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Siglec-7 is a CD33-related sialic acid-binding Ig-like lectin expressed strongly on NK cells, where it can function as an inhibitory receptor. Its sialic acid-binding activity on NK cells is masked by cis interactions with sialylated glycans, which are likely to be important for regulating the inhibitory function of Siglec-7, which exhibits an unusual preference for α2,8-linked disialic acids, a motif found in “b-series” gangliosides and some glycoproteins. To investigate the presence of α2,8-linked disialic acids on NK cells, T cells, monocytes, and B cells, we first analyzed their expression of all known α2,8-sialyltransferase genes by quantitative PCR. Unlike T cells, B cells, and monocytes, NK cells consistently expressed mRNA encoding ST8Sia VI, which creates α2,8-linked disialic acids on O-linked glycans of glycoproteins. All blood leukocytes expressed ST8Sia IV, implicated in polysialic acid synthesis, and NK cells variably expressed high levels of ST8Sia V mRNA required for GT3 expression. Two human IgM antibodies, Ha1 and Pi1, with specificity for the α2,8-disialyl motif reacted strongly with NK cells in a sialic acid-dependent manner and less strongly with T cells and monocytes. Antibody-induced clustering of Siglec-7 on NK cells resulted in partial colocalization with anti-Ha1. Finally, MALDI-TOF mass spectrometric analysis of isolated NK cell O-glycans revealed the presence of a peak at mass-to-charge ratio of 1619.4 mass units, corresponding to a putative α2,8-disialylated glycan. Together, these results suggest that NK cells are decorated with α2,8-disialic acid structures implicated in regulation of cellular activation via interactions with Siglec-7.

A subset of patients with recurrent high-grade gliomas benefits from the anti-angiogenic therapy bevacizumab (BVZ). An increased number of research efforts are performed to develop predictive biomarkers that allow for screening of patients. The immune system plays a crucial role in angiogenesis induction and maintenance. We analysed circulating immune cells in recurrent high-grade glioma patients treated with BVZ (10 mg/kg every 2 weeks) during a prospective bi-centric trial. Blood samples were collected before cycles 1, 3, 5 and 7 to perform flow cytometry analysis of monocytes (classical/intermediate/non-classical/ DRlow/ Tie2+/ VEGFR1+), Myeloid-Derived Suppressor Cells (MDSC) and regulatory T cells (Tregs) as well as a complete blood count. Thirty patients (29 Glioblastoma, 1 anaplastic astrocytoma) were enrolled between September 2012 and January 2014. Median OS and PFS were 9.1 months (6.7 – 18.4; 95% CI) and 3.3 months (1.8 – 5.1; 95% CI), respectively. During treatment results demonstrated a statistically significant increase in the median values of leukocytes, neutrophils, eosinophils, total and classical monocytes, as well as a statistically significant decrease of the percentage of Tregs among leucocytes. The strongest correlation between baseline levels of immune cells and OS were observed for neutrophils and Tregs. Patients with a percentage of Treg among leucocytes above the median (0.21 %) had an increased OS (19.8 [17.5-n.a] versus 5.8 [5.3–12.2] months, HR: 0.13 p

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.

Introduction:Glioblastoma multiforme (GBM) is the most lethal form of glioma with an overall survival at 5 years nearly null (< 5%). Increasing evidences point towards the RNase activity of IRE1 as a central player in GBM development, particularly in cancer cell invasion, tumor vascularization and recruitment of inflammatory or immune cells. Indeed, IRE1 RNase promotes XBP1 mRNA splicing, a well-described cytoprotective transcription factor that is required for cancer cell survival and this activity also contributes to the degradation of mRNA and microRNA, an activity called RIDD. Recent studies unraveled the presence of somatic mutations on the IRE1 gene in GBM that could play a driver role but without providing any functional information.Results:Here, we have sequenced IRE1 exons in 23 human GBMs and identified a new somatic mutation on the IRE1 gene. Interestingly, using biochemical approaches and an orthotopic tumor graft mouse model we show that this variant displays an altered IRE1 RNase activity. IRE1-dependent gene expression pattern alterations lead to major functional consequences in terms of tumor phenotype including (i) a magnified induction of the inflammatory and pro-angiogenic pathways based on transcriptome signatures from human tumors, (ii) an increase of the aggressiveness/infiltration potential of U87 derived tumors in mice and (iii) the modulation of immune cells recruitment to the tumor.Conclusions:This study provides the first evidence that the selectivity of IRE1 RNase can be modulated naturally and unravels the first mechanistic example of how a somatic mutation in the IRE gene can provide adaptive advantages to glioblastoma cells by reprogramming the tumor stroma.