Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-β expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-β-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1β, IL-6, and IL-23 but without TGF-β did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-β and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.

IntroductionNatural killer (NK) cell activation is initiated when NK cells form tight contacts with target cells. [1][2][3][4] However, this activation can be abrogated by interactions between major histocompatibility complex (MHC) class I molecules and inhibitory killer cell immunoglobulin-like receptors (KIRs). SHP-1 is then recruited to the KIR intracytoplasmic domain and a dominant inhibition of cytoskeletal and lipid raft polarization occurs, protecting MHC class I-positive targets from autologous NK cell-mediated cytotoxicity. [5][6][7][8][9][10][11] We have previously described another type of synapse (DCNK-IS) that regulates DC-mediated NK activation. 12 This dynamic network in which NK interacts with surrounding DC was also described in vivo during Leishmania major infection. 13 The DCNK-IS has been shown to enable optimal NK activation by IL-15 and to display a spatial organization distinct from the classic target/NK synapses. 12,14,15 However, the initial molecular events governing mature DC (mDC) and NK interactions remain to be identified. In particular, the mechanisms allowing autologous resting NK activation by mDCs in the presence of KIR on NK cells and self-MHC proteins on DCs, have not been studied. MethodsMice C57BL/6 mice were obtained from Janvier (Le Genest St Isle, France), and maintained in the IFR133 animal facility according to the guidelines of the Animal Ethics Committee. C57BL/6 CX3CR1-knockout mice 16 were provided by Bernhard Ryffel (GEM2358, CNRS, Orleans, France). Dendritic cell differentiationHuman DCs were generated from adherence-selected monocytes in AIMV complete medium containing 1000 IU/mL of both rhuGM-CSF and rhuIL-4 (Peprotech, Neuilly-sur-seine, Saint-Quentin, France). DCs were exposed to lipopolysaccharide (LPS; 1 g/mL, Sigma-Aldrich, France), characterized and used at day 6, as previously described. 12 Mouse bone marrowderived DCs (BMDCs) were differentiated as previously described. 12 Purification of NK cellsNK cells were negatively selected from Ficoll-separated peripheral blood mononuclear cells (PBMCs) using the Miltenyi NK-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) Slide preparation and confocal microscopy mDCs (5 ϫ10 4 ) and NK cells (10 5 ) were mixed and spread onto poly-Llysine-coated slides (Sigma, France) for 25 minutes at 37°C. Cells were fixed and permeabilized with 0.2% sodium dodecyl sulfate. After 20 minutes of blocking in 20% fetal bovine serum and washing, cells were stained For personal use only. on May 12, 2018. by guest www.bloodjournal.org From with the appropriate mAbs. Stacks of confocal images were collected with an Olympus FV1000 laser scanning confocal microscope. Cell labeling and antibodiesPhalloidin (Molecular Probes, Eugene, OR) and cholera toxin ␤ subunit (CTX; Sigma-Aldrich, St Louis, MO) were used to detect polymerized F-actin and raft-associated GM1 gangliosides. NK cells were imaged using anti-KIR2DL1 (T-20; Santa Cruz Biotechnology, Santa Cruz, CA) and PT-100 (Cell Signaling Technology, Beverly, MA). Anti-...