ACT and tumor immunotherapy

We implanted C57BL/6 with subcutaneous B16-OVA melanoma (1 × 10⁶ cells). At the time of ACT, 10 days after tumor implantation, mice (n ≥ 5 for all groups) were sub-lethally irradiated (500 cGy, XStrahl CIX2), randomized, and injected intravenously with a total of 2 × 10⁶ OT-1 Ly5.1 cells electroporated with scrambled sgRNA or Atp1a1-sgRNA. A third group was injected intravenously with 100 μL of PBS as a nontreatment control. Mice used to assess tumor-infiltrating lymphocytes (TIL) 5 to 7 days after transfer were intravenously injected with a mixture containing a total of 6 × 10⁵ OT-1 (3 × 10⁵ of Scramble/Atp1a1 sgRNA treated each) cells total. Both cohorts received intraperitoneal injections of rhIL2 in PBS (5 × 10⁴ IU in 0.5 mL) once daily for 2 days starting on the day of cell transfer. Mice supplemented with NAC were additionally administered with intraperitoneal injections of NAC at a dosage of 0.25 mg/g once daily until the study's endpoint. NAC intraperitoneal injections were prepared in PBS, neutralized to pH = 7.2–7.8, and sterile-filtered before administration. For longitudinal tumor curve studies, tumors were measured using digital calipers every 2–3 days for 28 – 35 days. Size was measured in a blinded fashion approximately every two days after transfer, and tumor area was calculated as length × width of the tumor. Mice with tumors greater than 400 mm² were euthanized. The products of the perpendicular tumor diameters are presented as mean ± s.e.m. at the indicated times after ACT.

For administration of Rag2^−/−CD45.2⁺ OT-1 T_N and VV-OVA to CD45.1⁺, CD45.2⁺ T_N cells were immediately prepared for transfer after FACS purification at a dosage of a total of 5×10⁴ cells per CD45.1⁺ recipient. These donor cells were administered via intravenous injection.