Constitutive activation of Wnt signaling favors generation of memory CD8 T cells

Abstract

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ralpha and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized beta-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized beta-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

Figure 1

Dynamic regulation of Wnt pathway-related genes during CD8 T cell responses. Naïve, effector (day 5 p.i.), and memory (day 135 p.i.) OT-I CD8 T cells were separated as detailed in “Materials and Methods”. Total RNA was extracted and analyzed for expression of indicated transcripts using quantitative PCR. The expression level of each transcript in naïve T cells was arbitrarily set to 1, and its relatively expression in effector and memory T cells was shown. Fold repression in effector T cells compared with naïve cells (italicized) and fold induction in memory T cells compared with effector cells were shown on top of the bar graphs. The data are means ± s.e.m. of triplicate measurements of a total of 3 samples from 2 independent experiments.

Figure 2

Constitutive activation of Wnt signaling favors formation of memory CD8 T cells. (A) and (D) WT, βCat-Tg, p45-Tg, and dTg mice were infected with actA^-LM-Ova and the CD8 T cell response to Ova_257-264 was monitored in the spleen at day 7 (A) and day 42 (D) post-infection using intracellular cytokine staining for IFN-γ. The percentages of IFN-γ⁺ CD8 cells are shown in the contour plots in the absence (shown for A) or presence of Ova peptide stimulation. IFN-γ⁺ cells were further analyzed for IL-2⁺, CD62L^high, IL-7Rα⁺, or CD27^high subsets, and their percentages were shown. Staining with isotype control for CD62L and IL-7Rα was displayed as dotted line in histograms showing IL-7Rα staining. Data are representative of 2 independent experiments with at least 3 mice examined in each experiment. (B) Frequency of IFN-γ⁺ cells in CD8 and (C) frequency of CD8 cells in splenocytes on day 7 post-infection. (E) Frequency of IFN-γ⁺ cells in CD8 and (F) frequency of CD8 cells in splenocytes on day 42 post-infection. Fold changes of mean values for each transgenic strain versus WT mice are shown for (B-C) and (E-F). (G) Kinetics of Ova-specific CD8 T cell responses shown as total numbers at indicated time points. Data are means ± s.e.m. in one of the two independent experiments with similar results (n = 3). (H) Survival rate of Ova-specific CD8 T cells at contraction and memory phases. The mean value of Ova-specific CD8 T cell numbers at days 14 and 42 (as in G) was normalized to respective peak response on day 7. The percentage of survived cells is shown. *, p<0.05; **, p<0.01 by t-test for each group of Tg mice versus WT controls.

Figure 3

Memory CD8 T cells generated in the presence of constitutively active Wnt signaling manifested enhanced functionality. (A) Frequency and (B) numbers of IL-2 producing Ova-specific memory CD8 T cells. The numbers were calculated from the frequency of IL-2⁺IFN-γ⁺ cells (A) and the absolute number of Ova-specific CD8 T cells on day 42 post-infection as in Fig. 2G. (C) and (D) Secondary antigen-specific CD8 T cell responses. Mice of indicated genotypes were first immunized with act^-LM-Ova as in Fig. 2, and detection of Ova-specific CD8 T cells on day 42 post-immunization was confirmed in periphery blood leukocytes by intracellular staining for IFN-γ (data not shown). The immunized mice were then infected with virulent LM-Ova, and CD8 responses to Ova were determined. Ova_257-264-specific CD8 T cells at day 3 and day 42 after secondary infection were detected as Thy1.2⁺IFN-γ⁺ CD8 cells, with the percentages shown in (C). Total numbers of antigen-specific CD8 T cells per spleen in each group are shown in (D) as means ± s.e.m. (E) and (F) Clearance of secondary bacterial infection by primary CD8 memory T cells. Naïve or immunized mice were infected with virulent LM-Ova and 3 days later, livers and spleens were harvested and CFUs were determined. Data are reported as CFU numbers per gram of liver (E) or per spleen (F). LOD, limit of detection. Each symbol represents one mouse. All data are from one of 2 independent experiments with similar results. *, p<0.05; **, p<0.01 by t-test for each group of Tg mice versus WT controls.

Figure 4

Detection of BrdU uptake and caspase activation in antigen-specific effector CD8 T cells. (A) Proliferation rate of effector CD8 T cells at early contraction phase. WT and dTg mice were infected with actA^-LM-Ova, and given BrdU on day 7 via intraperitoneal injection and in drinking water for 2 days. Percentages of BrdU⁺ cells in IFN-γ⁺ CD8 effectors are shown. (B) Activation of caspase-3/7 in effector CD8 T cells at early contraction phase. Splenocytes were isolated from WT and dTg mice on day 9 post-infection, and activated Caspase-3/7 were detected using the FLICA methodology. Percentages of cells having activated Caspase-3/7 are shown. Representative flow cytometric profiles were shown on upper panels and accumulative data from 2 independent experiments are shown in lower panels. **, p<0.01 by t-test.

Figure 5

Constitutive activation of Wnt signaling enhances memory CD8 T cell formation in response to viral infection. (A) Ova-specific memory CD8 T cells after VacV-Ova infection. WT and dTg mice were i.p. infected with vaccinia virus expressing Ovalbumin, and the Ova-specific CD8 T cells on day 42 post-infection were determined by intracellular staining for IFN-γ. (B) Antigen-specific memory CD8 T cells after LCMV infection. WT and dTg mice were i.p. infected with LCMV-Armstrong, and 42 days later memory CD8 T cells specific for dominant epitopes GP_33-41, NP_396-404, GP_276-286, and subdominant epitopes NP_205-212 were determined by ICS for IFN-γ. Fold changes of mean values were shown. *, p<0.05; **, p<0.01 by t-test.

Figure 6

Constitutively active Wnt signaling increases primary but not secondary memory CD4 T cells. Mice were infected as in Fig. 2, and the CD4 responses to LLO_190-201 were determined on various days post-infection. (A) and (D) Detection and characterization of LLO_190-201-specific CD4 T cells at day 7 (A) and day 42 (D) post-infection. The percentages of Thy1.2⁺IFN-γ⁺ CD4 cells are shown in the contour plots in the absence (shown for A) or presence of LLO peptide stimulation. IFN-γ⁺ CD4 cells were further analyzed for IL-2⁺, CD62L^high, IL-7Rα⁺, or CD27^high subsets in memory CD4 cells, and their percentages were shown. Staining with isotype control for CD62L and IL-7Rα was displayed as dotted line in histograms showing IL-7Rα staining. Data are representative of 2 independent experiments with at least 3 mice examined in each experiment. (B) Frequency of IFN-γ⁺ cells in CD4 and (C) frequency of CD4 cells in splenocytes on day 7 post-infection. (E) Frequency of IFN-γ⁺ cells in CD4 and (F) frequency of CD4 cells in splenocytes on day 42 post-infection. Fold changes of mean values for each transgenic strain versus WT mice are shown for (B-C) and (E-F). (G) Kinetics of LLO-specific CD4 T cell responses shown as total numbers at indicated time points. (H) Secondary antigen-specific CD4 T cell responses. Mice were first infected with act^-LM-Ova and 45 days later re-challenged with virulent LM-Ova as in Fig. 3C. CD4 responses to LLO_190-201 were determined on days 3, 5, 14 and 42 after the secondary infection. Total numbers of antigen-specific CD4 T cells per spleen in each group are shown as means ± s.e.m. Data are representative of 2 independent experiments with at least 3 mice examined in each experiment. *, p<0.05; **, p<0.01 by t-test for each group of Tg mice versus WT controls.