Siglec-7 Tetramers Characterize B Cell Subpopulations and Identify Immunomodulatory Ligands on Malignant Cells
Authors: Mang Philippa , Gieseke Friederike , Viebahn Susanne , Gondesen Inga , Kruchen Anne , Erbacher Annika , Pfeiffer Matthias , Handgretinger Rupert , Müller Ingo 
Published: October 2019

Abstract
Abstract 1728
Cell surface glycoconjugates have important functions in the modulation of immune cell maturation, activation and homeostasis. Glycans on cell surfaces are generally sialylated. Sialic acids are predominantly found on the non-reducing end of oligosaccharide chains of glycoconjugates. They are recognized by sialic acid-binding immunoglobulin-like lectins (siglecs). CD33-related siglecs are receptors predominantly expressed on immune cells. Most of the CD33-related siglecs display a tyrosine-based inhibitory motif (ITIM) known to downregulate effector cell functions. So far, identification of ligands for siglecs was hampered by low affinity between proteins and glycans, but by glycan array analysis α2,8-, α2,3- as well as α2,6-linked sialic acids were identified as the carbohydrate part of the ligand.
In order to analyse the expression of physiological siglec ligands on different lymphocyte subpopulations, the extracellular three domains of siglec-7 were cloned. To allow for mammalian glycosylation, the protein was expressed in 293T cells. To overcome the low affinity of lectins, siglec-7 was enzymatically biotinylated and oligomerized using streptavidin in analogy to HLA tetramers. Flow cytometric analysis of lymphocytes revealed reproducible binding patterns of siglec-7 tetramers. Specificity was ensured by treatment of PBMC with neuraminidase, specifically cleaving terminal sialic acids in α2,3, α2,6 and α2,8 linkages. On T cells, the density of siglec-7 ligands increased 3.5-fold during activation. This is contradictory to results using plant lectins, like Maackia amurensis lectin and Sambucus nigra lectin, which indicated an 1.8- and 1.5-fold decrease, respectively. Activation of NK cells did not affect the siglec-7 ligand expression. Interestingly, B cells could be divided in a siglec-7 ligand positive and a siglec-7 ligand negative population. This may reflect different differentiation or activation stages, which were not found with any antibody combination directed against typical protein B cell markers.
Malignant transformation is often associated with aberrant cell surface glycosylation. In acute lymphoblastic leukemia (ALL), blasts of the more aggressive T-ALL express 17 fold higher amounts of siglec-7 ligand than blasts of the B cell lineage (cALL), where the siglec-7 ligand was nearly not detectable. These findings may contribute to the poor prognosis of T-ALL by a possible immune escape achieved by siglec-ligand binding induced inhibition. Since immune evasion mechanisms are also described for other malignancies, we additionally analysed rhabdomyosarcoma (RMS) cells. Siglec-7 ligand could be found on all tested RMS cell lines in different expression levels, independent of the classification.
Siglec-7 is a known inhibitory receptor expressed by NK cells and T cells. The effect of siglec-7 ligand expressed by malignant cells on NK cell cytotoxicity was analysed. By the removal of all terminal sialic acids on the surface of RMS cells by neuraminidase treatment, the NK cell-induced cytotoxicity could be increased by 20%. Coating of siglec-7 ligands on target cells by preincubation with recombinant siglec-7 protein also resulted in an increased cytotoxicity.
These results show that sialic acids play an important role in the immune system as physiologic ligands. However, the expression of siglec-7 ligands on malignant cells may contribute to immune evasion mechanisms.

Disclosures:
No relevant conflicts of interest to declare.