Expanded Natural Killer Cells As Research Tool | Ignyte Bio
Natural killer (NK) cells are an important subset of lymphocytes for providing our body’s first line of defense. NK cells were originally described for their capacity to spontaneously kill tumor cells without prior sensitization which distinguishes them from cytotoxic T lymphocytes (CTLs).
Mature NK cells express a series of transmembrane receptors: activating receptors that allow them to recognize stress-induced ligands, and inhibitory receptors that prevent them from attacking normal cells. Upon ligand binding, these activating receptors can activate down-stream kinases, leading to NK cell degranulation and direct cytotoxicity of target cells as well as secretion of inflammatory cytokines. Binding of inhibitory receptors, on the other hand, leads to inhibition of NK cell functions. NK cell activity is tightly regulated through the balance between inhibitory and activating signals transduced by these receptors.
In addition to direct cytotoxicity, NK cells are capable of killing target cells through antibody-mediated cell cytotoxicity (ADCC) mechanism. Human NK cells can express both Fc receptors: FcγRIIC (CD32c) and FcγRIIIA (CD16a), which bind to the Fc portion of human immunoglobulins. Upon binding, through signal transduction mechanisms, NK degranulation, cytokine secretion and target cell lysis occur.
NK cell’s capabilities of tumor cytotoxicity and inflammatory cytokine production enable NK cells to play an important role in different settings of cancer immunotherapy.
Expanded Human NK Cells
Based on their important role in cancer immunotherapy, there has been a lot of research interest in NK cells, including their use in cell-based assays (e.g., NK cytotoxicity and ADCC). Researchers often experience logistic challenge when procuring fresh human NK cells for their cell-based assay. The steps taken prior to starting the assay, from ordering and receiving raw materials (e.g., blood or leukapheresis material) to NK isolation procedures, analysis of isolated NK etc. are very time consuming.
Ignyte Bio offers both cryopreserved isolated NK cells (negative selection) and our newest product: cryopreserved human expanded NK cells as “thaw and use” alternatives. Our expanded NK cell products have the following superior characteristics to support your assay:
- High viability (>85%)
- High purity (>85%) CD56+, CD3- population (figure 1)
- Relevant donor information, including FcγRIIIA-158 genotype, is included in each COA
- Can be used in assay directly after thawing
- Post-thaw cells are active and functional, as demonstrated in direct cell killing of target cells (e.g. K562, and ADCC (figure 2)
Figure 1. Phenotype of Cryopreserved Expanded Human NK Cells. Cryopreserved expanded human NK cells were thawed and incubated with anti-human CD14 FITC, CD19PE, CD56 Pacific Blue and CD13 APC, or CD314 (NKG2D) FITC, CD16 Pacific Blue and CD56 APC, or with isotype controls. Samples were analyzed in the Beckman CyAn flow cytometer.
Figure 2. Assays to Demonstrate Functional Activity of Expanded NK Cells. (a) Recognition and lysis of K562. Cryopreserved isolated and expanded NK cells from donor 102 were thawed and cultured in the presence of K562 target cells at various NK to target cell ratios. After an overnight incubation period, % dead target cells were analyzed using 7-AAD viability dye and plotted against NK to target cell ratios. Data presented shows increased recognition and lysis of K562 by expanded NK cells compared to isolated NK cells (b) ADCC of B-LCL target cells in the presence of anti-CD20 monoclonal antibody. B-LCL target cells were labelled with Tag-It Violet dye (Biolegend) and incubated in the absence or presence of 1 ug/mL Rituximab biosimilar (Biolegend) or human IgG1 isotype control (Biolegend). Expanded NK cells were thawed and added at various NK to target ratios. After an overnight incubation, % dead target cells were analyzed using 7-AAD viability dye and plotted against NK to target cell ratios