Fig. 2: Superior efficacy of MCJ-deficient TCR-antigen specific CD8 cells for the treatment of melanoma in vivo.
a B16-OVA tumor cells (4 × 105/mouse) were s.c. injected on the flank to WT mice (n = 7 biologically independent animals per group). WT and MCJ KO OT-I CD8 cells were activated with anti-CD3/anti-CD28 Abs as in Fig. 1, expanded with IL-2 for two expansions, and (5 × 105 cells/mouse) i.v. administered to the B16-OVA tumor-bearing mice 10 days post-implantation when the tumors were palpable. Tumor size was followed for 8 days (when a large fraction of mice needed to be euthanized based on tumor size), and it is presented as fold-increase relative to the size of the tumor at the time of the CD8 cell adoptive transfer. b Immunostaining for CD8 (red) and DAPI as nuclear marker (blue) of histological sections from B16-OVA tumors harvested 8 days after the adoptive transfer of WT or MCJ KO OT-I CD8 cells, as in (a). c–e WT and MCJ KO OT-I CD8 cells were activated and expanded as in (a), and injected into WT mice (n = 5 biologically independent animals per group) bearing B16-OVA melanoma tumors. Tumors were harvested 10 days after CD8 cell adoptive transfer and tumor cell homogenate was examined by flow cytometry for the presence of total CD8 cells (c) or OT-I CD8 cells (d) within the leukocyte (CD45+) population, as well as the expression of CD44 and PD1 on OT-I CD8 cells within the OT-I CD8 cells (e). f WT and MCJ KO OT-I CD8 cells were activated and expanded as in (a), and injected into WT mice (n = 4 biologically independent animals per group) bearing B16-OVA tumors. 7 days after CD8 cell adoptive transfer tumors were harvested to isolate tumor infiltrating CD8 cells. Pooled infiltrated CD8 cells were and activated ex vivo with anti-CD3/anti-CD28 Abs for 24 h and IFNγ levels in supernatant was examined by ELISA (n = 3 replicate wells per group). p was determined by 2-way ANOVA analysis (a) and two-sided unpaired t test (c–f). Mean ± SD is shown for (a, c–f).
