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Flow Cytometry-based Assay for the Monitoring of NK Cell Functions
Published on: October 30, 2016
Production and characterization of a novel monoclonal antibody inhibitory for murine natural killer cell activity
1Department of Microbiology, Dalhousie University, Halifax, Nova Scotia.
Abstract:
Natural killer (NK) cells are considered to play an important role in tumor surveillance. The killing of tumor target cells by NK cells is the result of a complex series of sequential binding, signal processing and lytic events. However, the mechanism which NK cells use to recognize tumor targets is poorly understood. To further study the cell-surface molecules involved in tumor recognition, we immunized rats against cloned murine T cells with NK activity (DBA/2.1) and generated rat-mouse hybridomas which were screened for the ability to block lytic activity of DBA/2.1 effector cells. Culture supernatants from one IgM-producing hybridoma, designated S1C4, were found to consistently inhibit DBA/2.1-mediated lysis of YAC-1 target cells. Endogenous splenic NK activity was also diminished in the presence of S1C4 monoclonal antibody (mAb) while alloantigen-specific cytotoxic T lymphocyte (CTL) activity was not affected. S1C4 mAb appears to react with effector cell-surface structures involved in the recognition/adhesion phase of NK activity since pretreatment of effector cells with mAb S1C4 inhibits their ability to bind to YAC-1 target cells. ELISA studies revealed that the S1C4 antigen is expressed by a range of lymphoid cell lines, as well as by DBA/2.1 cells and fresh splenic NK cells. S1C4 mAb were shown to react with 22, 24, 30, and 46 kiloDalton (kDa) DBA/2.1 cell membrane components on immunoblots performed under reducing conditions. These structures do not correspond to any known recognition/adhesion molecules, suggesting that mAb S1C4 defines novel cell membrane components involved in NK cell function.
Insights
This study identifies novel cell surface molecules involved in natural killer (NK) cell tumor recognition. The S1C4 monoclonal antibody blocks NK cell binding and lysis of tumor targets, suggesting new targets for immunotherapy.
Area of Science:
- Immunology
- Cell Biology
- Cancer Research
Background:
- Natural killer (NK) cells are crucial for tumor surveillance through cell-mediated cytotoxicity.
- The precise mechanisms of NK cell target recognition remain incompletely understood.
- Identifying cell-surface molecules involved in NK cell recognition is key to understanding their function.
Purpose of the Study:
- To investigate novel cell-surface molecules mediating NK cell recognition of tumor targets.
- To characterize the function of these molecules in the NK cell cytotoxic pathway.
Main Methods:
- Generation of hybridomas producing monoclonal antibodies against NK effector cells.
- Screening of antibodies for inhibition of NK cell-mediated lysis.
- Characterization of antibody-defined antigens using ELISA and immunoblotting.
Main Results:
- A novel monoclonal antibody, S1C4, was identified that inhibits NK cell lysis and binding to tumor cells.
- S1C4 specifically affects NK cell activity, not cytotoxic T lymphocyte (CTL) activity.
- Immunoblotting revealed S1C4 reacts with 22, 24, 30, and 46 kDa cell membrane components.
Conclusions:
- The S1C4 antibody defines previously unrecognized cell membrane components critical for NK cell recognition and adhesion.
- These novel molecules represent potential targets for enhancing NK cell-based cancer therapies.
