NK T (NKT) cells play important roles in the regulation of diverse immune responses. However, little is known about the mechanisms that regulate homeostasis and activation of these cells. Thymic NKT cells up-regulated the chemokine receptor CXCR6 following positive selection and migrated toward CXCL16 in vitro. However, CXCR6 was not essential for thymic development or maturation. In contrast, liver and lung NKT cells were depleted in CXCR6+/− and CXCR6−/− mice. The reduction in liver and lung NKT cells coincided with an increase in bone marrow NKT cells, suggesting a redistribution of NKT cells in CXCR6−/− animals. In wild-type mice, CXCL16 neutralization reduced accumulation of mature NK1.1+, but not immature NK1.1− NKT cell recent thymic emigrants in the liver. Given that thymic NKT cells are preferentially exported as NK1.1− cells, this suggests an additional role for CXCR6/CXCL16 in maturation or survival of immature liver NKT cells. CXCL16 blockade did not deplete resident NK1.1+ NKT cells, indicating that CXCR6/CXCL16 are not required to retain mature NKT cells in the liver. Cytokine production by liver and spleen NKT cells was impaired in CXCR6−/− mice following in vivo stimulation with α-galactosylceramide, implicating a novel role for CXCR6 in NKT cell activation. Reduced IFN-γ production was not due to an intrinsic defect as production was normal following PMA and ionomycin stimulation. Preformed transcripts for IL-4, but not IFN-γ, were reduced in CXCR6−/− liver NKT cells. These data identify critical roles for CXCR6/CXCL16 in NKT cell activation and the regulation of NKT cell homeostasis.

Invariant NKT (iNKT) cells can induce potent antitumor responses in vivo. However, the mechanisms that regulate the effects of iNKT cells are unclear. The chemokine receptor CXCR6, and its ligand CXCL16, have been shown to play critical roles in iNKT cell homeostasis and activation. Thus we investigated the role of CXCR6 in protection against experimental metastasis of B16-F10 melanoma (B16) and Lewis lung carcinoma (LLC) cells to the liver and lungs. Wild-type and CXCR6−/− mice exhibited no differences in tumor cell metastasis to the lungs. However, metastasis of LLC and B16 tumor cells to the liver was enhanced in CXCR6−/− mice. Liver metastasis was also increased in wild-type mice treated with a CXCL16 neutralizing Ab. As Ab treatments did not alter iNKT cell numbers, this implicates a direct role for CXCR6/CXCL16 in regulating antitumor immunity. Cytokine induction was significantly attenuated in CXCR6−/− mice upon systemic iNKT cell activation with the glycolipid Ags α-galactosylceramide (α-GalCer), α-C-GalCer (a Th1 polarizing derivative), or OCH (a Th2 polarizing derivative). Despite differences in the levels of cytokine production, liver and lung metastasis were inhibited significantly in both wild-type and CXCR6−/− mice treated with glycolipids. Single doses of α-GalCer, α-C-GalCer, or OCH were sufficient to prevent liver metastasis and subsequent doses failed to elicit optimal cytokine responses. Our findings implicate a role for CXCR6 in natural immunosurveillance against liver metastasis. However, CXCR6 deficiency could be overcome by systemic iNKT cell activation, demonstrating that even suboptimal iNKT cell activation can protect against metastasis.

IntroductionMetastasizing cancer cells are thought to overcome the endothelial barrier through mechanisms commonly observed in leukocyte trafficking, including the up-regulation of adhesion molecules, apoptosis induction, cytoskeletal reorganization, and disruption of normal endothelial intercellular contacts. 1-5 Both cancer cell transmigration and leukocyte trafficking are increased by proinflammatory cytokines, such as interleukin-1␤ and tumor necrosis factor (TNF), which up-regulate adhesion molecules, including P-selectin, 5-7 and E-selectin. 1,8 Given the parallels between these processes, we hypothesized that a therapy directed at the endothelium to reduce leukocyte trafficking might also be beneficial in preventing tumor cell metastasis. The serine protease activated protein C (aPC) has been used to treat severe sepsis. 9 It possesses anti-inflammatory and cytoprotective activities mediated through engagement of its receptor, endothelial protein C receptor (EPCR). [10][11][12] This interaction down-regulates endothelial proinflammatory and proapoptotic gene expression and decreases vascular permeability. 13,14 In this report, we assessed the ability of melanoma cells to adhere to and transmigrate through endothelial monolayers treated with recombinant human aPC (rhaPC). Furthermore, we investigated the role of the aPC/EPCR pathway in a murine model of hematogenous B16-F10 melanoma metastasis by characterizing liver and lung metastases in EPCR-overexpressing mice and by administering rhaPC to wild-type (WT) mice. Study design Animals and cell linesFemale Tie2-EPCR transgenic mice were generated on a C57BL/6 background as previously described. 15 Wild-type C57BL/6 mice from The Jackson Laboratory (Bar Harbor, ME) and transgene-negative littermates were used as controls. Experiments were performed in accordance with the Canadian Council on Animal Care guidelines. B16-F10 mouse melanoma and bEnd.3 mouse brain microvascular endothelial lines were purchased from ATCC (Manassas, VA), and rhaPC was purchased from SigmaAldrich (Oakville, ON). Research ethics approval was obtained for all animal studies from the Dalhousie University Committee on Laboratory Animals in accordance with the Canadian Council on Animal Care. Adhesion and transendothelial migration assaysFor adhesion assays, confluent bEnd.3 monolayers grown in 24-well polystyrene plates (BD Biosciences, Mississauga, ON) were pretreated with rhaPC (0, 5, 10, 25, 100 nM) for 3 hours then washed. B16-F10 cells (2 ϫ 10 5 ) labeled with CFDA-SE (Invitrogen, Burlington, ON) were added to each well. 4 One hour later, wells were washed and adherent fluorescent cells were counted. For transmigration assays, bEnd.3 monolayers on polycarbonate Transwell inserts (8-m pore-size, 6.4-mm diameter; CorningCostar, Corning, NY) were pretreated with rhaPC. To induce chemotaxis, DMEM supplemented with 15% FCS was added to the lower chambers. CFDA-SE-labeled B16-F10 cells (2 ϫ 10 5 ) in DMEM were added to the upper chambers. After 16 hours at 37°C/5% CO 2 , fluorescent cell...

Introduction: Engagement of endothelial protein C receptor (EPCR) by activated protein C (aPC) decreases expression of endothelial adhesion molecules implicated in tumorendothelium interactions.