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.

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.

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.

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.