Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9 + NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cellmediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

SUMMARY Primary infection with the human oncogenic Epstein Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion, which predisposes for the development of distinct EBV-associated lymphomas. It remains unclear why some individuals experience this symptomatic primary EBV infection, while the majority acquires the virus asymptomatically. Using a mouse model with reconstituted human immune system components, we show here that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis, mainly due to loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies.

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IntroductionNatural killer (NK) cells are innate lymphocytes that are primarily thought to curb viral infections and tumor cell expansion until antigen-specific adaptive immune responses can be primed to eradicate these threats to human health. 1 In contrast to adaptive lymphocytes like T and B cells, NK cells recognize their targets through germ line encoded receptors. These receptors transmit either activating or inhibitory signals. 2,3 The activating receptors recognize primarily stress-induced molecules on infected and transformed cells, including major histocompatibility complex (MHC) class I-like molecules that serve as ligands for the activating NK-cell receptor NKG2D, PVR and Nectin-2 as ligands for the activating NK-cell receptor DNAM-1, and B7-H6, as well as ligands of still poorly defined identity for the natural cytotoxicity receptors (NCRs) NKp30, NKp46, and NKp44. 4,5 Ligands for these activating receptors are up-regulated upon for example DNA damage or heat shock, 6,7 but are also constitutively present on some hematopoietic cells, including myeloid dendritic cells (DCs), 8 microglia, 9 and activated macrophages. 10 These activating signals are balanced by inhibitory receptor engagement, recognizing classical and nonclassical MHC class I molecules. In humans, killer immunoglobulin-like receptors (KIRs) recognize polymorphic determinants of classical MHC class I molecules, and C-type lectin receptors like the CD94/NKG2 heterodimer engage the nonclassical MHC class I molecule human leukocyte antigen (HLA)-E. 11 The balance of transmitted activating and inhibitory signals decides if NK cells will mount effector functions against conjugated target cells.The main effector characteristics of NK cells are cytokine secretion and cytotoxicity, 12 and humans carry NK-cell subsets that preferentially mediate one or the other of these functions. CD56 bright CD16 Ϫ KIR Ϫ NK cells respond primarily with production of interferon-␥ (IFN-␥), tumor necrosis factor, and granulocytemacrophage colony-stimulating factor to activation, and only exert cytotoxicity after prolonged activation. 13 In contrast, CD56 dim CD16 ϩ KIR ϩ NK cells are constitutively loaded with perforin and granzymes and are the primary human cytotoxic NK-cell subset. 14 While the latter population constitutes the majority of peripheral blood (PB) NK cells, CD56 bright CD16 Ϫ KIR Ϫ NK cells are enriched in human secondary lymphoid organs. 15,16 They have been proposed to limit pathogen invasion and polarize adaptive immune responses at these sites. 12,17 Thus cytotoxic NK cells patrol primarily the periphery, while immunoregulatory NK cells support Th1 polarization in secondary lymphoid organs.The developmental pathways leading to the functionally distinct human NK-cell subsets are still being defined. 18 So far 3 alternative pathways have been proposed. Originally, it was proposed that NK cells develop exclusively in the bone marrow from which they populate the periphery as constitutively reactive innate lymphocytes. 1 After the discovery that t...

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.