JJN3 were treated with 10 nM of ATRA in combination with 300 M of SIA prior to treatment with Dara and cocultured with NK cells as described previously. Percent specific lysis was calculated using the following formula: .05; ** .01; *** .001; **** .01; ns, not significant. Targeted desialylation of MM cells potentiates NK cell activation and tumor cell lysis To investigate the potential role of hypersialylation in regulating NK cell cytotoxicity, MM cells were selectively desialylated using either NEURA or SIA. NEURA Pipamperone treatment resulted in a near complete abolishment of Siglec-7L and Siglec-9L expression on K562, H929, and JJN3?cells (supplemental Physique 2A). CD38 expression can be treated with all agglutinin was from Vector Mmp27 Laboratories (B-1265), 3Fax-Peracetyl Neu5Ac was from Merck (566224-10mg), daratumumab (Dara) (Janssen) was supplied by University Hospital Galway, and neuraminidase (was supplied by Roche (11 080 725 001). Carboxyfluorescein diacetate succinimidyl ester (CFSE) and cell fixation and permeabilization kit were from Thermo Fisher Scientific (“type”:”entrez-nucleotide”,”attrs”:”text”:”C34554″,”term_id”:”2370695″,”term_text”:”C34554″C34554 and 00-5523-00). Tag-it dye was from Biolegend (425101). GolgiStop was from BD Biosciences (554724). Propidium iodide (PI) was from Sigma. (Antibody details are located in Supplementary Materials.) Siglec ligand staining on MM cell lines and primary samples MM cell lines or isolated primary MM cells were stained with Siglec-7 and Siglec-9 chimeras and then washed and stained with APC AffiniPure F(ab)2 fragment goat anti-human immunoglobulin G, Fc- fragment specific. Dead cells were excluded using PI staining. Siglec-7 ligand pulldown and analysis using mass spectrometry MM1S, JJN3, and H929 MM cell pellets were lysed, untreated or treated, with neuraminidase (NEURA). Lysates were then incubated with magnetic beadCSiglec-7 Fc chimera complexes, and bound ligands were subsequently isolated and analyzed using mass spectrometry. Full methodology for Siglec-7 ligand pulldown and mass spectrometry analysis is available in Supplementary Pipamperone Materials. NK cell growth Fresh peripheral blood or buffy coats were obtained from healthy donors after informed consent. Primary NK cells were isolated by unfavorable selection (Miltenyi) and expanded using NK cell growth media (Miltenyi) supplemented with 5% heat inactivated human AB serum (Sigma) and 500 IU/mL of IL-2. Expansions were cultured at 37C, 5% CO2. MM cell desialylation assays Briefly, JJN3, H929, MM1S, K562, and primary MM cells were pretreated with 0.1 U/mL of the sialidase NEURA or glycobuffer (GLYCO) control or treated with 300 M of 3Fax-Peracetyl Neu5Ac (sialyltransferase inhibitor, SIA) or DMSO control for 72 hours and subsequently cocultured with CFSE-stained KHYG-1 or primary NK cells (na?ve, IL-2 activated, and expanded) at indicated effector/target (E:T) ratios in 4-hour cytotoxicity assays in flat-bottomed 96-well plates. Total MM cell death (CFSE- cells) was determined by PI staining. In cytotoxicity assays using Dara, JJN3 and H929 were treated with SIA as described previously and treated with 10 g/mL of Dara for 30 minutes prior to being cocultured with specific NK cells. JJN3 were treated with 10 nM of ATRA in combination with 300 M of SIA prior to treatment with Dara and cocultured with NK cells as described previously. Percent specific lysis was calculated using the following formula: .05; ** .01; *** .001; **** .01; ns, not significant. Targeted desialylation Pipamperone of MM cells potentiates NK cell activation and tumor cell lysis To investigate the potential role of hypersialylation in regulating NK cell cytotoxicity, MM cells were selectively desialylated using either NEURA or SIA. NEURA treatment resulted in a near complete abolishment of Siglec-7L and Siglec-9L expression on K562, H929, and JJN3?cells (supplemental Physique 2A). Additionally, significantly enhanced NK cellCmediated lysis of desialylated MM cells was observed by naive, IL-2 activated, and expanded NK cells (Physique 3A-C). Additionally, enhanced KHYG-1-mediated cytotoxicity was observed in cocultures with NEURA-treated JJN3 (supplemental Physique 2B). NEURA treatment was observed to be toxic to MM1S (supplemental Physique 2C). Thus, a need for a gentler approach to desialylate MM cells was required. Using the cell-permeable sialic acid analog SIA, sialyltransferase activity was inhibited in MM1S cells without toxicity, and.
