Siglec‐7 tetramers characterize B‐cell subpopulations and leukemic blasts

Abstract

Cell surface glycosylation has important regulatory functions in the maturation, activation, and homeostasis of lymphocytes. The family of human sialic acid-binding immunoglobulin-like lectins (siglecs) comprises inhibitory as well as activating receptors intimately involved in the regulation of immune responses. Analyses of the interaction between siglecs and glycans are hampered by the low affinity of this interaction. Therefore, we expressed siglec-7 in eukaryotic cells, allowing for glycosylation, and oligomerized the protein in analogy to MHC tetramers. Using this tool, flow cytometric analysis of lymphocytes became possible. Sialic acid-dependent binding of siglec-7 tetramers was confirmed by glycan array analysis and loss of siglec tetramer binding after neuraminidase treatment of lymphocytes. In contrast to most lymphocyte subpopulations, which showed high siglec-7 ligand expression, B-cell subpopulations could be further subdivided according to different siglec-7 ligand expression levels. We also analyzed blasts from acute lymphoblastic leukemias of the B-cell lineage as well as the T-cell lineage, since malignant transformation is often associated with aberrant cell surface glycosylation. While pediatric T-ALL blasts highly expressed siglec-7 ligands, siglec-7 ligands were barely detectable on cALL blasts. Taken together, oligomerization of recombinant soluble siglec-7 enabled flow cytometric identification of physiologic lymphocyte subpopulations and malignant blasts.Keywords: B lymphocytes r Cell surface glycosylation r Sialic acid r Siglec (sialic acid-binding immunoglobulin-like lectins) Supporting Information available online IntroductionThe cell surface is not only comprised of proteins and lipids, but is also further diversified by glycosylation of these macromolecules. In the immune system, cell surface glycosylation, and in particCorrespondence: Dr. Friederike Gieseke e-mail: gieseke@kinderkrebs-forschung.de ular sialylation, characterizes different lymphocyte lineages and differentiation stages as well as activation-dependent changes [1]. Sialic acids are most often located at the reducing ends on N-and O-linked glycans or glycosphingolipids. About 20 different sialyltransferases conjugate sialic acids in the α2,3-, α2,6-, or α2,8 position to the subterminal carbohydrate. These sialic acids * These authors contributed equally to this work. It has been shown that engagement of CD33-related receptors inhibits cellular proliferation [6, 7], raises the cellular activation threshold [8], and induces apoptosis [9,10] as well as induces the release of anti-inflammatory cytokines [11]. The importance of siglecs in setting the threshold for effector cell activation has been demonstrated for both B cells and T cells [8,12]. Additionally, CD33-related siglecs can act as endocytosis receptors, which could play an important role in the clearance of sialylated antigens and may be involved in antigen presentation [2]. One of the most widely studied siglecs is siglec-7, which was described as a NK cell inhib...

A versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands

“…Several efforts have been made to increase multivalency of Siglecs. For example, biotinylation of monomeric Siglec-7 to create tetramers with Streptavidin 33 , pre-complexing Siglec-Fcs with αhIgG1 12 , and pentameric comp constructs of Siglecs to detect Siglec-8 and -9 ligands 13 . Taking this a step further, we have demonstrated that tetramerization of dimeric Siglec-Fc chimeras provides greatly enhanced sensitivity for detecting sialic acid ligands on cells and tissues.…”

Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7

“…typically assessed by studying binding of recombinant GBPs to synthetic arrays containing a broad collection of defined glycan structures chemically linked to an inert surface (18,19). Glycan arrays are useful tools that can point toward a class of isolated glycan structures that may form components of a biological binding motif.…”

Interactions between Siglec-7/9 receptors and ligands influence NK cell–dependent tumor immunosurveillance

“…Knowledge about Siglec expression and function on NK cell subsets is still limited in terms of NK cell differentiation and maturation, and the role of Siglec ligand-receptor interactions in disease remains to be explored. Whether Siglec-7 and -9 ligands in human cancer are expressed on malignant or healthy cells is a subject of controversy (3,(25)(26)(27)(28), as is the related question as to whether absence of inhibitory Siglecligand interactions promotes or inhibits tumor progression, either by uncoupled immune stimulation or by inhibition of immune defense (10,(25)(26)(27). Here, we found that Siglec-7 and -9 ligands were significantly overexpressed on a broad range of human malignant cells of different histological types and that surface Siglec-7 and -9 ligand expression determined cytotoxicity of human primary NK cells against both NK cell-susceptible and -resistant tumor cells.…”