Il 2 212 12

Spleens were isolated from mice at 13-14 days post B16F10 cell transplant or 7-8 days post-cryoablation. Spleens were mashed, subjected to RBC lysis, and single cell suspensions were prepared in a T-cell media comprising RPMI media (61870-010, gibco) with 10% FBS, 1% pencillin-streptomycin, 1% minimal essential media (11140-035, gibco), 1% sodium pyruvate (11360-039, gibco), 1%HEPES (15630-056, gibco) and 0.1% beta-mercapitoethanol (11140-035, gibco). Splenocytes were cultured at 5% CO₂ and 37°C. At day 3 and 6 of culture, 300units/ml of IL-2 (212-12, peprotech) was added to stimulate the proliferation of T-cells. On day 8, cultured cells were harvested and subjected to CD8⁺ T-cell enrichment by CD8 T-cell isolation kit (130-104-075, Miltenyi Biotec). CD8⁺ T-cells were seeded into 96-well Interferon-γ (IFN-γ) elispot plates at 200,000 cells/well. Murine trp-2 peptide (SVYDFFVWL, LICR) of 0.2µg was added to each well as a tumor antigen peptide. For the control wells, ovarian ID-8 tumor derived peptide (YACENGQTDV, LICR) was added as an irrelevant peptide. Freshly isolated and irradiated splenocytes of 10,000 cells/well were added as a source of antigen presenting cells. Elispot assay was performed as per the manufacturer’s instructions (3321-4HST-10, MABTECH).

CD4⁺ T cells were isolated from spleens of FoxP3⁺GFP⁺ (B6) mice and magnetically enriched for CD4⁺ T cells through magnetic isolation (EasySep Mouse T cell Isolation kit, STEMCELL Technologies) with anti-CD8a biotin (catalog 13-0081-82, clone 53–6.7, Invitrogen). FoxP3⁺GFP⁺ cells were sorted using a MACSQuant Tyto cell sorter to a purity of greater than 99%. APCs were isolated from WT B6 splenocytes and irradiated at 30 Gy. The sorted Tregs were then stained with CellTrace Violet (C34571, Invitrogen) and plated with irradiated APCs, soluble anti-CD3 (14-0031-85, eBioscience), and IL-2 (212-12, PeproTech) with or without erdafitinib in the cell culture. Cells were cultured for 3 days, stained with Zombie NIR (423105, BioLegend) and CD4-PE antibody (catalog 12-0042-82, clone RM4-5, Invitrogen), and FACS analyzed.