Fig 1: Targeted knockout of Siglec-7 using CRISPR/Cas9 enhances NK cell–mediated cytotoxicity toward Siglec-7L+/CD38+ MM cell lines. Siglec-7 was targeted for KO using the CRISPR/Cas9 system and the MaxCyte GT transfection system. NK cells were isolated and expanded from peripheral blood of healthy donors for 10 to 14 days prior to transfection. Cytotoxicity assays using CRISPR/Cas9-targeted NK cell were carried out 6 to 9 days after transfection. Siglec-7 KO was determined using flow cytometry, and efficiency was analyzed and displayed as a histogram (A) representative of n = 1 Siglec-7 KO readout on viable NK cells and bar graphs (B) of mock electroporated and CRISPR-targeted NK cells representing complete KO of Siglec-7 in n = 7 donors. Functionality of Siglec-7 KO NK cells was measured in cytotoxicity assays against the Siglec-7L+ MM cell lines H929 (C) and JJN3 (D). (E) CD16 expression was recorded on Mock and Siglec-7KO NK cells using flow cytometry. (F) Mock and Siglec-7KO NK cells were cocultured with Dara-treated CD38+ MM1S MM cells in cytotoxicity assays. Cytox assays were carried out for 4 hours; graphs represent mean specific lysis + SEM. n = 7 biological replicates (B-C); n = 6 (F). (B-D) Data analyzed using Student’s paired t-test; (F) data analyzed using one-way ANOVA. (C-D) Data represent individual values recorded in n = 7 biological replicates and fold change in specific lysis by both mock and Siglec-7KO NK cells. *P < .05; **P < .01; ****P < .0001.
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 expression is increased on NK cells from BMAs of patients with MM, while Siglec-9 expression is decreased. CD56+/CD3- NK cells sourced from peripheral blood of healthy donors (black) and from BMAs of patients with MM (red) were stained for the expression of (A) Siglec-7 and (B) Siglec-9. Data analyzed using Mann-Whitney unpaired t-test. Graphs represent both mean Siglec-7+ NK cells and mean MFI of Siglec-7+ NK cells as well as individual values for each sample. n = 5 for healthy donor peripheral blood-derived NK cells, and n = 6 for MM patient BMA-derived NK cells. **P < .01; ns, not significant.
Fig 4: 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 5: Erythrocytes induce chSiglec-7 signaling, accompanied by chSiglec-7 clustering. a Erythrocytes were either left untreated or treated with sialidase. Siglec-7 ligands were assessed by a recombinant human Siglec-7 Fc staining or respective IgG1 Fc control and measured by flow cytometry (n = 3). A representative figure of three independent experiments is shown. b chSiglec-7 or chSiglec-7R124A expressing Jurkat/MA cells were co-cultured with untreated or sialidase treated erythrocytes (1:2), after which luminescence was assessed using a luciferase assay, nested data of 5 donors are shown as mean ± SD. Figure represents nested data of 5 experiment, each containing data from one donor. c Erythrocytes were stained using Far Red Cell Trace and a co-culture was performed as described in (b). Subsequently, the cells were stained for Siglec-7 membrane expression (n = 2). d Same as in (c), but erythrocytes were treated with sialidase (n = 2). e Same as in (c), but chSiglec-7R124A expressing Jurkat/MA cells were co-cultured with untreated erythrocytes (n = 2). c–e Representative images are presented from two independent experiments. Scale bars: 25 µm. Signal in (c–e) was differently enhanced to visualize chSiglec-7 or chSiglec-7R124A distribution
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