Fig 2: Desialylation of MM cells increases NK cell degranulation and surface-expressed CD107a after coculture. (A) IL-2 activated NK cells were cocultured with K562, JJN3, or H929 ± desialylation using NEURA or GLYCO for 1 hour, after which cells were collected and CD107a expression was measured on bulk NK cells. Histogram representative of n = 1 biological repeat for each cell under each condition. (B) CD107a expression on IL-2 activated primary NK cells exposed to NEURA-treated K562, JJN3, and H929 was determined and compared with CD107a expression on NK cells cocultured with GLYCO-treated controls. (C) NK cells were subdivided based on Siglec-7/Siglec-9 expression and subset degranulation were measured after coculture with JJN3 treated with either GLYCO or NEURA. (D) CD107a expression was measured on expanded primary NK cells exposed to NEURA-treated K562, JJN3, and H929 and compared with CD107a expression on NK cells cocultured with GLYCO-treated controls. (E) TNF-α and IFN-γ expression within NK cells was measured after coculture with NEURA-treated K562, JJN3, and H929 and compared with the expression of TNF-α and IFN-γ when cocultured with GLYCO-treated controls. (B-E) Data analyzed using Student’s paired t-test; graphs represent mean CD107α/TNF-α/IFN-γ positive NK cells +SEM. An individual repeat of n = 7 donors (A), n = 7 (B-C), n = 5 (D-E). *P < .01; ***P < .001; ****P < .0001; ns, not significant.

Fig 3: Siglec-7 and Siglec-9 ligands, and their concomitant receptors, are expressed by MM and NK cells, respectively. Using recombinant Siglec-7 and Siglec-9 chimeras, primary MM cells isolated from BMAs supplied by MGUS and newly diagnosed (ND) patients (A) and a panel of commonly used MM cell lines (B) were stained for Siglec-7L and Siglec-9L expression. (C) Using a fluorescently labeled anti–Siglec-7 antibody primary NK cells from BMAs of patients with MM were screened for the expression of Siglec-7. (D) Primary NK cells (IL-2 activated and expanded) were stained for Siglec-7 and Siglec-9 expression. (E) To elucidate the identity of Siglec-7L in MM, mass spectrometry was carried out on proteins bound to Siglec-7 Fc chimera-magnetic bead complexes after incubation with MM1S, H929, and JJN3 cell lysates untreated or treated with neuraminidase. (A-D) Combined data represented as bar graphs and an representative dot blot from one individual MM BMA sample or biological repeat. Data in E are represented as a volcano plot. n = 8 independent samples for ND. (A) n = 3 for MGUS. (B) n = 3 biological replicates. (C) n = 11; (D) n = 7; (E) n = 3.

Fig 4: A cis-binding Siglec-9 agonist (pS9L) inhibits R848-induced NETosis via Siglec-9 and SHP-1. (a-c) Primary neutrophils were cotreated with R848 (10 µM) and glycopolypeptide (500 nM) in IMDM supplemented 0.5% hiFBS containing the membrane impermeable DNA intercalators Cytotox Green or Red (250 nM). Images were acquired by fluorescence microscopy every 15 min for 12 h. The area of all green fluorescent objects >300 µm2 was quantified and the total area was averaged across three images per well. Relative NETosis was determined by normalizing to the maximal NET area from R848 treatment alone (t = 8 h). (a) Representative phase contrast and fluorescence images from t = 8 h. Scale bars indicate 40 µm. (b) Quantitation of NETosis over time as area under the curve in (c). Error bars represent SD. (c) NET formation and degradation as a function of time. Error bands represent SEM. (d) Treatment of R848-stimulated neutrophils with various glycopolypeptides. Error bars represent SD. (e) pS9L is a mucin-like glycopolypeptide that bears high affinity and specific ligands for Siglec-9 and is functionalized with a membrane-tethering lipid tail. (f) HL-60 cells were transfected with siRNAs against SIGLEC9 (encoding Siglec-9), PTPN6 (encoding SHP-1), or a scrambled control and then grown for two days. Cells were then cotreated with R848 (10 µM) and vehicle or pS9L (500 nM). Relative NETosis is determined as in (b), except all objects >200 µm2 were quantified and the R848 maximum in dHL-60’s was observed at 2.5 h post induction. Error bars represent SD. Statistics were determined by two-way ANOVA (b) or one-way ANOVA (c,d,f). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig 5: SIGLEC7, SIGLEC9 and SIGLEC10-positive myeloid infiltration is increased in breast tumors and correlates with aggression. A. UMAP projection of immune cells from scRNAseq data of 34 breast tumors [20]. B. Dot plot of SIGLEC expression in immune cell clusters in breast tumors. C. Quantification of i. SIGLEC7, ii. SIGLEC9, and iii. SIGLEC10-positive inflammation as a percentage of total inflammation in normal breast and breast cancer subtypes. D. Quantification of myeloid infiltration as a percentage of total inflammation in normal breast and breast cancer subtypes. E. Heatmap showing the expression of gene signatures in RNAseq data from breast tumors from TCGA. Patients were ordered by the immunosuppressive Siglec signature.