Figure 2.

OT-I cells found in tumors 24 h after transfer were activated in situ and are localized within tumor parenchyma. (A) C57BL/6 mice bearing established B16-F1 lung tumors were challenged with B16-cOVA via the lateral tail vein, followed 24 h later by Thy1-mismatched OT-I cells. Lungs/tumors, LN, and spleen were harvested 24 h later, and lymphocytes were stained for flow cytometry. Results represent three independent experiments. n = 3. (B) C57BL/6 mice lacking LN after in utero ablation were splenectomized and injected s.c. with B16-cOVA 3 wk later. After tumors were established, mice were injected with Thy1.1⁺ OT-I cells. Tumors and indicated tissues were harvested 24 h later, and lymphocytes were stained for flow cytometry. Results are an example of n = 4 animals in 2 independent experiments. All plots in (A and B) are gated on Thy1.1⁺ CD8⁺ OT-I lymphocytes, and numbers indicate the percentage of OT- I cells that are single positive for CD69 or CD25, or positive for both markers. Axes on plots are displayed in Logicle scale. (C and D) Naive OT-I T cells were transferred into mice C57BL/6 mice bearing established lung (C) or s.c. (D) B16-cOVA tumors. Localization of OT-I cells within tumors was visualized via their Thy1.1 staining and CD31 staining on tumor vasculature. Images are representative of multiple fields and magnifications of three lung and s.c. tumors in three independent experiments. Bars, 200 µm.