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. 2019 Jun;68(6):937-949.
doi: 10.1007/s00262-019-02332-w. Epub 2019 Apr 5.

Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma

Kim C M Santegoets  1 , Paul R Gielen  1 , Christian Büll  1 , Barbara M Schulte  1 , Esther D Kers-Rebel  1 , Benno Küsters  2 , Sandra A J F H Bossman  3 , Mark Ter Laan  3 , Pieter Wesseling  4   5 , Gosse J Adema  6
Affiliations

Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma

Kim C M Santegoets et al. Cancer Immunol Immunother. 2019 Jun.

Abstract

Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid-Siglec interactions between glioma cells and MDSCs in the tumor microenvironment.

Keywords: Glioma; Myeloid-derived suppressor cells; Sialic acids; Siglecs.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Siglec expression on blood MDSCs from glioma patients and healthy donors. a Gating strategy for MDSCs from blood, showing M-, PMN- and e-MDSC subsets. b, c qPCR analysis of Siglec expression in M-MDSCs (b) and PMN-MDSCs (c) from glioma patients. Bar diagrams show mean relative Siglec expression normalized to GAPDH ± SEM (n = 5). d–h Cell surface expression of Siglecs on MDSC subsets measured by flow cytometry. Representative histograms show cell surface Siglec expression on M-MDSCs (left panel) and PMN-MDSCs (right panel) (d). The gray histograms represent the isotype control. Dot plots show median fluorescence intensity (MFI) of cell surface Siglec expression of M-MDSCs and PMN-MDSCs derived from blood of glioma patients (n = 11–13; 6 lower-grade gliomas (open circles) and 7 glioblastomas (filled circles)) (e, f) as well as from healthy donors (n = 10–14) (g, h). Horizontal lines show the mean Siglec expression
Fig. 2
Fig. 2
Siglec expression on glioma-infiltrating PMN-MDSCs. a–c Expression of Siglecs on the surface of PMN-MDSCs and myeloid cells in glioma tissue. Gating strategy shows CD45+ MHC II+ CD11b+ myeloid cells and CD45+ MHC II− CD11b+ CD15+ PMN-MDSCs (a, left panels). Representative histograms show Siglec expression on myeloid cells (a, upper panel) and PMN-MDSCs (a, lower panel). The gray histograms represent the isotype control. b, c Bar diagrams show mean Siglec expression ± SEM on glioma-infiltrating PMN-MDSCs (b) and myeloid cells (c) from three glioma patients
Fig. 3
Fig. 3
Expression of sialic acids on blood MDSCs from healthy donors and glioma patients. a, b Binding of sialic acid-binding lectins MALII (α2,3-linked sialic acid) and SNA-I (α2,6-linked sialic acid) and galactose-binding lectin PNA to MDSCs isolated from blood of healthy donors. a Representative histograms show lectin binding to M-MDSCs (upper panel) and PMN-MDSCs (lower panel) as determined by flow cytometry. The gray histograms represent the unstained control b) Bar diagram shows MFI ± SEM of lectin binding to both MDSC subsets (n = 3). c, d Binding of recombinant Siglec Fc chimera to MDSCs from healthy donors (n = 3). Bar diagrams shows MFI ± SEM of Siglec Fc binding to M-MDSCs (c) and PMN-MDSCs (d). e, f Siglec ligand expression on PMN-MDSCs obtained from the blood of glioma patients (n = 5). Bar diagram shows Siglec Fc binding as MFI ± SEM (e) and histograms show representative Siglec Fc binding (f). The gray histograms represent the isotype control
Fig. 4
Fig. 4
Glioma cell lines and freshly resected glioma cells express trans ligands for Siglecs. a Three glioblastoma cell lines were stained with recombinant Siglec Fc chimera and analyzed by flow cytometry. Bar diagrams show mean MFI ± SEM of Siglec Fc chimera binding to U-87, T98G and U251 cells of three independent experiments. b, c Expression of Siglec ligands on glioma cells obtained from freshly resected tumor tissue (glioblastoma n = 1, oligodendroglioma n = 3). Representative histograms show binding of Siglec Fc chimera to CD45− cells isolated from glioma tissue (b). Bar graphs show mean fluorescent intensity ± SEM of Siglec Fc binding (c). d Immunohistochemistry for Siglec-7 and Siglec-9 ligand expression in paraffin-embedded glioma tissue untreated (upper row) or treated with sialidase (lower row). Data are representative of 4 glioblastoma patients
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