Pediatric T-ALL but Not cALL Blasts Express Ligands for Siglec-7 Inhibiting NK Cell Mediated Lysis.

Abstract

Changes in the glycosylation pattern of cell surface proteins occur during early stages of malignant transformation. These alterations in the glycan repertoire may contribute to numerous processes of tumor cell survival such as adhesion, migration and immune evasion. Sialic acid is the most common terminal carbohydrate moiety of glycan structures in humans binding to a family of eleven receptors termed sialic acid binding Ig-like lectins (siglecs). Siglecs are expressed on virtually all effector cells of the immune system and several members function as inhibitory receptors. Siglec-7 is known as an inhibitory receptor expressed by NK cells and T cells. Therefore, siglec-7 ligands may contribute to immune evasion of malignant cells.In order to analyze the expression of siglec-7 ligands by malignant cells, we cloned the extracellular domain of human siglec-7. The recombinant protein was expressed in 293 cells to allow glycosylation, which is supposed to be important for specificity. As the interaction of a single glycan and lectin is of low affinity, we tetramerized siglec-7 in analogy to MHC-tetramers to increase avidity. This strategy allowed for reliable use of siglec-7 fusion protein in flow cytometry. Analysis of PBMC from healthy donors revealed that siglec-7 ligands are expressed on all subpopulations of PBMC: expression was detected on CD4+ and CD8+ T-cells as well as on monocytes and NK cells. B cells could be subdivided into a positive and negative population after staining with siglec-7 tetramers.Subsequently, we analyzed primary lymphatic blasts from childhood leukemias. Interestingly, T-ALL blasts expressed ligands for siglec-7, whereas these ligands were absent from cALL blasts. As cALL blasts are known to be better targets for NK cell-mediated cytotoxicity than T-ALL blasts, we hypothesized that siglec-7 ligands on the surface of leukemic blasts inhibit NK cells. To test the functional relevance of siglec-7 ligand expression for NK cell-mediated cytotoxicity, we used soluble monomeric siglec-7 as competitive inhibitor in cytotoxicity assays. In these competition assays, NK cell-mediated specific lysis of leukemic cells increased roughly twofold in the presence of soluble siglec-7.These results show that engagement of inhibitory siglecs on the surface of effector cells might contribute to immune escape mechanisms of malignant cells. It underlines the function of siglec-7 as a non-MHC-specific inhibitory receptor on NK cells and the important role of altered glycans expression on malignant cells in tumor immunology.