T cell factor (Tcf) 1 and lymphoid enhancer-binding factor (Lef) 1 are downstream transcription factors of the Wnt/β-catenin signaling pathway. Tcf1 and Lef1 are required for normal thymic T cell development, but less is known about Wnt function in mature T cells^2,4. Although experiments using multimerized TCF/LEF binding site reporter system have revealed that Wnt signaling is active in mature CD8⁺ T cells, the impact of this pathway to this cell population has yet to be fully elucidated⁵. At least three lines of evidence indicate that Wnt signaling might regulate the maturation of post-thymic T lymphocytes: i.) CD8⁺ T cells from Tcf7^−/− mice, which are missing the gene that encodes for TCF1, display a more differentiated phenotype (CD44^high and CD62L^low) than WT T cells⁶; ii.) expression of Lef1 and Tcf7, decreases with progressive differentiation of CD8⁺ T cells from naive (T_N) → central memory (T_CM) → effector memory (T_EM) inhumans⁷ and mouse (Supplementary Fig. 1); and iii.) high levels of Ctnnb1 (which encodes β-catenin), Lef1, Tcf7 have been detected in T cells with increased potential to form memory in vivo^8,9. Thus, triggering the activities of the Wnt signaling transcription factors TCF1 and LEF1 could promote stem-like self-renewal capacity in mature T cells.

To test the impact of Wnt/β-catenin signaling on mature CD8⁺ T cells, we primed T_N in the presence of the 4,6-disubstituted pyrrolopyrimidine TWS119, a potent inhibitor of the serine/threonine kinase glycogen-synthase-kinase-3β (Gsk-3β)¹⁰. Gsk-3β blockade mimics Wnt signaling by promoting the accumulation of β-catenin, the molecule that tethers the TCF and LEF transcription factors to targeted DNA². TWS119 triggered a rapid accumulation of β-catenin (mean 6.8 +/− SD 1.7-fold increase by densitometry; p < 0.05) (Fig. 1a), augmented nuclear protein-TCF/LEF oligonucleotide interaction (Fig. 1b) and sharply up-regulated Tcf7¹¹, Lef1¹² and other Wnt target genes including Jun¹³, Frizzled7 (Fzd7)¹⁴, Nemo-like-kinase (Nlk)¹⁵ (Fig. 1c). By contrast, T cell activation in the absence of the Gsk-3β inhibitor resulted in the down-regulation of the Wnt/β-catenin pathway at these steps of the signaling cascade (Fig. 1a–c). Thus, TWS119 activated the Wnt/β-catenin pathway in naive T cells and reversed the physiological down-regulation of Tcf7 and Lef1 induced by T cell activation⁷.

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Figure 1

TWS119 activates Wnt signaling in CD8⁺ T cells

Naive CD8⁺ T cells were primed in vitro with anti-CD3 (2 μg ml⁻¹) and anti-CD28 (1 μg ml⁻¹) specific antibodies with or without 7 μM TWS119. a, Western blot analysis of β-catenin and Gapdh in CD8⁺ T cells treated with or without TWS119. b, Electrophoretic mobility shift assay of nuclear extract from CD8⁺ T cells treated with or without TWS119 using P³²-labeled oligonucleotide probes designed from the TCF/LEF binding region of TCF1 target gene Fzd 7. Unlabeled oligonucleotide probes were used as competitor. c, Quantitative RT-PCR analysis of the expression of Tcf7, Lef1, Jun, Fzd7, and Nlk in CD8⁺ T cells treated with or without TWS119. Data are represented as mean +/− SEM. All data are representative of at least two independently performed experiments.

We sought to assess the effect of Wnt signaling on CD8⁺ T cell differentiation and proliferation. We stimulated CFSE-labeled CD8⁺ T cells from pmel-1 TCR transgenic mice¹⁶ with the cognate antigen, gp100, in the presence of titrated doses of TWS119 and analyzed them for the expression of the differentiation markers CD44 and CD62L. CD44 expression is known to increase with T cell differentiation while CD62L is progressively lost¹⁷. TWS119 increased the frequency of T cells that retained CD62L expression in a dose-dependent manner, indicating that it inhibited CD8⁺ T cell differentiation (Fig. 2a). Interestingly, 46% of CD8⁺ T cells cultured in the presence of the highest concentration of Gsk-3β inhibitor failed to up-regulate CD44, maintaining a “naive” CD44^lowCD62L^high phenotype (Fig. 2a). Low doses of TWS119 (≤ 1 μM) preserved CD62L expression without affecting T cell proliferation, while higher drug concentrations promoted a dose-dependent inhibition of cell cycling (Fig. 2b). Arrested differentiation and proliferation of CD8⁺ T cells by TWS119 was not secondary to the impact of the drug on dendritic cells (DC), because we observed similar results stimulating purified CD8⁺ T cells in a DC-free system (Supplementary Fig. 2a,b). Similar to TWS119, we found that the structurally unrelated Gsk-3β inhibitor, 6-bromo-substituted indirubin, BIO^18,19, inhibited T cell differentiation (Supplementary Fig. 3a) and induced the expression of the Wnt transcription factors Tcf7 and Lef1 (Supplementary Fig. 3b). The use of an analog, BIO-acetoxime¹⁹, with a greater Gsk-3β kinase inhibitory specificity, retained the observed activity while the use of N-methylated analog (Methyl-BIO)¹⁹, a kinase inactive control, had no effect (Supplementary Fig. 3a,b). These results are in contrast with those obtained using lithium chloride²⁰ as a Gsk-3β inhibitor, which is less active and specific than the inhibitors used in the present study¹⁹. Because Gsk-3β regulates several signaling pathways other than Wnt, we sought to more directly test whether the impact of the pharmacological blockade of Gsk-3β was dependent on mimicking the downstream signals of the Wnt/β-catenin pathway. We primed CD8⁺ T cells in the presence of Wnt3A, a Wnt protein that has been shown to promote stem cell self-renewal and pluripotency via β-catenin accumulation in the cell nucleus²¹. Like TWS119, we found that Wnt3A itself inhibited T cell differentiation and proliferation (Supplementary Fig. 4). Thus, T cell proliferation and differentiation could be restrained through the activation of the Wnt/β-catenin pathway by the naturally-occuring ligand, Wnt3A, and by the pharamcologic inhibition of Gsk-3β downstream. Neverthelss, our data did not rule out the possibility that Gsk-3β inhibitors were regulating T cell differentiation by affecting other pathways in addition to Wnt.

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Figure 2

Wnt signaling inhibits CD8⁺ T cell proliferation and effector differentiation

a–c,CFSE-labeled naive pmel-1 CD8⁺ T cells were primed in vitro with CD8⁺ T cell depleted splenocytes pulsed with 1 μM hgp100_25–33, in conjunction with 10 ng ml⁻¹ IL-2 and titrated doses of TWS119. Four days following T cell activation, phenotypic (a) CFSE dilution assays (b) and cytokine and ⁵¹Cr release assays (c) were performed. Data in panel c are represented as mean +/− SEM. d, Quantitative RT-PCR analysis of the expression of Eomes in CD8⁺ T cells after priming with anti-CD3 and anti-CD28 specific antibodies with or without 7 μM TWS119. Data are represented as mean +/−SEM. e, Flow cytometry and enumeration of T cell subsets six days after vaccination with or without TWS119. WT mice received adoptive transfer of 10⁶ naive pmel-1 thy1.1⁺ CD8⁺ T cells in conjunction with recombinant fowlpox-based hgp100 vaccine. Mice received four daily doses of TWS119 (at 30 mg kg⁻¹) from day 0 to day 3 or DMSO as control. All data are representative of at least two independently performed experiments.

We sought to evaluate whether the phenotypic differences of pmel-1 CD8⁺ T cells induced by TWS119 were associated with functional changes. It has been previously shown that differentiating CD8⁺ T cells lose the capacity to produce IL-2 as they acquire the ability to kill target cells and release high levels of IFN-γ^17,22. We found that TWS119 induced a dose-dependent decrease in T cell-specific killing and IFN-γ release associated with a preservation in the ability to produce IL-2 (Fig. 2c). These functional data confirmed the phenotypic findings that TWS119 was a negative regulator of T_EFF differentiation. We next assessed if the inhibition of T_EFF development was associated with the suppression of eomesodermin (Eomes), a master regulator of CD8⁺ T-cell effector function²³. We found that the exposure of cells to TWS119 abrogated the induced expression of Eomes that occurred within 8h after T cell priming, indicating that TWS119-mediated suppression of the effector program was an early event (Fig. 2d). Altogether, phenotypic, functional and molecular data indicated that induction of Wnt signaling during T cell priming resulted in an inhibition of CD8⁺ T cell differentiation into T_EFF cells. Given our in vitro findings, we tested whether TWS119 could influence the qualities of adoptively transferred pmel-1 CD8⁺ T cells in response to fowlpox-based gp100 immunization and prevent the induction of highly differentiated, senescent T cells, a major pitfall of current T cell-based vaccines²⁴. We found that there were no differences in frequency or numbers of pmel-1 T cells in spleens six days after vaccination, but the development of T_EFF was inhibited (Fig. 2e). More importantly, TWS119 augmented the numbers of CD44^high, CD62L^high T_CM in responding CD8⁺ T cells in the post-vaccination setting(Fig. 2e). Although the present approach does not enable the dissection of the cellular mechanism of action in vivo, TWS119 effectively altered CD8⁺ T cell differentiation after immunization.

We observed a cell population that expressed low levels of CD44 and high levels of CD62Lwhen TWS119 was administered in vitro and in vivo (Fig. 2a,e) but it was unclear whether these cells remained naive after antigen encounter or had entered into a primordial memory state that retained some phenotypic traits of T_N. Using a murine model of graft-versus-host disease, Zhang et al. have described population of CD44^low CD62L^high memory CD8⁺ T cells that expressed high levels of stem cell antigen-1 (Sca-1), B-cell lymphoma protein 2 (Bcl-2), and common IL-2/IL-15 receptor β chain (CD122)²⁵. Because these cells displayed robust self-renewal and the multipotent capacity to derive T_CM, T_EM, and T_EFF they were designated “T memory stem cells” (T_SCM)²⁵. We sought to explore whether the CD44^low CD62L^high pmel-1 T cells generated in vitro with TWS119 were T_SCM. We found that TWS119-induced CD44^low CD62L^high T cells had undergone up to four divisions as manifested by CFSE dilution, and uniformly expressed high levels of the core phenotypic traits of T_SCM, namely Sca-1, CD122 and Bcl-2 (Fig. 3a). By contrast, T_N expressed these markers at low levels (Fig. 3a). Similarly, Sca-1, CD122 and Bcl-2 were up-regulated in CD44^low, CD62L^high pmel-1 T cells generated in vitro in the presence of BIO-acetoxime (Supplementary Fig. 5) or Wnt3A (Supplementary Fig. 6) and in vivo after specific-vaccination and TWS119 administration (Supplementary Fig. 7). These activation/memory T cell markers were not exclusively expressed by T_SCM but instead only defined T_SCM in the context of cells the were also CD44^low CD62L^high (not shown). Antigen-experienced memory T cells can be distinguished from T_N not only by phenotype but also by a number of functional properties: i.) rapid acquisition of effector functions upon antigen re-challenge^26–28; ii.) pronounced cell cycling capacity²⁹; iii.) robust homeostatic proliferation³⁰; and iv.) independence of MHC class I for persistence³¹ and anti-tumor activity³². We found that, unlike T_N, TWS119-induced CD44^low, CD62L^high T cells rapidly released cytokines (IFN-γ and IL-2) upon antigen encounter (Fig. 3b), had undergone more cell division after adoptive transfer into a lymphoreplete host (Fig. 3c) or into sublethally irradiated or genetically lymphodepleted Tcra^−/− (T cell-deficient) or Rag1^−/− (T and B cell-deficient) hosts (Fig. 3d), and persisted and mediated tumor destruction (P < 0.05) in B2m^−/− mice (which are MHC class I-deficient) (Fig. 3e,f). Altogether, these findings indicated that Wnt signaling induced the generation of a T_SCM-like population that possessed the rapid recall ability, proliferative capacity and MHC class I independency that are distinct from naive T cells and characteristic of memory T cells.

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Figure 3

Wnt signaling promotes the generation of T_SCM

CFSE-labeled, naive pmel-1 CD8⁺ T cells were primed in vitro with CD8⁺ T cell-depleted splenocytes pulsed with 1 μM hgp100_25–33, in conjunction with 10 ng ml⁻¹ IL-2 and 7 μM TWS119. a, Flow cytometry analysis of TWS119-treated and naive pmel-1 cells four days following T cell activation. b, Cytokine release assay of sorted CD44^lowCD62L^high TWS119-treated and naive pmel-1 cells five days after antigenic stimulation. Data are represented as mean +/− SEM. c and d, CFSE dilution of sorted CD44^lowCD62L^high TWS119-treated and naive thy 1.1⁺ (c) or ly5.1⁺ (d) pmel-1 cells one month after transfer into WT (c) and sublethally-irradiated WT, or Tcra^−/−, or Rag1^−/− mice (d). Data are shown on thy 1.1⁺ (c) or ly5.1⁺ (d) CD8⁺ lymphocytes. e, Flow cytometry analysis of sorted TWS119-treated and naive ly5.1⁺ pmel-1 cells one month after transfer into WT or B2m^−/− mice. Data are shown on ly5.1⁺, CD8⁺ lymphocytes. f, Tumor treatment of myeloablated WT or B2m^−/− mice bearing B16 tumors established for 7 days. Mice received age-matched, lineage-depleted bone marrow cells. On the following day, 10⁶ CD44^lowCD62L^high TWS119-treated or naive pmel-1 cells were transferred in conjunction with exogenous IL-2.g and h, Flow cytometry analysis of CFSE-labeled, sorted CD44^lowCD62L^high TWS119-treated and naive ly5.1⁺ pmel-1 cells one month after transfer into sublethally-irradiated WT mice. Data are represented as the percentage of CD44^lowCD62L^high cells as a function of CFSE dilution of two independent experiments (g) and as fraction of cells with any given number of divisions (h). All data are representative of at least two independently performed experiments.

The concept of “stemness” encompasses the capability to both self-renew and generate more differentiated, specialized cells. We sought to determine the “stemness” of TWS119-generated CD44^low, CD62L^high T cells by evaluating their fate after adoptive transfer cells into sublethally irradiated mice. Four weeks later, we found that high percentages of TWS119-generated cells preserved their original CD44^low, CD62L^high phenotype even after multiple cell divisions, while T_N rapidly acquired CD44 as a function of CFSE dilution (P < 0.02) (Fig. 3g,h). The differentiation of TWS119-induced CD44^low, CD62L^high T cells was not arrested however, because they acquired CD44^high expression, albeit at a slower pace. Unlike T_N, TWS119-derived CD44^low, CD62L^high, Sca-1^high Tcells robustly proliferated (by 28 days, 95% had undergone cell division) (Fig. 3h), enabling their re-isolation and transfer to a secondary recipient (Supplementary Fig. 8). After four weeks, we found that the secondarily transferred CD44^low, CD62L^high, Sca-1^high Tcells had again regenerated all T cell subsets. Importantly, 43% of the cells retained the T_SCM phenotype in spite of a second round of robust proliferation in vivo (Supplementary Figure 8). These findings indicated that Wnt signaling promoted the generation of self-renewing, multipotent T_SCM.

Having established that the CD44^low, CD62L^high T cells generated in the presence of TWS119 were bona fide T_SCM and not merely naive, we sought to compare them with the well defined memory T cell subsets, T_CM and T_EM, in secondary challenge experiments. Highly-purified pmel-1 thy1.1⁺ memory T cell subsets were adoptively transferred into sublethally-irradiated micein combination with recombinant vaccinia-based gp100 vaccine and IL-2. As previously reported by our group and others^33,34, the in vivo recall responsesof T_CM were greater than T_EM (Fig. 4a). T_SCM, however, robustly expanded by approximately 200-fold in the spleen alone. These levels were ≈ 10-times higher than T_CM and ap; 30-times higher than T_EM (Fig. 4a). Furthermore, T_SCM exhibited an enhanced survival capacity as revealed by the frequencies of pmel-1 cells in the spleens of vaccinated mice one month after transfer (Fig. 4b). T cell proliferative and survival capacities have been correlated with tumor responses in mice and humans receiving adoptive T cell-based therapies^35,36. To assess whether the enhanced replicative capability of T_SCM would result in superior anti-tumor activity, we adoptively transferred limiting numbers of pmel-1 memory T cell subsets into sublethally-irradiated hosts bearing B16 tumors, in combination with gp100 vaccine and IL-2. As we previously reported³³, T_CM conferred superior anti-tumor immunity compared with T_EM (tumor regression P < 0.01; overall survival P = 0.0644) (Fig. 4c). It was striking that minuscule numbers (4 × 10⁴) of T_SCM were able to trigger the destruction of bulky tumors (1 cm³ containing ap; 10⁹ cells) and to improve survival (T_SCM vs.T_CM, P < 0.005; T_SCM vs. T_EM P < 0.005) (Fig. 4c). Thus, adoptive transfer of T_SCM in combination with tumor-antigen vaccination and exogenous IL-2 produced a far more robust and therapeutically significant secondary response compared with the other memory T cell subsets.

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Figure 4

T_SCM possess enhanced in vivo recall response and anti-tumor activity compared to T_CM and T_EM

Pmel-1 naive CD8⁺ T cells were primed in vitro with splenocytes pulsed with 1 μM hgp100_25–33, in conjunction with 10 ng ml⁻¹ IL-2 with or without 7 μM TWS119. Five days after antigenic stimulation, T_SCM, T_CM or T_EM were sorted based on the phenotype. Sublethally-irradiated WT mice received 5 × 10⁴ pmel-1 T_SCM, T_CM or T_EM in conjunction with a recombinant vaccinia virus encoding hgp100 and exogenous IL-2. a, Absolute numbers of adoptively transferred pmel-1 cells (identified by CD8⁺ thy1.1⁺ lymphocytes) in the spleens of treated animals. Data are represented as mean +/− SEM. b, Flow cytometry analysis for the expression of CD8 and thy1.1. c, Tumor treatment and survival of sublethally-irradiated WT mice bearing B16 tumors established for 10 days (n = 5 for all groups) receiving 4 × 10⁴ pmel-1 T_SCM, T_CM or T_EM in conjunction with a recombinant vaccinia virus encoding hgp100 and exogenous IL-2. Data are represented as mean +/− SEM. All data shown are representative of at least two independently performed experiments.

Arrest of lymphocyte differentiation in order to maintain long-lived, self-renewing antigen-experienced T cells with the stem cell-like properties has been postulated as the basis of the continual generation of effector T cells³⁷, but the transcription factors that regulate this process have not been fully elucidated. Although the physiologic role of Wnt signaling in post-thymic T cell development remains unknown, our data indicate that Wnt can regulate the “stemness” of CD8⁺ T cells by suppressing their differentiation into T_EFF. In addition, other circumstantial evidence supports our findings. We recently reported that high levels of the Wnt transcription factors Tcf7 and Lef1 were expressed by T cells whose development into T_EFF had been arrested using IL-21⁸. Conversely, Tcf7^−/− mice reportedly have an increased frequency of T cells displaying a CD44^high, CD62L^low T_EFF/T_EM phenotype⁶. Paralleling our own observations in CD8⁺ T cells, expression of a stabilized form of β-catenin inhibited the proliferation and effector function of CD4⁺ T cells in a model of inflammatory bowel disease³⁸. Furthermore, productive CD4⁺ T cell memory precursors overexpressed Ctnnb1 and Tcf7 as well as Bcl2 and Il2rb⁹. These factors were all implicated in the formation and maintenance of T_SCM in the present study.

Our findings also have parallels in stem cell biology where Wnt signaling plays a pivotal role in promoting self-renewal while limiting proliferation and differentiation^2,3. Using mice overexpressing the Wnt signaling inhibitor Dickkopf1 in the hematopoietic stem cell niche, Fleming et al., have found that hematopoietic stem cells exposed to a Wnt-inhibitory environment were less quiescent and had profound defects in their ability to reconstitute the host after serial transplants³. T cell lineage commitment to effector versus memory subsets has been recently linked to the asymmetric segregation and inheritance of proteins that control cell fate specification³⁹. Although we do not know the role of Wnt in the asymmetric division of CD8⁺ T cells, unequal localization and activities of Wnt signaling component including β-catenin have been implicated in cell fate specification in Caenorhabditis elegans⁴⁰.

The ability to pharmacologically induce T_SCM has significant implications for the field of adoptive immunotherapy. The findings that small numbers of T_SCM in conjunction with specific vaccination and IL-2 can cause the regression of large established vascularized tumors could reduce the cost and complexity of highly effective therapies based on the adoptive transfer of anti-tumor T cells³⁵. Using existing technology³⁶, it may be possible to genetically engineer the human counterpart of CD8⁺ T_SCM to express T cell receptors or chimeric antigen receptors while pharamacologically mimicking Wnt signaling. This might ultimately allow for the widespread application of adoptive immunotherapy based on multipotent, highly proliferative, tumor-specific T_SCM. Finally, the modulation of Wnt signaling to induce long-term T cell memory might be important for T cell-based vaccines designed to target intracellular pathogens. Increasing data indicate that protection against intracellular pathogens correlates with the induction and maintenance of T_CM responses, but these responses have not been reproducibly obtained using current vaccination strategies^24,28,34. In vivo administration of small molecule agonists of Wnt might be useful to consistently achieve this goal.