NRG mice were inoculated i.d. with CMS5 cells 7 days prior to adoptive transfer of DUC18 Tcm (10⁶ cells/mouse). One day after Tcm transfer, mice were vaccinated i.v. with VSV-ErkM (2 × 10⁸ PFU/mouse). Mice receiving VSV-ErkM alone, Tcm alone, or PBS were included as controls. (A) Tumor volume and (B) survival of treated mice is shown at the indicated dpt. (C) The numbers of transferred (Thy1.1⁺) CD8⁺ T cells in the peripheral blood of TB and TF NRG mice on days 3, 5, 12, and 19 after combination therapy were determined by flow analysis. WT mice surviving initial CMS5 tumor challenge after treatment with Tcm plus VSV-ErkM were rechallenged with CMS5 relapse (CMS5r) cells 60 days later, and subsequent tumor growth (D) and survival (E) are shown. Naive mice that received CMS5 were included as controls. Data are shown as representative results of 3 (A and B) or 2 (C–E) independent experiments with n = 5 per group. Data were analyzed using repeated measures 2-way ANOVA with Holm-Šidák correction for multiple comparisons (C) and log-rank (Mantel-Cox) test (B and E). *P < 0.05; **P < 0.01. (F) Schematic of the PCR product resulting from genomic DNA amplification of the ERK2 gene is shown with the SfcI recognition sequence generated by the ErkM mutation displayed in uppercase letters. Expected fragments generated from SfcI digestion of PCR amplicons from WT ERK (356bp bands for ErKwt) and mutant ERK alleles (260 and 96 bp bands for ErKM) are shown with dotted brackets. (G) Restriction digestion of PCR products amplified from CT26 (negative control), CMS5, and CMS5r cell line genomic DNA is shown as well as (H) chromatogram of sequencing result from PCR products.