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The molecular mechanisms that regulate mature T cell fate and enable cells to differentiate into memory T cells are largely unknown. Memory T cells share certain key features with stem cells, they both have the ability to self-renew and are long-lived. The Wnt-β-catenin signaling pathway is a key player in regulating stem cell self-renewal and differentation. We generated a conditional knock-out mouse that specifically lacks β-catenin in mature T cells and report here that β-catenin is not involved in regulating effector versus memory T cell differentiation. β-catenin deficient memory T cells were phenotypically and functionally indistinguishable from control cells and made normal recall responses. β-catenin deficiency does not affect T cell migration, T cell function in a model of chronic infection or lymphopenia induced proliferation. Together, our data suggest that self-renewal and differentiation is regulated differently in memory T cells compared to epithelial and hematopoietic stem cells.
Mature T cells harbor an incredible proliferative potential that is displayed when a naïve T cell is activated and acquires an effector phenotype during the course of 15 or more rounds of cell division (1). While T cells with an effector phenotype are rather short-lived, a population of long-lived memory T cells emerges after the peak of the expansion phase. These memory T cells have the capacity to self renew by slow homeostatic turnover (2) and share certain transcriptional patterns with hematopoietic stem cells (HSCs)(3). The Wnt-β-catenin pathway was identified as a key pathway in regulating self-renewal and differentiation in epithelial cells (4)and has since been proposed to play the same role in HSCs(5). In the absence of Wnt signals, β-catenin is continuously marked for degradation by GSK3β. Binding of a member of the Wnt ligand family to its receptor leads to inhibition of GSK3β activity and allows β-catenin accumulation and translocation to the nucleus, where it interacts with TCF/LEF transcription factors. In addition to the Wnt signaling pathway, β-catenin release to the nucleus is also affected by E-cadherin(6).
Wnt signaling has been studied in several gain-of-function studies and in a very limited number of loss-of-function studies examining hematopoietic stem cell function as well as lymphocyte development (7). Although there are some discrepancies in these gain-of-function studies (7), the data tend to confirm the original notion of a key role for β-catenin in epithelial cell self-renewal(4)to be true in HSCs as well(5, 8). However, since data from loss-of-function studies are limited and conflicting, this conclusion remains disputed (9, 10). The role of β-catenin in T cell development is still controversial as well (11, 12). The reason for the different outcome is still unclear and while the different timing of deletion might play a role, a compensatory role of γ-catenin when β-catenin is deleted early in HSCs was not found(13).
The role of β-cateninin mature T cell differentiation has not been addressed by deleting the genein T cells post-thymic selection in vivo, although one study used an elegant in vitro approach to delete β-catenin in mature T cells(14). Two groups reported that TCF-1 deficient T cells have an impaired ability to generate memory T cells (15, 16), but since TCF-1 plays a crucial role in thymic selection (17)and TCF-1 deficient T cells in the periphery have an altered phenotype (18), it is not possible to pinpoint the defect as a result of improper development in the thymus or as a mature T cell differentiation defect. Loss-of-function studies that exclude secondary effects caused by changes in thymic selection are ultimately necessary to confirm these conclusions, but still missing.
We asked if β-catenin expression is required for the generation of functional memory cells in vivo. In order to circumvent a possible effect of β-catenin deficiency during thymic selection on mature T cell function, we bred β-catenin-flox mice (19) to mice that express Cre under the control of the distal lck promoter that is turned on after positive selection and a YFP reporter (20). We tested T cell memory generation in various conditions including after viral and bacterial infection and in lymphopenic conditions. We found that β-catenin was not necessary for memory T cell generation and function and conclude that β-catenin function is not congruent in epithelial cells and mature T cells.
C57BL/6 mice and RAG1-deficient mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in specific pathogen-free conditions in the animal facilities at the University of Washington. β-catenin-flox mice were provided by Kris Hogquist (University of Minnesota) and originally came from Rolf Kemler (Max Planck Institute, Freiburg). dlck-cre transgenic mice bred to Rosa-YFP reporter mice were provided by Pam Fink (University of Washington) and originally came from Nigel Killeen (University of California, San Francisco). Data shown are representative of results from at least two independent experiments. All experiments were done in accordance with IACUC guidelines.
Listeria monocytogenes expressing a secreted form of OVA (LM-OVA) was grown as previously described(21). For primary infections, mice were injected i.v. with 2×103 cfu LM-OVA or intra-nasally with 1×107 actA-deficient LM-OVA. For rechallenge experiments, mice received 2×105 cfu LM-OVA i.v. and were euthanized 5 days later. For priming with LCMV Armstrong and LCMV Clone13 mice were injected i.p. with 2×105 pfu and i.v. with 2×106 pfu, respectively.
TWS119 (Merck/EMD) experiments were performed as previously described (22). Cells were stained with LIVE/DEAD cell stain kit (Invitrogen) prior to FACS analysis. Wnt3a experiments and Eomes expression analysis were performed as previously described (15). Data shown are combined from two experiments and analyzed in Prism using the t-test.
Cells and tissues were prepared for staining and assays, and analyzed as previously described(21). In some experiments cells were labeled with CellTracker Violet (Invitrogen) according to manufacturer’s instructions. When applicable, YFP+ cells were sorted using a FACSAria.
Three experimental groups of mice were used: the first group lacked expression of dlck-cre (WT). The other 2 groups expressed both, the dlck-cre transgene and the YFP reporter. Of these latter two groups of mice, one group had one wild-type and one deleted (floxed) allele (het) and the other group had β-catenin deleted on both (floxed) alleles (KO). This setup allowed for proper control of possible Cre mediated toxic effects that are independent of the deletion of the target gene. Our YFP gating strategy, confirmation of β-catenin deletion and abrogation of the Wnt-signaling pathway in β-catenin deficient cells are shown in Suppl. Fig. 1. Data from the WT group are shown once and omitted for brevity in ensuing experiments. We infected all three groups with a recombinant Listeria monocytogenes strain (LM-OVA) that allowed us to track a specific CD8 T cell epitope. We examined T cell numbers and phenotype at the peak of the response on day 7. All experimental groups generated an equivalent primary CD8 T cell response to OVA as measured by tetramer staining (Fig. 1a) with indistinguishable surface marker (CD62L, KLRG1) phenotypes (Suppl. Fig. 1d and data not shown). Contrary to what has been reported in epithelial cells (23), CD44 expression was not regulated by β-catenin activity in T cells as CD44 expression on antigen specific T cells increased in all experimental groups (Fig. 1).
A recent study proposed a role for Wnt signals in mediating effector cell migration (24). We speculated that a systemic infection could mask a potential role for β-catenin in T cell migration. We infected mice intra-nasally with LM-OVA and examined T cell numbers in spleen and lung. T cells of all three groups showed equivalent expansion and migration(Fig. 1b). Thus, we exclude a general role for β-catenin mediated migration of effector cells, though this does not exclude the possibility that β-catenin signaling might be involved in migration under certain situations or in specific tissues.
It is not known whether memory cells give rise to effector cells in a true stem cell fashion, where one daughter cell differentiates into an effector cell while the other daughter retains a memory phenotype. We hypothesized that if β-catenin was required to balance effector versus memory differentiation, a rechallenge would reveal any defects in β-catenin deficient T cells. We determined the number of antigen specific memory cells during the memory phase (at least 30 days post infection)and found equivalent CD8 memory T cell levels in all experimental groups (Fig. 2a and data not shown). To examine the ability of β-catenin deficient memory cells to expand after rechallenge, we infected mice with a rechallenge dose of LM-OVA. CD8 memory T cells expanded equally well 5 days after the rechallenge and had identical functional properties independent of β-catenin expression (Fig. 2b). WT, het and KOCD8 T cells expressed identical levels of granzyme B and showed no evidence of any statistically significant changes in function, migration or phenotype caused by β-catenin deficiency (Fig. 2c and data not shown). Similary, we did not observe any differences in the re-expansion of the CD4 memory T cell compartment (data not shown). A loss of regulation mediated by Tregs could theoretically mask a potential β-catenin dependent phenotype, however we could not find any evidence for an impaired Treg population. Generation of Foxp3+ cells in in vitro polarization assays by β-catenin deficient cells was normal (data not shown), but even the 50% of CD4 T cells that do not delete β-catenin (Suppl. Fig. 1a) would be sufficient for normal Treg function(25).
We went on to test the ability of β-catenin deficient T cells to proliferate in a lymphopenic environment. We speculated that the different stimuli that drive T cell proliferation and acquisition of a memory phenotype in a lymphopenic environment might more closely resemble the turnover of epithelial cells and could reveal a dependency on β-catenin. Proliferation in a lymphopenic host is largely driven by non-inflammatory cytokines, availability of self-ligands and is independent of co-stimulation (26). We transferred CellTracker labeled lymph node cells into RAG deficient recipient hosts and analyzed them 12 days later. Again, we found that the lack of β-catenin did not impair the ability of T cells to proliferate and acquire a memory phenotype (Fig. 3 and data not shown). The majority of cells were CellTracker negative after 12 days, while another population had divided less than 7 times and still contained the dye. Both populations were present in all three groups at similar numbers. Together, these data suggest that β-catenin is not involved in memory T cell generation and maintenance regardless of the signals that mediated T cell activation.
While β-catenin is dispensable for the generation of memory cells, we speculated that T cell exhaustion caused by chronic infection such as LCMV-Cl13 might be more profound in β-catenin deficient mice, if β-catenin expression is required for self-renewal of a T cell. We infected mice from all three experimental groups either with LCMV-Armor LCMV-Cl13. T cells from LCMV-Arm infected mice mounted a normal primary response regardless of β-catenin expression (Fig. 4a, left panel) consistent with our data using Listeria immunization (Figure 1). In addition β-catenin sufficient and deficient T cells showed a similar response and phenotype on day 8 after LCMV-Cl13 infection (Fig. 4a, right panel). Mice that were infected with LCMV-Arm generated a functional, stable T cell memory pool (Fig. 4b, left panel). T cells from mice infected with LCMV-Cl13 showed impaired cytokine production and thus signs of functional exhaustion, regardless of their genotype (Fig. 4b, right panel). Furthermore, antigen specific KO and het CD8 T cells expressed an equivalent amount of PD-1 on their surface (Fig. 4c). Together these data suggest that the lack of β-catenin does not result in any functional or proliferative changes in a situation of chronic T cell stimulation and exhaustion.
Finally, we addressed whether the reported generation of memory stem cells by a drug that inhibits GSK3β is truly β-catenin dependent (22). Regardless of the genotype of cells, we found that TWS119 blocked in vitro stimulated T cell proliferation starting at a dose that exceeded the specific target IC50 (30nM according to manufacturer) by 100-fold (3μM, Fig. 5 and Suppl. Figure 2), but had little to no effect if used at a lower dose(Suppl. Figure 2), an effect also apparent in the original paper. Treatment of T cells with a high dose of TWS119 prevented clustering of stimulated T cells so that only a fraction of cells were activated and divided (Fig. 5 and Suppl. Fig. 2). The previously reported “stem cell phenotype” of CD44 low, CD62L high TWS119 treated cells was found in the undivided cell population, but not in the divided cell population (Fig. 5). Importantly, this was the case regardless of β-catenin expression (Suppl. Fig. 2). The reported stem cell like features of these treated cells can be explained by the fact that the TWS119 treated cells contained a population that had been at least partially activated, and are thus superior to the naive control cells used in these experiments (22). Thus, our data support and extend the conclusions and concerns raised by Gajewsky and colleagues regarding TWS119 treatment and suggest that the data from Restifo’s group need to be reevaluated (14).
In summary, we used an in vivo loss-of-function system to address the role of β-cateninin various differentiation and self-renewal processes of mature T cells. We show that effector and memory T cell differentiation occurs unperturbed in the absence of β-catenin and argue against a model where β-catenin stabilization and translocation to the nucleus is required to direct memory T cell self-renewal and differentiation. While WT T cells responded to Wnt3a stimulation in vitro (Suppl. Fig. 1c)(15), the response of β-catenin deficient T cells to Wnt3a was abrogated suggesting that there is no compensation mechanism by other catenin family members in place. Why would memory T cells have the signaling machinery in place but not use it to regulate self-renewal? HSCs are located in specialized niches, while effector and memory T cells can be found in lymphoid and non-lymphoid tissues, including fat tissue. Recent data suggest that HSC renewal is specifically regulated by one Wnt family member, Wnt3a, in a non-redundant manner (27). A Wnt-independent way of self-renewal might allow for equal memory T cell development in all tissues regardless of Wnt availability, a feature not necessary for HSCs which are located in specialized niches of the bone marrow. While it is clear that T cells can respond to Wnt-mediated signals in vitro (15, 24) (Suppl. Fig. 1c), further experiments will be required to determine if there are physiological settings in which Wnt signaling participates in regulating aspects of T cell fate or function in vivo, similarly to what has recently been reported in DCs(28).
This work was supported by NIH grants AI 019335 (to M.J.B.) and AI 079159 (to M.P.) and the Howard Hughes Medical Institute(to M.J.B.) and AI 079159 (to M.P.).