Negative selection magnetic beads

After 6 days in culture, BMDCs (50,000/well) were pulsed overnight with 10-1,000ng/ml NP68 peptide (ASNENMDAM, H-2D^b) or control H-Y peptide (WMHHNMDLI, H-2D^b) in 24 well-plates. The next day, CD8a+ cells were purified from the spleens of F5 TCR.Tg mice using negative selection magnetic beads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. Isolated F5 CD8+ cells were added to the 24-well plates at 10,000 cells/well. 10 μg/ml of HuIgG1 or avelumab was also added at this time, together with 0-1,000 pg/ml NHS-muIL12 in a final volume of 1ml/well. After 5 days of in vitro T cell activation, supernatant samples were collected and stored at -20°C. Supernatant IFN-γ concentrations were later determined using a standard ELISA kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Sample optical densities at 450nm were measured using a Synergy HT plate reader (BioTek, Winooski, VT). Sample IFN-γ concentrations were extrapolated from a standard curve.

Splenocytes from female CD45.1+ OT-1 mice were activated with 1 μM SIINFEKL peptide and treated with vehicle (water), 0.25 mM aminoxyacetic acid (AOA, Sigma), 1 mM oxaloacetate (OAA, Sigma) or 0.25 mM AOA + 1 mM OAA in the presence of IL-2 (100 units/ml). After 48 hours, CD8+ OT-1 T cells were purified using magnetic negative selection beads (Miltenyi) and cultured for 5 further days with vehicle (water), 0.25 mM aminoxyacetic acid (AOA, Sigma), 1 mM oxaloacetate (OAA, Sigma) or 0.25 mM AOA + 1 mM OAA in the presence of IL-2 (100 units/ml). On day 7, one million OT-1 T cells were transferred intraperitoneally into tumor-bearing CD45.2+ C57BL/6J mice that had been subcutaneously injected with 500,000 B16 F10 OVA cells 7 days prior and then four days after that received an intraperitoneal injection of 300 mg/kg cyclophosphamide to achieve lymphodepletion. The expansion of OT-1 T cells in peripheral blood was evaluated from tail vein blood samples taken 7 days following OT-1 transfer. Tumor growth was evaluated until a humane endpoint was reached. Tumors were measured blindly every 2-3 days (daily evaluations during injections) with digital calipers and tumor volume was calculated using the formula: 0.5 x (a₂ x b) where a is the width and b is the length of the tumor.