Tumor models

Murine B16 melanoma cells expressing the surrogate tumor antigen chicken ovalbumin (B16-OVA) were cultured in DMEM supplemented with 10% FBS, and the tumor cells (5 × 10⁵) were injected s.c. into 8-week-old WT or Tbkbp1-KO mice. The challenged mice were monitored for tumor growth, and tumor size was expressed as tumor area. For survival rate calculation, mice with a tumor size reaching 225 mm² were considered lethal and sacrificed based on the protocol approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson. To minimize individual variations, age- and sex-matched (mostly littermate) WT and Tbkbp1-KO mice were used.

For depletion of CD8⁺ T cells, 8-week-old WT or Tbkbp1-KO mice challenged with murine B16 melanoma cells were administered i.p. with rat anti-mouse CD8α (clone 53–6.7) antibody or isotype control rat IgG2a (2A3) at the dose of 150 μg per mouse on days −2, 0, 2, 4, 6, 8, and 10. The challenged mice were monitored for tumor growth and sacrificed on day 12 to check the efficiency of CD8 T-cell depletion by flow cytometric analysis of draining lymph node cells.