Figure 3.
CXCL12 excludes CD8+ T cells. A) Isolated primary CD8+ T cells were cocultured with SW480 (control or CXCL12 overexpressed) for 3 h, and T cell migration was analyzed (lower right panel). B) The lower chambers of transwell were treated with 0, 50, and 1000 ng mL−1 rhCXCL12 and cultured with isolated primary naïve or CD3/CD28 activated CD8+ T cells for 3 h, T cell migration was subsequently analyzed. C) Jurkat T cells were cultured with the CM from SW480 cells (control or CXCL12 overexpressing cells) for 3 h and then T cell migration was analyzed. D) Isolated primary CD8+ T cells were cocultured with SW480 (control, ROS treated, CXCL12 overexpressing or ROS/shCXCL12) for 3 h and then T cell migration was analyzed. E) Ex vivo culture. p16INK4A positive CRC tissues were cocultured with GFP lentivirus infected isolated primary CD8+ T cells in media containing CXCL12 neutralizing antibody for 24 h and then stained for p16INK4A and GFP. The number of GFP positive cells was counted, and the results are presented as a bar graph. F and G) MC38 (control or mCXCL12 overexpressing) or CT26 (control or mCXCL12 overexpressing) mouse colon cancer cells were transplanted into 7 week old C57BL/6 or BALB/c mice, respectively. The mice were euthanized after 3 weeks, and the tumor size was analyzed. The tumor growth is presented as line graph. Results are presented as mean ± SD. Tumor tissues were stained with CXCL12 or CD8 antibodies. The arrow indicates the CD8+ cells. Mouse CD8 mRNA expression was analyzed and presented as dot graph. The infiltration of CD8+ T cells in tumors was analyzed (200×). Three randomly selected areas of the tumor tissue per animal were photographed and analyzed for CD8+ T cell infiltration; the results were averaged and then presented as a dot graph. In vitro cell proliferation assay. MC38 (control or mCXCL12 overexpressing) or CT26 (control or mCXCL12 overexpressing) cells were cultured, and the cell number was analyzed. Results are presented as mean ± SD. The p value was calculated by the Mann–Whitney U test (A, C, E, F, and G) or Kruskall–Wallis test (A, B, and D). N and n indicated the number of cases and independent experiments, respectively.