other cKOs and wt littermates (Figure 3D). Further analysis demonstrated tumor-infiltrating cells from GSK-3 double cKO mice to have higher levels of granzyme B expression compared to the single cKO mice. All three strains of GSK-3 cKO had increased levels of IFNγ expressing TILs compared to wt littermates. Finally, cell surface PD-1 expression on TILs showed a clear reduction in the double cKO mice cells (Figure 3E), some reduction in the Gsk3b cKO and little change in the Gsk3a cKO, in agreement with Pdcd1 gene expression changes observed in spleen (Figure 3C). The data from both the EL4 (Figure 2) and B16 (Figure 3) flank tumors demonstrate non-redundant activity of the GSK-3α and GSK-3β isoforms, with genetic inactivation of GSK-3β resulting in enhanced rejection of both tumor types in the flank. Human cutaneous melanoma metastasizes to multiple sites, including the lungs and the B16 model can seed tumors in the lung following tail vein injection. We therefore analyzed the requirement for T cell GSK-3 isoforms in B16 pulmonary metastasis in our cKO mice. Luciferase tagged B16 tumor cells were injected intravenously into the different cKO and control mice. At day 24, mice were injected intraperitoneally with luciferin and scanned by IVIS Lumina imaging; data from five mice from each group are shown along with the total flux measurements from these groups (Figure 4A). As found in the EL4 and B16 flank models, rejection of B16 pulmonary tumors was enhanced by deletion of GSK-3β, revealed by the Gsk3b cKO and double cKO mice but not the Gsk3a cKO mice (Figure 4A). This was shown further by tracking tumor spread within the lung at various timepoints (Figure 4B). This method also aided in the tissue collection from mice, ensuring that spleen and tumor samples were taken when tumors were apparent in all the different strains (Day 18). As found in the flank models, no difference was seen in the number of splenic CD4+ or CD8+ T cells between the different cKO mice (Figure 4C). Splenic CD8+ T cells from the double cKO and Gsk3b cKO mice showed the expected reduction in Pdcd1 gene expression and all three strains of cKO mice showed increased Tbx21 expression (Figure 4D). Analysis of TILs revealed increased infiltration of CD4+ and CD8+ T cells in the Gsk3b cKO mice and all three strains of cKO mice exhibited an increased frequency of CD8+ cells expressing granzyme B or IFNγ compared to controls (Figure 4E). Collectively, these data demonstrate a key role for the GSK-3β isoform in controlling T cell mediated anti-tumor immunity. Mice injected intravenously with B16 melanoma cells were culled 18 days later and lungs removed for immunohistochemistry (Figure 5). This timepoint was chosen to ensure that all mice were tumor bearing as confirmed prior to harvest by in vivo imaging. Staining showed CD4+ and CD8+ T cells to be scattered throughout the tumor region (Figures 5A and 5B, respectively) however, significantly increased numbers of CD4+ T cells were apparent in the Gsk3b cKO sections as determined using Imagescope software (Figure 5A, right panel). Further staining demonstrated all strains to contain PD-1 expressing cells throughout the tumor (Figure 5). A slight decrease in PD1+ cells was observed in the Gsk3b cKO, but this reduction was only significant when both isoforms were depleted in the double cKO. Interestingly, Foxp3 expression was significantly increased in all three cKOs compared to wt littermates. T cell distributions within the peribronchial regions (areas surrounding the tumors rather than within the tumor itself) were also examined revealing accumulations of CD4+ T cells in the double cKO and Gsk3a cKO but not the Gsk3b cKO mice (Figure 6A). Furthermore, there was a significant reduction in CD8+ T cells in the Gsk3b cKO mice (Figure 6B). This reduction in peribronchial CD4+ and CD8+ T cells in the Gsk3b cKO mice was also reflected in lower numbers of Foxp3 and PD1 expressing cells (Figures 6C and 6D). Taken together, these data demonstrate a clear difference in T cell distribution in response to the presence of tumor growth with a higher level of tumor-infiltrating T cells being seen in the Gsk3b cKO mice. Furthe" @default.
• W3163101880 created "2021-05-24" @default.
• W3163101880 creator A5003708582 @default.
• W3163101880 creator A5008035303 @default.
• W3163101880 creator A5014918178 @default.
• W3163101880 creator A5019635842 @default.
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• W3163101880 date "2021-06-01" @default.
• W3163101880 modified "2023-10-02" @default.
• W3163101880 title "Non-redundant activity of GSK-3α and GSK-3β in T cell-mediated tumor rejection" @default.
• W3163101880 cites W1264856134 @default.
• W3163101880 cites W1485363052 @default.
• W3163101880 cites W1518972222 @default.
• W3163101880 cites W1567624342 @default.
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