tions in Daoy only supernatants and increased to higher levels in proportion to the degree of confluency of the Daoy cells. Next, we determined the source of RANTES in the tumor/ endothelium coculture. At the protein level, murine RANTES shows 88% homology with human RANTES and exhibits species cross reactivity on human and mouse cells. [33,34] The RANTES probe in our chemokine protein multiplex assay cross reacted with both human and murine RANTES. Therefore we determined whether the RANTES we detected was human- (tumor cell) or murine- (endothelial cell) derived by re-measuring RANTES levels using a murine-specific ELISA. Our species-specific assay showed high activity of murine endothelium-derived protein but not of its human counterpart (Figure 4B). To test if the signal derived from the tumor–endothelial coculture system attracts T lymphocytes: we collected these supernatants daily and used them to test T lymphocyte migration in a transwell system. Culture supernatants from tumor or endothelial cells alone grown for six days induced minimal migration of T lymphocytes. By contrast, we observed avid migration of T lymphocytes when we used supernatants from the combination of tumor and endothelial cells. (Figure 4C) The degree of migration paralleled the concentration of RANTES protein in the supernatants, becoming detectable after 48 hours of coculture, and progressively increasing to plateau from day 6. To determine the role of RANTES and other chemokines in inducing T lymphocyte migration, we incubated 6 day Daoy and bEND.3 coculture supernatants with RANTES-blocking antibody (Ab) or IL8- or MIF-blocking Ab, as controls. RANTES Ab blocked the migration to the coculture supernatant by up to 60% while control IL8- and MIF-Ab failed to do so (Figure 4D). Tumor-derived ‘‘Macrophage Migration Inhibitory Factor (MIF)’’ is the primary tumor chemokine signal triggering RANTES release from the endothelium Our chemokine array analysis had revealed that all 7 primary medulloblastomas and Daoy secreted MIF, and that neither secreted RANTES (Figure 3B and Figure S1 and S2). To investigate if MIF acts directly on endothelial cells to release RANTES, we incubated bEND.3 cells with increasing concentra- tions of recombinant human MIF (rhMIF), a molecule that is 90– 98% homologous to murine MIF and biologically active on both human and murine cells. [14,35] Using a murine ELISA, we observed that supernatants from such cultures contained RANTES in a MIF-dose-dependent concentration (Figure 5A). We thus concluded that, in vitro, MIF can directly trigger the release of RANTES from endothelial cells. To further confirm in vivo that MIF was necessary for triggering RANTES production from endothelial cells, we generated MIF- knock down (KD) Daoy medulloblastoma cells using a retroviral vector encoding a MIF-specific shRNA. The MIF-specific shRNA reduced MIF production by 80% in comparison to the parental cell line (Figure 5B). To corroborate the link between expression of MIF and RANTES in vivo, we established tumors from the parental cell line Daoy, from MIF-KD Daoy cells, and from IL-8- KD Daoy cells (control). RNA was extracted from the tumor and the adjacent brain matter analyzed by qPCR for RANTES and two housekeeping genes (GAPDH and b-actin). While IL-8-KD Daoy tumors induced murine RANTES RNA levels similar to those of un-manipulated Daoy, MIF-KD Daoy tumor RNA showed a 38-fold decrease in RANTES expression compared to parental Daoy tumors. Control gene expression was similar among Daoy, MIF-KD and IL8-KD Daoy (Figure 5C). Hence, MIF-KD tumor cells failed to induce murine RANTES in vivo; confirming Tumor/Endothelium Crosstalk Attracts T Cells that MIF released by tumor cells is the primary chemokine that triggers the release of RANTES from endothelial cells. Finally, we studied, in vivo, the requirement of the MIF- RANTES axis for T lymphocyte migration to the tumor site. Ten days after establishing tumors derived from MIF-KD Daoy in the right caudate nucleus of mice, we stereotactically injected firefly luciferase-expressing T lymphocytes into the left caudate nucleus. Histological examination of a subset of animals showed that Daoy and MIF-KD Daoy xenografts had similar volumes (not shown). Firefly luciferase-expressing T lymphocytes that migrated briskly to established Daoy tumors failed to migrate to the contra-lateral MIF-KD Daoy tumors (Figure 5D). IHC of tumors explanted from the right hemispheres confirmed the absence of T lymphocytes. Discussion We have shown that the interaction between medulloblastoma cells and their associated endothelium triggers T lymphocyte migration into the tumor microenvironment. Medulloblastoma cells constitutively release the chemokine MIF which itself cannot recruit T lymphocytes, but triggers the release of a second chemokine, RANTES, from endothelial cells in the tumor stroma. This in turn attracts T lymphocytes since they express RANTES- cognate chemokine receptors. [36] We thus demonstrate that ‘cross-talk’ between the tumor cells and endothelium creates a chemotactic gradient triggering T lymphocyte migration to the tumor microenvironment. Leukocyte migration to disease sites has been extensively studied in research models of inflammation and autoimmune diseases. [14–24] These studies have shown that in the presence of chemokines induced by the pathological process, leukocytes are activated and directed out of the vasculature into the sites of tissue injury. The role of cells in the tumor microenvironment, including endothelial cells, in creating a gradient for leukocyte migration is well characterized in these models. [14–24] In contrast, little is known about their role in the process of T lymphocyte migration in the context of malignancies. Decoding such mechanistic events would increase our understanding of T lymphocyte migration to the tumor microenvironment. Our work describes a previously uncharacterized dimension to the phenomenon of intra-parenchymal T lymphocyte migration that involves a third party, the tumor endothelium. Coculture of medulloblastoma tumor cells and endothelial cells resulted in a new chemokine signature dominated by the progressive secretion of endothelium- rather than tumor-derived RANTES, a strong T lymphocyte chemo-attractant that is not produced by either cell type culture alone. Since CCR3 is strongly expressed on migrating T lymphocytes, we suspected that RANTES was the major chemoattractant of T lymphocytes to the tumor microenviron- ment. This was confirmed by the observed inhibition of T lymphocyte migration after RANTES neutralization. While both MIF and IL-8 were secreted by the majority of primary tumors as well as by Daoy cell line, T lymphocytes migrated to two different experimental tumors derived from primary cells that secreted only MIF. MIF, a chemokine-like inflammatory mediator, triggers leukocyte recruitment by binding to CXCR2 and CXCR4 [37,38]. While CXCR4 was expressed on T lymphocytes tested in this study, its chemotactic effect is contingent on forming heterodimers with cell surface-expressed CD74. [39] The latter was undetectable on the T lymphocytes, explaining their failure to migrate to MIF in transwell systems. We further demonstrated that knocking down MIF but not IL-8 in the tumor cell line results in failure of T lymphocytes to migrate to PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20267