In Vitro and In Vivo Killing of OVA-Expressing E0771 Cells

Control and ICAM-1 KO E0771 cells were stably transduced with an OVA-mCherry-encoding construct cloned in the pHR OVA/p2a/mCherry-CaaX lentiviral vector (Addgene, cat. 113030). Cells expressing the mCherry reporter were sorted and their expression of the OVA SIINFEKL peptide complexed with cell surface MHC-I (H-2Kb) was measured by 25-D1.16 mAb staining (32)). Control or ICAM-1 KO was seeded in a 96-well plate and cultured O.N. OT-1 effector CTLs were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and overlaid on the tumor cells at different effector to target ratios. One day later, the number of viable E0771 cancer cells was determined by FACS. For in vivo killing analysis, 10³ control or ICAM-1 KO E0771 cells (suspended in Matrigel, 1:1 in PBS) were implanted in the mammary fat pad. Approximately 3 days later, 10⁷ effector OT-I CTLs were injected intravenous (i.v) and the tumor size was measured 12, 14, and 16 days later.