Wnt ligands are lipid-modified secreted glycoproteins that regulate embryonic development, cell fate specification, and the homeostasis of self-renewing adult tissues. In addition to its well-established role in thymocyte development, recent studies have indicated that Wnt/β-catenin signaling is critical for the differentiation, polarization, and survival of mature T lymphocytes. Here, we describe our current understanding of Wnt signaling in the biology of post-thymic T cells, and discuss how harnessing the Wnt/β-catenin pathway might improve the efficacy of vaccines, T-cell–based therapies, and allogeneic stem cell transplantation for the treatment of patients with cancer. Clin Cancer Res; 16(19); 4695–701. ©2010 AACR.
Wnt ligands are secreted lipid-modified glycoproteins that are essential in diverse cellular processes, including stem cell maintenance, cell fate decision, cell proliferation, survival, migration, and polarity determination. These manifold functions are accomplished by a large number of possible ligand-receptor combinations between 19 Wnt proteins, 10 Frizzled (Fzd) receptors, two coreceptors, and non-Fzd receptors such as the receptor tyrosine kinase-like orphan receptor and the Ryk receptor-like tyrosine kinase (1). These ligand-receptor interactions trigger a diversity of downstream signaling pathways, including the “canonical” Wnt/β-catenin pathway, the Wnt/Planar cell polarity pathway, and the Wnt/Ca2+ pathway (1).
Here, we focus our discussion on the role of the Wnt/β-catenin pathway in T-cell immunity and cancer immunotherapy. Following a detailed review of the specifics of this signaling pathway, we describe its role in the differentiation, polarization, and memory formation of post-thymic T lymphocytes. We conclude by envisioning how the Wnt/β-catenin pathway might be harnessed to develop potent new immunotherapeutic approaches for the treatment of patients with cancer.
Molecular aspects of the Wnt/β-catenin signaling pathway
In the absence of Wnt signal, the cytoplasmic levels of β-catenin are regulated by the “destruction complex,” which consists of the scaffold proteins adenomatosis polyposis coli (Apc) and axin, and the serine-threonine kinases glycogen-synthase kinase 3β (Gsk-3β) and casein kinase 1 (Ck1; Fig. 1). After binding axin and Apc, β-catenin is phosphorylated by Ck1 at S45, and sequentially by Gsk-3β at T41/S37/S33 (Fig. 1). Phosphorylated β-catenin is further polyubiquitinated by β-transducin-repeat–containing protein and Wilms tumor suppressor protein complex, and targeted for proteasome-mediated degradation (Fig. 1; refs. 2–5).
Upon Wnt binding to Fzd and the low density lipoprotein receptor-related protein 5 or 6 (Lrp5/6) coreceptor, Disheveled is recruited to the Wnt/receptor complex to promote phosphorylation of the Lrp5/6 coreceptor by Ck1 and Gsk-3β (Fig. 1; ref. 6). Phosphorylated Lrp5/6 in turn attracts axin to the intracellular domains of Wnt/receptor complex resulting in the disruption of the “destruction complex” and accumulation and nuclear translocation of β-catenin (Fig. 1; ref. 6).
In the nucleus, β-catenin interacts with diverse DNA binding partners to remodel chromatin and orchestrate transcriptional programs (Fig. 1). The best characterized β-catenin–binding partners are transcription factors of the T-cell factor (Tcf)/lymphocyte-enhancer–binding factor (Lef) family, including Tcf1 (encoded by Tcf7), Lef1, Tcf3, and Tcf4 (7). In the absence of β-catenin, Tcf/Lef inhibit gene expression by interacting with repressors such as transducin-like enhancer proteins (Tle) and C-terminal binding protein (CtBP), which mediate histone deacetylation and chromatin compaction (Fig. 1; refs. 8–10). β-catenin replaces Tle to interact with Tcf/Lef and recruits several coactivators, including B-cell chronic lymphocytic leukemia (CLL)/lymphoma 9, pygopus, and the histone acetyltransferase CREB binding protein and E1A binding protein p300 (Ep300) to promote specific gene expression (Fig. 1; refs. 11, 12). More recently, PROP paired-like homeobox 1 (Prop1; ref. 13) and the special AT-rich binding protein 1 (Satb1; ref. 14) have been identified as DNA-binding transcription factors that recruit β-catenin to mediate Wnt signaling (Fig. 1). Prop1–β-catenin signaling has been implicated in the regulation of pituitary gland development by simultaneously promoting the transcription of the cell-lineage–determining factor Pou Class 1 Homeobox 1, and suppressing the expression of the transcriptional repressor Hesx Homeobox 1 via Tle/Reptin/Histone deacetylase 1 corepressor complexes (13). Satb1 is a chromatin organizer enriched in the T-cell lineage that is critical for the expression of a large number of genes involved in T-cell proliferation and development (14, 15). Satb1 acts predominantly as a repressor on most of its target genes; however, upon Wnt/β-catenin signaling it undergoes deacetylation, which facilitates its association with β-catenin and conversion into a transcriptional activator (14). Thus, at least three sets of DNA binding proteins, Tcf/Lef, Prop1, and Satb1, have been found to interact with β-catenin to regulate gene transcription.
The Wnt/β-catenin pathway is subjected to extensive regulation at various levels of the signaling cascade. Wnt-Fzds-Lrp5/6 interactions are modulated by the secreted frizzled-related protein, dickkopf (Dkk), and wise/sclerostin family members as well as Wnt inhibitory factor 1, and Apc down-regulated 1 (6, 16). Nuclear chibby homolog 1 and inhibitor of β-catenin and Tcf (ICAT) inhibit the interaction of β-catenin with Tcf/Lef, repressing β-catenin-Tcf/Lef–mediated transactivation (17–19). β-catenin-Tcf/Lef binding can also be inhibited by naturally occurring Tcf and Lef short isoforms (25 to 40 kD), which lack the N-terminal β-catenin binding domain and act as dominant negatives (20, 21). In addition, posttranslational modification such as phosphorylation, acetylation, sumoylation, and ubiquitination affect the binding affinity of Tcf/Lef to β-catenin or Tle, thus further regulating either transcriptional activation or repression (20). Finally, expression of Wnt signaling components such as Fzds, Lrp6, Axin2, Tcf, Lef, and Dkk1 is also regulated by β-catenin-Tcf in either positive or negative feedback loops (1, 6). The high degree of complexity and the level of regulation involved in the Wnt/β-catenin signaling pathway reflect its centrality to cellular processes.
Wnt/β-Catenin Signaling in T-Cell Immunity
Several studies using gain- and loss-of-function approaches indicate that Wnt/β-catenin signaling is a key regulator of T-cell development at various stages of thymocyte differentiation (reviewed elsewhere by Staal and colleagues, ref. 22, and Yu and colleagues, ref. 23). In vivo studies using either transgenesis (24) or recombinant retroviruses (25) to complement Tcf1 deficiency, perhaps provide the clearest evidence that Wnt/β-catenin signaling is critical for T-cell development. Mice deficient in Tcf1 have impaired thymocyte maturation with defects at double-negative and immature single-positive stages of T-cell development (26, 27). This defect could be rescued by the Tcf1 p45 isoform, but not by the p33 isoform, which lacks the N-terminal β-catenin–binding domain (24, 25). Although the role of Wnt/β-catenin signaling in T-cell development has been extensively scrutinized in the past, its function in the biology of mature T lymphocytes has just begun to be explored.
Modulation of CD8+ T-cell differentiation and memory formation
Early studies investigating a possible role of Wnt/β-catenin signaling in mature CD8+ T cells revealed that Tcf1-deficient CD8+ T cells had no defects in proliferative responses to mitogens or allogeneic antigens and could acquire cytotoxic effector functions similar to wild-type controls (27). These findings led to the surprising notion that Tcf1, which in adult mammals is exclusively expressed by T cells (Supplementary Fig. S1; ref. 28), might not have a role in post-thymic CD8+ T-lymphocyte function. However, new findings clearly indicate that Wnt/β-catenin signaling is functionally important in mature T cells in vivo.
Tcf/Lef reporter systems (29) and gene expression analyses (30–32) indicate that Wnt signaling is functionally active and dynamically regulated in mature T cells in vivo. Tcf7 and Lef1 are highly expressed by naïve CD8+ T (TN) cells, but their levels rapidly drop following a productive encounter with antigen as they undergo massive expansion and differentiation into cytotoxic effector T cells (30–32). The minority of cells that persist as long-lived memory T cells after the effector phase expresses intermediate levels of these Wnt transcription factors (32), but heterogeneity occurs within the T-cell memory compartment. High levels of Tcf7 and Lef1 expression are found in “central memory” T (TCM) cells (30, 31), which express the lymphoid-homing molecules CD62L and CCR7, have long teleomeres, high proliferative capacity, and possess the stem-like quality for plasticity and self-renewal (33–36). Conversely, low levels of Tcf7 and Lef1 are found in CD62Llow and CCR7low “effector memory” T (TEM) cells (30, 31), which have poor replicative potential and have acquired the capacity to produce high amounts of immune interferon (IFN-γ; refs. 33–36). The tightly regulated expression of Wnt signaling transducers indicate that this pathway is associated with the maintenance and function of less-differentiated TN and TCM cells, but causality has only recently been tested.
To directly assess the impact of Wnt/β-catenin signaling on mature CD8+ T-cell differentiation, we activated TN in the presence of Wnt3A or inhibitors of Gsk-3β to stabilize β-catenin and mimic downstream events of the Wnt-signaling cascade (31, 37). Activation of the Wnt/β-catenin pathway withheld T cell proliferation and effector differentiation while promoting the generation of TCM and a novel T-cell memory population named CD8+ memory stem (TSCM) cells, which possess enhanced self-renewal capability in serial transplant experiments, have the multipotent capacity to generate TCM, TEM, and effector T cells, and a proliferative potential that exceeds other effector and memory T-cell subsets (31, 38).
Our work provides the first evidence that enforced Wnt/β-catenin signaling can favor CD8+ T-cell memory formation by suppressing their maturation into terminally differentiated effector T cells (Fig. 2), but there is now evidence that Wnt has similar effects in the physiologic setting (39, 40). Consistent with previous findings (27), Tcf1 deficiency did not impair the ability of CD8+ T cells to undergo effector differentiation in response to viral and bacterial infections (39, 40). Rather, effector differentiation was enhanced, as manifested by the increased numbers of T cells expressing granzyme B (40) and the terminal differentiation and senescence marker Klrg1 at the peak of the immune response (39, 40). This unrestrained effector differentiation prevented CD8+ T cells from forming TCM and severely impaired the ability of CD8+ T cells to mediate secondary immune responses to pathogen re-challenge (39, 40). Regulation of CD8+ T-cell differentiation and memory formation by Tcf1 was dependent on its binding to β-catenin, because genetic complementation with the Tcf1 p45, but not the p33 isoform, inhibited terminal effector differentiation and restored memory recall responses (39). Secondary responses were also depressed in CD8+ T cells lacking both β- and γ-catenin (39). Furthermore, long-term maintenance of CD8+ memory T cells was severely impaired in the absence of Tcf1 due to reduced responsiveness to IL-15 and diminished expression of the antiapoptotic molecule Bcl-2, possibly through an Eomesodermin-dependent mechanism (40). Taken together, these findings indicate that the Wnt/β-catenin pathway is essential for CD8+ T-cell memory formation under physiologic conditions in vivo (39, 40).
Zhao and colleagues came to the same conclusion that the Wnt/β-catenin pathway promotes CD8+ T-cell memory formation using transgenic mice to constitutively activate Wnt signaling, rather than to ablate it (32). Echoing our findings, enforced expression of stabilized β-catenin and Tcf1 inhibited the expansion of antigen-specific CD8+ T cells in the effector phase of the immune response and enhanced CD8+ T-cell memory formation and function in a variety of infection models, including Listeria monocytogenes, LCMV, and vaccinia virus (32).
A number of interesting findings further support a role for Wnt/β-catenin signaling in CD8+ T-cell differentiation and memory formation. First, enforced expression of ICAT, which specifically disrupts β-catenin–Tcf interactions, impairs the survival of CD8+ T cells following in vitro activation (41). Secondly, increased CD8+ T-memory formation was observed in mice after administration of metformin (42), an agonist of adenosine monophosphate kinase that can, among other substrates, phosphorylate and stabilize β-catenin, promoting downstream Tcf/Lef transcriptional activity (43). Finally, gene expression data reveal that high levels of Tcf7 and Lef1 were found in CD8+ T cells with increased potential to form memory in vivo such as in T cells lacking B-lymphocyte–induced maturation protein 1 (Blimp-1; ref. 44) and interleukin (IL)-2Rα (45, 46), two key drivers of terminal effector differentiation. Tcf7 and Lef1 were also high in T cells programmed in the presence of IL-21, a cytokine that inhibits effector differentiation and enhances functional memory (47, 48).
Arrest of lymphocyte differentiation to maintain long-lived antigen-experienced T cells with stem cell–like properties was postulated by Fearon almost a decade ago as a way to continually generate effector T cells, but the molecular mechanisms regulating this process were poorly understood (35). Given the weight of the evidence described above, it is now clear that Wnt/β-catenin is a key signaling pathway governing arrested differentiation in CD8+ T cells. These findings have parallels in stem cell biology, in which Wnt signaling has a pivotal role in promoting stem cell self-renewal while limiting proliferation and differentiation (22, 49). Many unanswered questions remain to be addressed: (i) What Wnt ligands if any are modulating the differentiation and memory formation of CD8+ T lymphocytes in vivo?; (ii) What are the cellular sources and anatomic niches in which Wnt ligands are provided to CD8+ T cells and when does this signaling take place relative to antigen priming?; (iii) What are the molecular events orchestrated by Wnt/β-catenin signaling that restrain effector differentiation in order to enhance memory formation?
Regulation of CD4+ T-cell polarization and survival
Like their CD8+ T-cell counterparts, mature CD4+ T cells can respond to Wnt signals (Fig. 2; ref. 14). Following antigen activation, naïve CD4+ T cells polarize into T helper (Th) cell populations secreting distinct sets of cytokines (50). Classically, CD4+ T-cell polarization has been viewed as a result of diverse transcriptional programs triggered by the cytokine signals that T cells experience at the time of encounter with antigen (50). However, new findings indicate that Wnt proteins can also influence the lineage choice of naïve CD4+ T cells (Fig. 2; ref. 14). The predominance of data implicates an active role of Wnt/β-catenin signaling in the differentiation of Th2 cells (14, 51), which express IL-4 and IL-13 under the control of the master transcription factor GATA binding protein 3 (Gata 3; ref. 52). Wnt-dependent accumulation of β-catenin has been observed in CD4+ T cells undergoing Th2 polarization (14). Blockade of Wnt/β-catenin signaling by the Wnt antagonist Dkk1, or by small interfering RNA (siRNA)–mediated silencing of β-catenin, resulted in decreased expression of Gata3 and low levels of Th2 cytokine secretion (14). Conversely, enforced expression of stabilized β-catenin was found to enhance Gata3 transcription and IL-4 production (51). These results indicate that Wnt signaling critically regulates Th2 polarization by promoting Gata3 expression.
The mechanism whereby Wnt/β-catenin signaling modulates Gata3 transcription has been studied in considerable detail (14, 51). Notani and colleagues have shown that Wnt signaling enhances Th2 polarization by recruiting Satb1, β-catenin, and the histone acetyltransferase Ep300 to the Gata3 promoter (14). Studies by Yu and colleagues (51) have instead focused on Tcf1–β-catenin interactions in the initiation of Th2 cell differentiation. Chromatin immunoprecipitation and luciferase reporter assays indicated that Tcf1 and β-catenin bind to the Tcf1-binding site upstream of Gata3 exon-1b to activate Gata3-1b transcription (51). Tcf1 deficiency diminished Gata3-1b expression and IL-4 production in CD4+ T cells and impaired in vivo Th2 responses in a mouse model of allergic asthma (51). Impairment in Gata3-1b expression and IL-4 production was also observed upon enforced expression of ICAT, showing a requirement for Tcf1 and β-catenin interaction for Th2 polarization (51). Interestingly, the authors found that Tcf1 overexpression also inhibited Th1 fate and the production of the paradigmatic type 1 cytokine IFN-γ, although in a β-catenin–independent fashion (51).
The role of β-catenin has also been studied in naturally occurring CD4+CD25+ T regulatory (nTreg) cells. Enforced expression of β-catenin did not alter immunosuppressive functions in nTreg cells, but enhanced their survival (Fig. 2), resulting in more effective protection against inflammatory bowel disease (53). In addition, stabilized β-catenin impaired the ability of CD4+CD25− T cells to produce IFN-γ and mediate autoimmune colitis. The authors concluded that β-catenin stabilization caused T-cell anergy, but in light of Yu and Notani's results, Wnt/β-catenin might have had simply skewed CD4+ T-cell fate toward less aggressive Th2-type immune responses (14, 51, 54).
In summary, new studies indicate that the Wnt/β-catenin pathway is critical in controlling important aspects of CD4+ T-cell biology, including T-cell survival and lineage fate decisions. It is now clear that Wnt/β-catenin signaling can favor Th2 over Th1 polarization and can enhance the persistence of nTreg, but its impact on the function and differentiation of other CD4+ T-cell subsets remains to be investigated.
Therapeutic strategies targeting Wnt/β-catenin have thus far focused on the suppression of the pathway because of its involvement in carcinogenesis (55, 56). However, the discovery that Wnt/β-catenin signaling is a key regulator of T-cell immunity now raises the possibility that potentiating Wnt signaling could be used to improve cancer therapies through immune-based mechanisms.
The generation of a robust population of memory T cells is critical for effective vaccine and T-cell–based therapies to prevent and treat cancer (34). Although only a minority of patients receiving current therapeutic cancer vaccines experience objective tumor regression, and virtually none are cured by existing vaccines (57), recent phase III randomized trials have shown benefits in overall or disease-free survival in patients with refractory prostate cancer (58), lymphomas (59), and melanoma (60). Some cancer vaccines might fail because they drive T cells to terminal differentiation and senescence rather than generating stem-like T-memory populations (34) that have been shown to be effective in both viral (61) and tumor models (31, 62). Generation of these potent memory populations might be enhanced if vaccinations were done in concert with the immunomodulatory influences of agonists of Wnt/β-catenin signaling. For example, inhibitors of Gsk-3β, which mimic Wnt signaling, have been shown to augment T-cell memory formation in response to a self and/or tumor antigen (31). This strategy could be rapidly translated in the clinical setting because many Gsk-3β inhibitors are already approved for human use or are in clinical evaluation for other indications (63).
The use of agonists of Wnt/β-catenin signaling also has immediate implications for the improvement of immunotherapies based on ex vivo manipulation of lymphocytes prior to adoptive transfer to patients. Allogeneic stem cell transplantation can be highly effective for the treatment of patients with refractory leukemias and lymphomas, but graft-versus-tumor (GVT) responses can come with the high price of graft-versus-host-disease (GVHD), which can be difficult to manage and even lethal (64). Treg and Th2 cells are being actively explored as a means of inhibiting GVHD while maintaining some antitumor effects (65), and the immunoregulatory effects of these CD4+ T-cell subsets might be considerably potentiated by enhancing Wnt/β-catenin signaling with drugs or gene therapy. Our own focus has been to enhance immunotherapies on the basis of the adoptive transfer of autologous tumor-specific CD8+ lymphocytes. Preclinical models have shown that Gsk-3β inhibitors enable the generation of stem-like memory CD8+ T cells, which can be highly effective in small numbers (31). This pharmacologic approach might be employed to efficiently prevent the detrimental terminal differentiation of antitumor T-cell populations that can occur when using current methods for ex vivo expansion (66, 67). Finally, human CD8+ T cells with stem-like properties generated under the influence of Wnt signaling, and genetically engineered to have tumor specificities (68, 69), might reduce the cost and complexity of the treatment and, ultimately, allow the widespread application of adoptive immunotherapies.
The role of the Wnt/β-catenin pathway in the function of post-thymic T cells has just begun to be realized, but it is clear that the modulation of this molecular machinery can be exploited to enhance CD8+ T-cell memory formation and alter CD4+ T-cell fate decisions. A deeper understanding of the biology of Wnt/β-catenin signaling in mature T lymphocytes might create new strategies for not previously imagined therapeutic intervention.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The authors would like to thank C. Klebanoff, P. Muranski, and M. Bachinski for critical review of this manuscript.
Grant Support: Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.