Figure 8. Radiation therapy and CD25-depleted adoptive cell transfer synergize with Gvax/αCTLA-4 to reject established tumors. To generate the DLIs, tumor-bearing mice were injected or not injected with anti-CD25 on day 4 after challenge with B16/BL6 melanoma, and then treated with Gvax/αCTLA-4 on days 8, 11, and 14. 19 d after tumor challenge, T cells were isolated from donor mice and injected into mice bearing 8-d-old tumors. All mice receiving DLI were also treated with radiation therapy (RT) 8 h before the DLI. (A–E) Recipient mice were killed 18 d after tumor challenge. (A–C) Spleens were analyzed for expression of CD8, CD4, and Foxp3 using flow cytometry. All recipient mice were treated with Gvax/αCTLA-4 as described in the Materials and methods section. The groups were as follow: no RT no DLI (recipients receiving only Gvax/αCTLA-4 without RT or DLI), +RT no DLI (recipients receiving RT without DLI and then Gvax/αCTLA-4), +RT+DLI (recipients receiving RT, DLI, and Gvax/αCTLA-4), and +RT+DLI (αCD25) (recipients receiving RT, DLI from CD25-depleted donors, and Gvax/αCTLA-4). The data are presented as number of CD8+ cells (A), number of CD4+Foxp3+ T cells (B), and ratio of CD8+ T cells to CD4+Foxp3+ T cells (C). In D, CD8+ T cells were purified from the different groups and restimulated in vitro with irradiated B16 melanoma or control TRAMP-C2 tumor cell lines for 24 h. IFN-γ production was determined by ELISPOT assay, as described in the Materials and methods section. (E) Tumors were harvested from mice in the different groups, and frozen sections were stained with anti-VCAM FITC (green), anti-CD31 PE (red), and anti-ICAM APC (magenta) in the top row. The bottom row shows tumor sections stained with anti-CD8 Alexa Fluor 488 (green) and anti-CD31 PE (red). All images were acquired with a 20× water immersion objective. Bar, 40 μm. (F and G) In a parallel set of experiments, tumor growth was followed over time. Whereas F shows tumor growth over time, G presents data as the percentage of mice surviving after tumor challenge and treatment. A–D shows cumulative data from 4 different experiments (n = 3 mice per group). In E, the data are representative of 4 independent experiments (n = 3 mice per group), and F and G are representative of 3 independent experiments (n = 10 mice per group).
Figure 8.

Radiation therapy and CD25-depleted adoptive cell transfer synergize with Gvax/αCTLA-4 to reject established tumors. To generate the DLIs, tumor-bearing mice were injected or not injected with anti-CD25 on day 4 after challenge with B16/BL6 melanoma, and then treated with Gvax/αCTLA-4 on days 8, 11, and 14. 19 d after tumor challenge, T cells were isolated from donor mice and injected into mice bearing 8-d-old tumors. All mice receiving DLI were also treated with radiation therapy (RT) 8 h before the DLI. (A–E) Recipient mice were killed 18 d after tumor challenge. (A–C) Spleens were analyzed for expression of CD8, CD4, and Foxp3 using flow cytometry. All recipient mice were treated with Gvax/αCTLA-4 as described in the Materials and methods section. The groups were as follow: no RT no DLI (recipients receiving only Gvax/αCTLA-4 without RT or DLI), +RT no DLI (recipients receiving RT without DLI and then Gvax/αCTLA-4), +RT+DLI (recipients receiving RT, DLI, and Gvax/αCTLA-4), and +RT+DLI (αCD25) (recipients receiving RT, DLI from CD25-depleted donors, and Gvax/αCTLA-4). The data are presented as number of CD8+ cells (A), number of CD4+Foxp3+ T cells (B), and ratio of CD8+ T cells to CD4+Foxp3+ T cells (C). In D, CD8+ T cells were purified from the different groups and restimulated in vitro with irradiated B16 melanoma or control TRAMP-C2 tumor cell lines for 24 h. IFN-γ production was determined by ELISPOT assay, as described in the Materials and methods section. (E) Tumors were harvested from mice in the different groups, and frozen sections were stained with anti-VCAM FITC (green), anti-CD31 PE (red), and anti-ICAM APC (magenta) in the top row. The bottom row shows tumor sections stained with anti-CD8 Alexa Fluor 488 (green) and anti-CD31 PE (red). All images were acquired with a 20× water immersion objective. Bar, 40 μm. (F and G) In a parallel set of experiments, tumor growth was followed over time. Whereas F shows tumor growth over time, G presents data as the percentage of mice surviving after tumor challenge and treatment. A–D shows cumulative data from 4 different experiments (n = 3 mice per group). In E, the data are representative of 4 independent experiments (n = 3 mice per group), and F and G are representative of 3 independent experiments (n = 10 mice per group).

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