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. 2012 Feb 7;3(2):144–157. doi: 10.18632/oncotarget.420

Figure 4. FTS decreases secretion of the immunosuppressive cytokine TGF-β from GL261 glioma cells and increases the proliferative and cytotoxic capacities of CTLs in vitro as well as accumulation of CTLs in the tumors in vivo.

Figure 4

(A) GL261 tumor cells were treated in vitro for 24 hours with FTS or CD8 or both, as described in Results. The cells were then washed thoroughly, and CFSE-labeled CD8+ T cells, (isolated from FTS- or vehicle-pretreated GL261 tumor-bearing mouse splenocytes) were added and cocultured with the FTS-pretreated GL261 cells for 96 hours. The rate of CTL proliferation was measured by flow cytometry. Statistical analysis of the results is presented as means ± SEM (n=8). ***, p<0.001 compared with vehicle-treated mice. (B) The experiment was performed as in A, except that the CD8+ T cells were now separated from the GL261 tumor cells by a transwell, preventing cell passage. The rate of CD8+ T cell proliferation was measured by flow cytometry. Statistical analysis of the results is presented as means ± SEM (n=8). ***, p<0.001 compared with vehicle-treated mice. (C) GL261 tumor cells were treated with the indicated doses of FTS or with vehicle (control) for 24 hours and then assayed for TGF-β (see Methods). The ELISA results are shown (means ± SEM, n=8). ***, p<0.001 compared with vehicle-treated control cells. (D) Isolated CD8+ T cells were cocultured with FTS-pretreated GL261 cells for 96 hours and their proliferation was analyzed, as described in Methods. Numbers of viable CTLs are presented as means ± SEM (n=8). *, p<0.05, ***, p<0.001 compared with vehicle-treated cells. (E-F) GL261 tumor cells were incubated with CFSE-labeled-CD8+ T cells, with or without TGF-β-blocking anti-TGF-β Ab, for 96 hours. The rate of CD8+ T cell proliferation was measured by flow cytometry (E), and viable GL261 cells were counted (F). Statistical analysis of the results is presented as means ± SEM (n=5). ***, p<0.001 compared with cells not treated with anti-TGF-β Ab. (G) C57bl/6 mice implanted s.c. with GL261 tumor cells were divided into two groups for treatment with FTS (n=10) or vehicle (n=10), as described in Methods. The mice were killed 21 days after the cells were implanted and their tumors and spleens were assayed for CD8+ T cells by flow cytometry (left and middle). Statistical analysis of the flow cytometry results (n=10) is presented (right). *, p<0.05 compared with vehicle-treated controls.