CD8 T cells activated within tumors acquire effector function. (A and B) Ex vivo analysis of tumor-specific T cell function. Mice bearing established B16-cOVA, B16-AAD, or LLC-OVA in the indicated locations received CFSE-labeled Thy1-mismatched OT-I or FH cells. FTY720 treatment was initiated at the time of T cell transfer and continued for the duration of the experiment. 4 (A) or 8 (B) d after T cell transfer, tumors were harvested and stained for flow cytometry after a brief ex vivo restimulation. Plots are gated on tumor-specific Thy1.1⁺ CD8⁺ OT-I cells. Numbers on plots indicate the total percentage of OT-I cells that are single positive for IFN-γ or CD107a or double positive for both markers. Axes on plots are displayed in Logicle scale. Graphs summarize the composition of the antitumor effector response (A) and compare the antitumor response at 4 and 8 d after T cell transfer (B). Results represent at least two independent experiments with at least three mice per tumor type. (C) Analysis of in vivo IFN-γ production by tumor-specific T cells. C57BL/6 mice bearing established i.p. B16-cOVA tumors were given CFSE-labeled OT-I cells. FTY720 treatment was initiated at the time of T cell transfer and continued for the duration of the experiment 4 or 8 d after T cell transfer, mice were treated with BFA, and 4 h later tumors were harvested and stained for flow cytometry. Dot plots are gated on Thy1.1⁺ CD8⁺ OT-I cells. Numbers on plots indicate the percentage of OT-I cells that are divided (CFSE negative) and IFN-γ⁺. Axes on plots are displayed in Logicle scale. Graph summarizes in vivo IFN-γ production and the number of OT-I cells in the tumor. Results represent 2 independent experiments, with n = 3 mice at each time point. Statistical analyses in (A and B) were performed using a two-tailed Student’s T test (Prism version 5.0; GraphPad Software, Inc.). (D) Granzyme B expression. Lymphocytes were isolated from s.c. tumors and tumor-draining LN of FTY720-treated animals 4 d after initial naive cell transfer. Gray histogram indicates isotype control staining and black histogram indicates staining on OT-I T cells. Histograms are gated on Thy1.1⁺ CD8⁺ lymphocytes. Axes on plots are displayed in Logicle scale. Graph summarizes the percentage of divided OT-I that express Granzyme B when activated in the tumor or the draining LN. Staining was performed with n = 6 mice in 1 experiment. (E) In vitro cytotoxicity of intratumorally primed OT-I cells. Thy1.1⁺ OT-I cells were isolated from i.p. tumors and tumor-draining LN of FTY720-treated animals 4 d after initial naive cell transfer, and then incubated with a mix of CFSE-labeled peptide-pulsed and unpulsed targets. Histograms are gated on CFSE-labeled target cells. Numbers on histograms represent the percentage of target cells that are CFSE^lo or CFSE^hi. Axes on plots are displayed in Logicle scale. Results represent pooling of OT-I cells from eight tumors and eight tumor-draining LN in each of three independent experiments.