Single cell suspensions from spleens and lymph nodes were prepared as described.6 (link) TIL were isolated from pooled tumors as described.26 (link) All flow cytometry experiments were performed at least 3 times. Single cell suspensions were incubated with mouse Fc receptor binding inhibitor for 10 minutes before staining with antibodies to CD45 (clone 30-F11), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD19 (clone eBio1D3), CD86 (clone GL1), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), CD69 (clone H1.2F3), CD44 (clone IM9), CD62L (clone MEL-14), and CD11c (clone N418; all from eBioscience, San Diego, CA) for 30 minutes. Flow cytometry was performed using FACS Calibur (BD Biosciences) and the lymphocyte population was selected by gating CD45+ cells. The data were analyzed using Flow Jo software (Tree Star, Ashland, OR). For tetramer staining, 10 μL of PE labeled HLA-A*02:01 Human HPV16 E7 tetramer (NIH Tetramer Core Facility) was added to 200 μL mouse lymphocyte suspension (1×106 cells per tube). After incubation for 30 minutes, cells were centrifuged and resuspended in phosphate-buffered saline with 1% paraformaldehyde and then analyzed by flow cytometry. PE labeled HLA-A*02:01 human mesothelin tetramer (NIH Tetramer Core Facility) was used as control.