Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion

Abstract

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human natural killer (NK) cell activation through the recruitment of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7). Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing, suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy.

Figure 1. A glycocalyx engineering approach to studying sialoside dependent NK inhibition

(a) In the presence of activating ligands and absence of inhibitory ligands on the target cell, NK cells are activated to release cytotoxic effectors and cytokines. Coating cancer cells with sialylated glycopolymers by membrane insertion can emulate cancer associated glycosylation changes that engage the Siglec family of inhibitory receptors. Localization of Siglecs to the site of activation enhances SHP-1/2 phosphatase recruitment to halt the phosphorylation cascade before cellular activation. (b) The methyl vinyl ketone (MVK) polymer consists of a polyketone backbone that is end-functionalized with a DPPE phospholipid. Oxime-linked polymers were generated from the chemoselective reaction of aminooxy compounds with the MVK scaffold (See Supplementary Information for abbreviations).

Figure 2. Glycopolymers enable controlled manipulation of cellular glycosylation status

(a) Fluorescence microscopy of CHO, Jurkat, and MCF-7 cells labeled with AF488 conjugated glycopolymers demonstrate good incorporation across cell types. Scale bars, 10 μm. (b) K562 cells were incubated with increasing concentrations of AF488-2,6-SiaLacNAc polymer for 45 min at room temperature or 1 μM polymer for increasing time points. Incorporation was measured by flow cytometry. a.u., arbitrary units. (c) Glycopolymers show concentration dependent labeling that correlates with Siglec-7-F_c lectin binding. Jurkat cells were coated with increasing concentrations of AF488-GD3 polymer at room temperature for 45 min and labeled with Siglec-7-F_c and anti-humanF_c-647 on ice. For (b) data are presented as mean ± s.d. (n = 3).

Figure 3. Sialoside glycopolymers protect target cells from NK cell-mediated cytotoxicity

(a) Glycopolymers protect target cells from NK-mediated cytotoxicity in a structure dependent manner. Jurkat cells were labeled with 1 μM indicated polymer and incubated with purified NK cells at an effector to target ratio of 3:1 in a 4 h cytotoxicity assay. (b) To assess if Siglec-7 binding correlated with protection seen in (a), Jurkat cells were treated with indicated polymer and Siglec-7-F_c binding was assessed by flow cytometry. (c) Dependence of Siglec-7 for Sia polymer protection was probed by preincubating purified NK cells with 10 μg/mL of Siglec-blocking or isotype antibody and mixing with Jurkat cells at a 4:1 NK cell to target ratio in a 4 h cytotoxicity assay. (d) K562 target cells were labeled with increasing concentrations of Sia polymer and labeled with Siglec-7-F_c and anti-hF_c-647 or incubated with PBMC at a 10:1 effector to target ratio. Data are presented as mean ± s.d. (n = 3; *P < 0.05, **P < 0.01 for polymer coated versus no polymer control). a.u., arbitrary units.

Figure 4. Siglec-7 provides a strong NK inhibitory signal in response to a sialic acid glycopolymer

(a) PBMC was incubated with Jurkat cells at a 1:1 effector to target ratio. CD107a and IFN-γ were detected by antibody labeling and reported as % positive on gated CD56⁺ cells. (b) Siglec-7 displayed recruitment to the NK synapse in fluorescence microscopy of NK-Jurkat target cell conjugates labeled with AF488-Sia polymers. Scale bars, 10 μm. (c) Western blot analysis of Siglec-7 activation. NK cells were incubated with K562 cells with or without Sia polymer and lysed at the indicated times followed by anti-Siglec-7 immunoprecipitation. An increase in SHP-1 recruitment and prolonged Siglec-7 phosphorylation was seen in NK cells treated with Sia polymer-coated K562 cells but not with untreated targets. (d) NK-92 cells were retrovirally transduced to overexpress FLAG-tagged Siglec-7 and incubated with polymer-treated Jurkat cells at various ratios. NK-92 cells expressing Siglec-7 were susceptible to Sia polymer inhibition while mock treated cells were uninhibited. (e) Western blot analysis of NK-92 cells stimulated for 5 min with target Jurkat cells coated with no, Sia, or SiaLe^x polymers. Only the Sia polymer coated targets elicited Siglec phosphorylation and increased SHP-1 in NK-92 cells overexpressing Siglec-7. Siglec-9 was phosphorylated in all instances. Data are presented as mean ± s.d. (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 for polymer coated versus no polymer control, two-tailed, paired analysis). Full blots are shown in Supplementary Note 2.

Figure 5. Sialylation status affects susceptibility to native and antibody-dependent NK cytotoxicity in multiple cancer lines

(a) Removal of cell surface sialic acid from cancer cell lines increases NK cell-mediated cytotoxicity. NK immunoprotection is recovered by treatment with the Sia polymer (Sia Pol). Cancer target cells were treated with VC sialidase for 1 h at 37 °C before polymer incorporation at room temperature and coculture with purified NK (5:1, effector:target) (b) Western blot analysis of Siglec-7 phosphorylation after target cell sialylation remodeling. Promyelocytic HL-60 cells treated with sialidase and coated with Sia polymer were cocultured with NK-92 cells overexpressing Siglec-7 at a 2:1 effector to target ratio and lysed immediately or at 5 min. (c) Cytotoxicity assays performed with Daudi B lymphoma and NCI-N87 gastric carcinoma cells in the presence of 10 μg/mL anti-CD22 or 2 μg/mL anti-HER2 antibody respectively in increasing NK:target ratios. Data are presented as mean ± s.d. (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 for Sia polymer coated versus no polymer control, two-tailed, paired analysis). Full blots are shown in Supplementary Note 2.

Figure 6. Primary xenogeneic porcine and allogeneic hematopoietic stem cells are protected by Sia polymer incorporation

Sia polymer cell surface incorporation protects transplant cell targets from NK cell-mediated cytotoxicity. (a) Bone marrow derived CD34+ hematopoietic stem cells (HSC) were treated with 4 μM Sia polymer for 1 h at room temperature followed by coculture with purified NK at indicated effector to target ratios in a 4 h cytotoxicity assay (b) The Sia polymer also dampened the NK cytotoxic response to pig aortic endothelial cells (PAOEC) at a 10:1 effector:target ratio. Data are presented as mean ± s.d. (n = 3; **P < 0.01 for polymer coated versus no polymer control, two-tailed, paired analysis).