Results T lymphocytes consistently infiltrate primary medulloblastoma T lymphocytes constitute one of the most important effector mechanisms of anti-tumor-immunity and often contribute to the tumor microenvironment. It is well established that T lymphocytic infiltrates are present in tumors outside the neuraxis, where they can have tumor inhibitory or promoting effects. [6–13] In contrast, less is known about these infiltrates in the context brain tumors. We therefore opted to study this in medulloblastoma, the most common pediatric brain tumor. We examined a series of primary medulloblastoma and found a T lymphocyte infiltration in 20 of 20 tumors examined using immunohistochemistry (IHC) for CD3. The degree of the T lymphocyte infiltrate varied between individual tumors and ranged between a modest perivascular pattern (#1%; 15 of 20 tumors); moderate perivascular infiltration with varying degrees of parenchymal spill (.1–10%; 4 of 20 tumors) to a uniform heavy perivascular and parenchymal infiltration (.10%; 1 of 20 tumors). Similarly, tumor sections from the same subjects were stained for CD8 using IHC and showed similar degrees and patterns of infiltration (Figure 1). T lymphocytes migrate and localize to distant tumor deposits in the brain We hypothesized that these infiltrates result from an innate avidity of T cells to the tumor cells and or the tumor complex. To test this hypothesis, we used a murine model in which human primary medulloblastoma (from subjects MB1277 and MB 1323) were orthotopically propagated in the right cerebellar cortex. Ten days after tumor establishment, mice were injected with T lymphocytes or sham injected into the right (ipsilateral) caudate nucleus. The injected T lymphocytes had a predominantly CD8- positive population. Six days later, the tumors were removed and examined by IHC for human CD3. Figure 2A shows that human CD3-positive cells could be detected only in animals receiving human T lymphocytes but not sham injected animals, suggesting that primary medulloblastoma can trigger T lymphocyte migra- tion from distant sites in the brain. To confirm T cell migration in the Daoy human medulloblastoma model by bioluminescence Tumor/Endothelium Crosstalk Attracts T Cells imaging, we injected luciferase-expressing human T cells into the left caudate nucleus of mice with established, 10 day old Daoy tumors in their contralateral caudate nucleus. T lymphocytes migrated within the brain to contralateral tumors in all experimental animals as shown in Figures 2B and 2C. Immuno- histochemistry using human CD3-specific antibodies demonstrat- ed columns of T lymphocytes migrating along the corpus callosum, the only solid connection between both hemispheres, and their clustering at the tumor periphery as well as within the tumor itself (Figure 2D). T lymphocytes that migrated to tumors in vivo fail to migrate to tumor cells in vitro T lymphocyte migration has generally been attributed to a response to a chemokine gradient generated by chemokine- secreting tumors. [28–32]. To examine the mechanism whereby tumor cells are attracting T lymphocytes, we performed a series of ex vivo transwell studies. Surprisingly, the same T lymphocytes that avidly migrated in vivo to tumor deposits failed to migrate in vitro when exposed to supernatants from cultures of the primary medulloblastoma samples in short-term culture or to the medulloblastoma cell line, Daoy (Figure 3A). Failure to migrate could not be explained by adverse in vitro cell culture conditions, since positive control B-lymphoblastoid cell line (LCL) superna- tants induced brisk migration of T lymphocytes in vitro. As anticipated, no migration was seen in response to media alone. To determine the mechanism responsible for failure of T lymphocyte migration to tumor cells in vitro, we characterized the chemokine profile of the tumor cells using a semiquantitative chemokine profiling system. We then used flow-cytometric analysis of T lymphocytes to determine their expression of the chemokine counter-receptors. Daoy medulloblastoma cells had a distinct chemokine signature, producing MCP-1; Gro-a; MIF; IL-8; Serpin E1; and IL-6 (Figure 3B and Figure S1). All 7 primary tumors secreted MIF, 4 tumors secreted IL-8, 3 tumors secreted MIP-1a and only one tumor secreted IL-13 and IL12. MB1277 and MB1323 secreted only MIF, the only molecule common to all samples tested. T lymphocytes, however, only expressed substantial levels of CXCR3, CXCR4 and CCR3 on their surface; CXCR1, CXCR2 and CCR1 were expressed by less than 10% of cells Figure 1. T lymphocytes consistently infiltrate primary medulloblastoma. Representative photomicrographs of CD3 IHC of human primary medulloblastoma demonstrating variable degrees of infiltration: modest perivascular pattern (#1%; 15 of 20 tumors), moderate perivascular infiltration with varying degrees of parenchymal spill (.1–10%; 4 of 20 tumors) and uniform heavy perivascular and parenchymal infiltration (.10%; 1 of 20 tumors). Similarly, tumor sections from the same subjects were stained for CD8 using IHC and showed similar degrees and patterns of infiltration. Magnification x100. doi:10.1371/journal.pone.0020267.g001 PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20267