Contributions
Abstract: PB2136
Type: Publication Only
Background
Multiple Myeloma (MM) is a malignant plasma cell disorder, accounting for 10% of all haematological malignancies. The immune-suppressive functions of sialylated glycans, abundant on the surface of myeloma cells, has not previously been addressed. We hypothesize that hypersialylation of MM enables evasion of Natural Killer (NK) cells within the bone-marrow niche. Disrupting the interactions between these sialylated glycans and their cognate sialic acid-binding lectin (Siglec) receptors on NK cells could lead to the development of novel immunotherapeutic strategies for treating MM.
Aims
Determine the expression of Siglec-7 ligands on a panel of MM cell lines and primary MM cells and determine the expression of Siglec-7 on primary bone marrow-derived NK cells. Determine the effect de-sialylating MM cell lines has on their sensitivity to NK-mediated cytotoxicity using both a sialidase and sialyltransferase inhibitor and the effect blocking Siglec-7/Ligand interactions has on NK-mediated cytotoxicty.
Methods
Siglec-7 ligand expression was measured by staining with a recombinant Siglec-7 Fc chimera (1.25µg/ml, R&D systems) and flow cytometry was used to determine Siglec-7 ligand positive cells. Siglec-7 expression was measured using an anti-Siglec-7 antibody (Miltenyi) and flow cytometry.
In order to desialylate MM both the sialidase Neuraminidase (Sigma-Aldrich) and sialyltransferase inhibitor P-3Fax-Neu5Ac (Merck) were used. Cells were either treated with 1X neuraminidase for 45mins prior to co-culture, or with 200µM 3Fax for 5 days.
To block Siglec-7 interacting with its concomitant ligands, RPMI8226 myeloma cells were treated with recombinant Siglec-7 Fc chimera (10 µg/ml, R&D systems) for 30mins prior to co-culture with KHYG-1 NK cells.
Results
We observed that Siglec-7 ligands (Siglec-7L) are highly expressed across a panel of MM cell lines. Furthermore, Siglec-7L expression was also observed on CD38+/CD138+ primary MM cells isolated from BM aspirates of newly diagnosed and relapsed MM patients (N=3,N=2 respectively). Immunophenotyping analysis revealed that the BM-derived NK cells of MM patients have significant cell surface expression of the cognate Siglec-7 receptor (Siglec-7R)(82±25%, n=5).
Cytotoxicity assays and flow cytometry analysis revealed that abolishing sialylated glycans from the cell surface of MM cells using a sialyltransferase inhibitor results in a 1.3-1.6 fold increase (p<0.05) in NK cell induced cell death in MM cell lines RPMI8226 and H929, but not MM1S. (Fig.1, n=3)
Furthermore, to validate our observations we pre-treated RPMI8226 cells with 10µg/ml of recombinant Siglec-7 Fc chimera to block Siglec-7 ligands on the cell surface. We observed statistically significant enhancements of NK cytotoxicity against the Siglec-7 Fc chimera treated RPMI 8226 versus an isotype Fc control and at all E:T ratios (P = 0.003 1:1, P = 0.001 2.5:1, P = 0.03 5:1).
Conclusion
We observed that Siglec-7 and it's cognate ligands are abundantly expressed on the surface of NK and MM cell lines respectively. Additionally, Siglec-7 is significantly expressed on primary NK cells circulating within the BM of MM patients. Functional assays revealed that Siglec-7L and Siglec-7R receptor interactions have significant immune suppressive effects on the cytotoxicity of NK cells towards MM cell lines. Thus, we can leverage the findings of this study to develop NK cell based cellular therapies to target Siglec-7 ligand- receptor interactions in MM.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Immunology, Immunosuppressive therapy, Multiple Myeloma, Natural killer
Abstract: PB2136
Type: Publication Only
Background
Multiple Myeloma (MM) is a malignant plasma cell disorder, accounting for 10% of all haematological malignancies. The immune-suppressive functions of sialylated glycans, abundant on the surface of myeloma cells, has not previously been addressed. We hypothesize that hypersialylation of MM enables evasion of Natural Killer (NK) cells within the bone-marrow niche. Disrupting the interactions between these sialylated glycans and their cognate sialic acid-binding lectin (Siglec) receptors on NK cells could lead to the development of novel immunotherapeutic strategies for treating MM.
Aims
Determine the expression of Siglec-7 ligands on a panel of MM cell lines and primary MM cells and determine the expression of Siglec-7 on primary bone marrow-derived NK cells. Determine the effect de-sialylating MM cell lines has on their sensitivity to NK-mediated cytotoxicity using both a sialidase and sialyltransferase inhibitor and the effect blocking Siglec-7/Ligand interactions has on NK-mediated cytotoxicty.
Methods
Siglec-7 ligand expression was measured by staining with a recombinant Siglec-7 Fc chimera (1.25µg/ml, R&D systems) and flow cytometry was used to determine Siglec-7 ligand positive cells. Siglec-7 expression was measured using an anti-Siglec-7 antibody (Miltenyi) and flow cytometry.
In order to desialylate MM both the sialidase Neuraminidase (Sigma-Aldrich) and sialyltransferase inhibitor P-3Fax-Neu5Ac (Merck) were used. Cells were either treated with 1X neuraminidase for 45mins prior to co-culture, or with 200µM 3Fax for 5 days.
To block Siglec-7 interacting with its concomitant ligands, RPMI8226 myeloma cells were treated with recombinant Siglec-7 Fc chimera (10 µg/ml, R&D systems) for 30mins prior to co-culture with KHYG-1 NK cells.
Results
We observed that Siglec-7 ligands (Siglec-7L) are highly expressed across a panel of MM cell lines. Furthermore, Siglec-7L expression was also observed on CD38+/CD138+ primary MM cells isolated from BM aspirates of newly diagnosed and relapsed MM patients (N=3,N=2 respectively). Immunophenotyping analysis revealed that the BM-derived NK cells of MM patients have significant cell surface expression of the cognate Siglec-7 receptor (Siglec-7R)(82±25%, n=5).
Cytotoxicity assays and flow cytometry analysis revealed that abolishing sialylated glycans from the cell surface of MM cells using a sialyltransferase inhibitor results in a 1.3-1.6 fold increase (p<0.05) in NK cell induced cell death in MM cell lines RPMI8226 and H929, but not MM1S. (Fig.1, n=3)
Furthermore, to validate our observations we pre-treated RPMI8226 cells with 10µg/ml of recombinant Siglec-7 Fc chimera to block Siglec-7 ligands on the cell surface. We observed statistically significant enhancements of NK cytotoxicity against the Siglec-7 Fc chimera treated RPMI 8226 versus an isotype Fc control and at all E:T ratios (P = 0.003 1:1, P = 0.001 2.5:1, P = 0.03 5:1).
Conclusion
We observed that Siglec-7 and it's cognate ligands are abundantly expressed on the surface of NK and MM cell lines respectively. Additionally, Siglec-7 is significantly expressed on primary NK cells circulating within the BM of MM patients. Functional assays revealed that Siglec-7L and Siglec-7R receptor interactions have significant immune suppressive effects on the cytotoxicity of NK cells towards MM cell lines. Thus, we can leverage the findings of this study to develop NK cell based cellular therapies to target Siglec-7 ligand- receptor interactions in MM.
Session topic: 13. Myeloma and other monoclonal gammopathies – Biology & Translational Research
Keyword(s): Immunology, Immunosuppressive therapy, Multiple Myeloma, Natural killer