Tumor/Endothelium Crosstalk Attracts T Cells Figure 4. Supernatant from medulloblastoma and brain endothelial cell coculture induces avid T lymphocyte migration in vitro through the induction of RANTES. (A) Semiquantitative measurement of chemokines using protein profiler membranes showed that while RANTES was near undetectable in supernatant from bEND.3 or in supernatant from Daoy grown for 6 days, its secretion substantially increased as early as 48 hours of Daoy and bEND.3 coculture and rapidly increased over time to plateau after 6 days. (B) ELISA for murine RANTES quantitatively confirmed these results and determined that RANTES is endothelium- rather than tumor-derived. (C) Supernatants from Daoy and b.END.3 coculture system were used to test T lymphocyte migration in transwell systems. In contrast to supernatants from tumor or that from brain endothelium cells, supernatants of tumor and endothelium cocultures induced avid migration of T lymphocytes. (D) Using 1,000 ng/mL and 10,000 ng/mL of RANTES blocking Ab decreased T lymphocyte migration by 30 and 60 %, respectively (p = 0.03 and 0.007, respectively) confirming the dominant role of RANTES in triggering T lymphocyte migration. Blocking Ab to IL8 and MIF up to 10,000 ng/mL failed to counteract the migration to Daoy and bEND.3 coculture supernatants. doi:10.1371/journal.pone.0020267.g004 dependent since it was preserved when tumor and endothelial cells were separated by a 0.2 mm pore membrane. Other chemokines/chemo-attractant molecules that were present in low concentrations but progressively increased in coculture included C5-a, MCP-1, Gro-a, sICAM-1, MIF and IL-8. These molecules, unlike RANTES, were also present at low concentra- PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20267