Nrf2 is dispensable for donor CD8⁺T-cell cytotoxicity and GVT capacity. (A) In vitro cytotoxicity assay. WT and Nrf2^−/− CD8⁺ T cells isolated from steady-state spleens were activated with anti-CD3 and anti-CD28 in vitro for 72 hours, followed by incubation with ⁵¹Cr-labeled allogeneic A20 tumor target cells for 18 or 19 hours (n = 2 independent experiments). (B) In vivo cytotoxicity assay. Lethally irradiated BALB/c recipients were transplanted with WT B6 TCD BM and a total of 0.5 × 10⁶ T cells, composed of B6 WT CD4⁺ cells mixed with WT or Nrf2^−/− CD8⁺ T cells at a 2:1 ratio. On day 7, recipients were challenged with a 1:1 infusion of CFSE-labeled BALB/c allogeneic and PKH26-labeled B6 syngeneic control targets. Elimination of targets was assessed in the spleen by flow cytometry 4 hours after challenge (n = 3 in BM group, n = 9 per group in BM + WT or Nrf2^−/− T groups; 2 independent experiments). (C) Lethally irradiated BALB/c recipients were transplanted with WT B6 TCD BM alone or with a total of 0.5 × 10⁶ T cells, composed of B6 WT or Nrf2^−/− CD4⁺ and CD8⁺ T cells mixed at a 2:1 ratio. Recipients were monitored daily for mortality (n = 20 per group; 2 independent experiments). (D-E) Lethally irradiated BALB/c recipients transplanted with WT B6 TCD BM alone or with 0.5 × 10⁶ WT or Nrf2^−/− T cells were challenged with 0.5 × 10⁶ A20-TGL cells. The whole-body distribution of tumor burden was tracked weekly using bioluminescent signal intensity. (D) Representative bioluminescence images showing pseudocolor images superimposed on conventional photographs. (E) Tumor burden in mice quantified as bioluminescence image flux. Data represent mean ± SEM from 2 independent experiments (n = 10 in BM group, n = 18 to 20 each in BM + WT or Nrf2^−/− T groups). (F) Lethally irradiated BALB/c recipients transplanted with WT B6 TCD BM alone or with 0.5 × 10⁶ WT or Nrf2^−/− T cells were challenged with 0.5 × 10⁶ A20-TGL cells. Recipients were monitored daily for mortality (n = 15 in BM group, n = 25 each in BM + WT or Nrf2^−/− T groups; 3 independent experiments). *P < .05, **P < .01. n.s., not significant.