contralateral tumor deposits. Furthermore, RNA extracted from established tumors showed substantial inhibition of RANTES in MIF-KD explants. This indicates that MIF, derived from tumor cells, is responsible for RANTES production by endothelial cells and subsequently, T lymphocyte migration. The vast majority of human chemokine molecules have a high degree of amino acid sequence homology to their murine counterpart. [14,34,35,40–44] These molecules exhibit complete cross-species activity on human and mouse cells but, depending on the antibody used for testing, could be immunologically distinct. [14,35,40,41] We thus took advantage of the xenogenic setup to determine the source of RANTES using both human and murine specific ELISA that employ antibodies specific to a non-conserved epitope on the molecule. To exclude the possibility that the observed induction of RANTES was due to a xenogeneic effect, we repeated the same experiment using co-culture of Daoy cells with primary Human Brain Microvascular Endothelial cells (HBMEC) and obtained similar results to those obtained with murine endothelial cells. (Figure S3) We thus propose a model of T lymphocyte migration as a result of the interaction between the tumor and endothelium as outlined in Figure 6. Rubin et al. has shown previously that endothelial cells attract medulloblastoma cells through the CXCL12/CXCR4 axis and that this interaction is critical for tumor establishment. [45] Our results now indicate that the tumor cell/endothelial cell interac- tions results in recruitment of T lymphocytes, which have the potential to further promote tumor growth. Indeed, on examining a number of primary medulloblastomas, we found an infiltrate of T lymphocytes that was typically perivascular with various degrees of parenchymal involvement. This finding has implication for how tumor niches are formed and suggests a ‘domino effect’ in which the recruitment of one cell type triggers the recruitment of another. Thus, interventions that inhibit the effects of MIF (to counteract the primary tumor-derived signal) [38] and or RANTES (to counteract the endothelium derived signal), or block their cognate receptors could possibly offset tumor growth by altering the tumor- induced niche. Conversely, the innate avidity of T lymphocytes to the tumor microenvironment could be exploited therapeutically by redirecting their specificity towards tumor-restricted antigens. Studies to test these therapeutic interventions by our group are currently underway. In summary, we have shown that the crosstalk between tumor and endothelial cells creates an effective chemotactic gradient triggering the in vivo migration of T lymphocyte to the tumor Figure 6. Proposed Model of T Lymphocyte Migration to Medulloblastoma Cells. MIF secreted by medulloblastoma cells induce endothelial cells to produce RANTES, which triggers the migration of T lymphocytes to the tumor microenvironment. doi:10.1371/journal.pone.0020267.g006 Tumor/Endothelium Crosstalk Attracts T Cells microenvironment. Manipulation or exploitation of these interac- tions may be of benefit to tumor control. Materials and Methods Blood donors, primary tumor cells and cell lines Studies were performed on Baylor College of Medicine IRB- approved protocols and informed written consent was obtained from all donors. The medulloblastoma line Daoy was purchased from ATCC (Manassas, VA). All cell lines were grown in DMEM (Invitrogen, Carlsbad, CA) with 10% fetal calf serum (FCS; HyClone, Logan, UT), with 2 mM GlutaMAX-I, 1.5 g/L sodium bicarbonate 0.1 mmol/L and 1.0 mmol/L sodium pyruvate (Invitrogen). T lymphocytes derived from PBMCs were activated on CD3 antibody-coated plates and were expanded in IL-2 (100U/mL)-containing RPMI 1640 with 10% FCS and 2 mM GlutaMAX-I. Primary Human Brain Microvascular Endothelial Cells (HBMECs) were obtained from CellScience (Carlsbad, CA) and were cultured in Endothelial Cell Medium, obtained from the same vendor. Tumor tissues were processed aseptically, and primary cell cultures were initiated using the supplemented DMEM. Cells were used within three days of plating. Retroviral construct, retrovirus production and transduction of T lymphocytes We used an SFG retroviral vector containing an eGFP-firefly luciferase fusion gene in order to generate firefly luciferase expressing T lymphocytes for the in vivo study. [46,47] To produce retroviral supernatant, 293T lymphoctyes were cotransfected with retroviral vector containing plasmid, pEQ.PAM-E plasmid encoding the sequence for MoMLV gag-pol, and plasmid pMEVSVg containing the sequence for VSV-G, using GeneJuice transfection reagent (EMD Biosciences, San Diego, CA) [40]. Supernatants containing the retrovirus were collected 48 and 72 hours later. VSV-G pseudotyped viral particles were used to transduce the FLYRD18 producer cell line for the production of RD114 pseudotyped viral particles [41]. T lymphocytes were transduced with retroviral vectors as described. [46,47] Briefly, peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep gradient centrifugation. 56105 PBMC per well of a 24-well plate were activated with OKT3 (OrthoBiotech, Raritan, NJ) at a final concentration of 1 mg/mL. On day 2, recombinant human IL-2 (Chiron, Emmeryville, CA) was added at a final concentration of 50 units/mL, and on day 3 cells were harvested for retroviral transduction. For transduction, we pre-coated a non-tissue culture treated 24-well plate with a recombinant fibronectin fragment (FN CH-296; Retronectin; Takara Shuzo, Otsu, Japan). Wells were washed with phosphate-buffered saline (PBS; Sigma, St. Louis, MO) and incubated twice for 30 minutes with retrovirus. Subsequently, 36105 T lymphocytes per well were transduced with retrovirus in the presence of 50 units IL-2 per mL. After 48– 72 hours, cells were removed and expanded in the presence of 50– 100 units IL-2 per mL for 10–15 days prior to use. Orthotopic xenogenic SCID mouse model of medulloblastoma All animal experiments were conducted on a protocol AN3949 approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC). Recipient NOD-SCID mice were purchased from Taconic (C.B-Igh-1b/IcrTac-Prkdcscid; FOX CHASE CB-17 SCIDTM ICR; Taconic, Hudson, NY). Male 9 week to 12 week-old mice were anesthetized with rapid sequence PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20267