Fig. 5: Effects of forced CD39 expression in a CD39⁻ T-cell line on ATP metabolism and antitumor immunity in vivo.

The CD39⁻ murine T lymphoblast cell line EG7-OVA was infected with a retrovirus vector expressing murine CD39 or mock, including a GFP-expressing site. a As in Fig. 3, after culture for the indicated number of hours, ATP consumption was measured in mock or CD39 transfected EG7-OVA cells. Bar heights indicate the mean ± SD of the % ratio of remaining ATP relative to the supplemented amount, from triplicate experiments. **P < 0.01, Student’s t test, compared with the value of mock transfected EG7-OVA. b Upper left panel: The experimental scheme of the tumorigenesis assay. We subcutaneously transplanted mock infected EG7-OVA cells (2 × 10⁶) into the left flanks of C57B/6 J mice, and the same amount of CD39-transfected EG7-OVA cells into the right flanks. On posttransplantation days 3 and 6 the mice were intraperitoneally injected with Poly(I:C) or PBS. Tumor sizes were observed from posttransplantation day 6.Upper right panel: Photographic image of typical tumors on Day 13. Tumors in the left flanks are from transplanted CD39⁻ EG7-OVA cells and those in the right flanks from transplanted CD39⁺ cells. PBS was administered to the left mouse and Poly(I:C) was administered to the right. Lower panels: Line chart indicates volumes of mock infected tumors (left panel; left flanks) and CD39⁺ tumors (right panel; right flanks). Dotted and continuous lines, respectively, indicate the tumor volumes in PBS-injected mice and Poly(I:C)-injected. The results are expressed as the mean ± SD from six mice. *P < 0.05 and **P < 0.01, Student’s t test comparing the values in PBS-injected and Poly(I:C)-injected mice. c Left panels: Immunostaining of each tumor sample. CD8 positive cells were stained with Alexa Fluor®594 and cell nuclei with DAPI. Yellow arrows indicate CD8⁺ cells. Green lines show the tumor borders. Right panels: CD8⁺ cells observed in each tumor sample. Plot shows the proportion of CD8⁺ cells to DAPI-stained cells in the boundary areas of tumors in each group of mice. Cell numbers were counted in each field of view. Horizontal bars indicate median values. P < 0.001.