a CD45.1 Pep Boy mice were randomized when EG7.OVA tumors reached ~230 mm3 (n = 8), treated with naïve OT-1 cells, and dosed with 2.5 × 108 mRBC-OVA-4-1BBL-IL-12. Seven out of eight mice cured of original EG7.OVA tumors and were rechallenged on day 66 with EG7.OVA. Age-matched naïve CD45.1 Pep Boy mice (n = 5) were treated on day 65 with 5 × 105 naïve OT-1 cells 1 day before challenge with EG7.OVA cells, as controls. b All previously cured mice rejected EG7.OVA rechallenge. c Representative flow cytometry plots showing OT-1 and endogenous OVA-specific T cells in 50 μL of peripheral blood 10 days after EG7.OVA rechallenge (Day 76). d OT-1 and e endogenous OVA-specific T-cell numbers in 50 μL peripheral blood 2 days before rechallenge (Day 64), 4 days post rechallenge (Day 70), and 10 days post rechallenge (Day 76). One-way ANOVA compared to day 64; OT-1: day 75 P = 0.0009; endogenous OVA-specific: day 75 P = 0.0079. Unpaired two-way Student’s t test compared to naïve; OT-1 day 75 P = 0.018; endogenous OVA-specific: day 75 P = 0.018. f At 61 days post-second EG7.OVA challenge on day 127, cured mice (n = 7) along with age-matched naïve control mice (n = 5) were challenged with EL4. Three out of seven cured mice had delayed EL4 growth and three out of seven rejected EL4. g TCRβ sequencing analyses of OT-1 frequency on days 65, 73, 126, and 136 in the blood. h The significantly expanded TCR clones after EL4 challenge were tracked throughout the tumor challenges. The log of the sum clone frequencies in individual mice is shown. Data are depicted as mean ± s.d. and are representative of two (b–f) or one (g, h) independent experiment. Source data are provided as a Source Data file.