Anti cd3 28 stimulation beads

OT1 CD8+ T-cells were activated and expanded using the H-2Kb-restricted OVA MHC class I epitope (OVA_257–264; SIINFEKL), according to previously published methods.^{[19 (link)]} Antigen-experienced T-cells were viably frozen and subsequently thawed for experiments, allowing cells to recover overnight. Cells were incubated for 6 d with 100 IU mL⁻¹ recombinant murine IL-2, anti-CD3/28 stimulation beads according to manufacturer protocol (Miltenyi, 130-095-925), and 1 µg mL⁻¹ recombinant murine PD-L1 protein (R&D systems, 1019-B7), with media and reagents changed out every 48 h. On day 7, T-cells were coincubated with B16-OVA tumor cells at a 10:1 effector to target ratio in 100 µL media for 3–6 h, along with 250, 50, 5, 0.5, or 0 µg of free aPD-1/aOX40 or equivalent dose of DINP. After coincubation, nonadherent T-cells were transferred onto an anti-IFN-γ coated ELISpot plate (BD, 551083) and incubated for 72 h before read-out, according to manufacturer protocol. Briefly, ELISpot plates were read out on an AID ELISpot reader (min. size 15, min. gradient > 1), reporting both absolute count and well activity. Well activity is an AID manufacturer-defined measurement of total cytokine release, dependent upon both absolute count and spot signal intensity. Activity values were normalized to the untreated control group.

OT1 CD8+ T-cells were activated and expanded using the H-2Kb-restricted OVA MHC class I epitope (OVA_257–264; SIINFEKL), according to previously published methods.^{[19 (link)]} Antigen-experienced T-cells were viably frozen and subsequently thawed for experiments, allowing cells to recover overnight. Cells were incubated for 6 d with 100 IU mL⁻¹ recombinant murine IL-2, anti-CD3/28 stimulation beads according to manufacturer protocol (Miltenyi, 130-095-925), and 1 µg mL⁻¹ recombinant murine PD-L1 protein (R&D systems, 1019-B7), with media and reagents changed out every 48 h. On day 7, T-cells were coincubated with B16-OVA tumor cells at a 0.25:1 effector to target ratio in 100 µL media for 48 h, along with 250, 50, 5, 0.5, or 0 µg of free aPD-1/aOX40 or equivalent dose of DINP. After coincubation, nonadherent cells were washed thrice from the plate, and remaining cell viability was measured with a CellTiter-Glo Luminescence kit (Promega, G7570), according to manufacturer protocol.