Assess NK Cell Function and Phenotype
Natural Killer (NK) cells perform immunological surveillance as one of the body’s first responders to infections and cancer. Understanding NK biology and activity is critical to fully exploit their immune response potential. Use Incucyte® live-cell imaging and analysis and iQue® 3 advanced flow cytometry for activities such as immunophenotyping, assessment of functional activity, cell viability, immunophenotyping, and profiling of cytolytic effector proteins, cytokines, and chemokines.
3D Immune cell killing
Measure ADCC in tumor spheroids with Incucyte®. Blended phase and fluorescent images of SKOV-3 Incucyte® NucLight Red spheroids in the presence immune cells, either untreated or in the presence of anti-CD3 (10 ng/ml) and IL-2 (10 ng/ml) or Herceptin (0.08 – 50 ug/mL). Cytotoxicity was quantified based on the red fluorescent intensity. Data demonstrates destruction of tumor spheroids by the activated T-cell population and a Herceptin-induced cell cytotoxicity.
Gain biological insights into NK cell mediated tumor cell killing in physiologically relevant models
Natural killer cell mediated-antibody-dependent cellular cytotoxicity induces concentration dependent target cell death, granzyme production.
Encoded Raji tumor cells (20K/well) were co-cultured with PBMCs (200K/well) from two separate donors. PBMCs were incubated with one of three anti-CD20 antibodies: Ab-1 (IgG1), Ab-2 (IgG1) or a negative control Ab-3 (IgA2). Concentration range was between 10ug/mL – 0.128 ng/mL.
At 4h, 10 µL samples were analyzed to assess tumor cell killing using the Human NK Cell Killing Kit and the iQue® 3, Granzyme A was also measured using an iQue® Human NK Cell Companion Kit. (A,B) Target cell killing by two donors show differential response to the antibodies. (C,D) Granzyme A production was both concentration and donor dependent. (E,F) Detection of CD16 on NK cells decreased with increasing concentration of anti-CD20 antibody. This could be due to CD16 shedding or competition of CD20 antibody.
Cytokine stimulation enhances direct NK cell-mediated killing of tumor cells. Encoded K562 tumor cells (20K/well) were co-cultured with enriched (negatively selected) human NK cells that had been incubated for 16-18 h in either media alone (Non-activated NK cells) or media containing 200 U/mL IL-2 & 100 ng/mL IL-15 (Activated NK cells) at an Effector:Target ratio of 1:1 or 5:1. At 4 h and 24 h, 10 µL samples were analyzed to assess tumor cell killing using the iQue® Human NK Cell Killing Kit and the iQue® 3. (A) Histogram depicting target cell viability following co-culture with non-activated or cytokine activated NK cells for 4 h. (B,C) Summary of percent target cell death following co-culture of tumor cells with non-activated or cytokine activated NK cells for (B) 4 h or (C) 24 h.
Simultaneously quantify NK cell surface protein & cytokine expression
NK cell activation is induced in the presence of target cells and further enhanced by cytokine stimulation. Encoded K562 tumor cells (20K/well) were co-cultured with enriched (negatively selected) human NK cells that had been incubated for 16-18 h in either media alone (Non-activated NK cells) or media containing 200 U/mL IL-2 & 100 ng/mL IL-15 (Activated NK cells) at an Effector:Target ratio of 5:1. Following 24 h of co-culture, 10 µL samples were removed and analyzed using the iQue® Human NK Cell Killing Kit and the iQue® 3 system. (A) Expression of activation markers, CD69 and CD25. (B) Production of IFNg and Granzyme B secretion. (C) Additional cytokines were assessed in parallel by combining iQue® Human NK Cell Companion Kits with the iQue® Human NK Cell Killing Kit. NK = Natural Killer cells. T = Target K562 tumor cells.
Activation with IL-2 and IL-15 caused increased activation marker expression and secretion of multiple effector proteins, such as Granzyme B and CCL5 (RANTES). These effects were further enhanced when NK cells were cultured with target cells.
For further reading:
1. Tarazona R et al. Current progress in NK cell biology and NK cell-based cancer immunotherapy. Cancer Immunol Immunother. 2020 May;69(5):879-899. doi: 10.1007/s00262-020-02532-9. Epub 2020 Mar 4
2. Hodgins JJ, Khan ST, Park MM, Auer RC, Ardolino M. Killers 2.0: NK cell therapies at the forefront of cancer control. J Clin Invest. 2019 Sep 3;129(9):3499-3510. doi: 10.1172/JCI129338
3. Waggoner SN et al. Roles of natural killer cells in antiviral immunity. Curr Opin Virol. 2016 Feb;16:15-23. doi: 10.1016/j.coviro.2015.10.008. Epub 2015 Nov 16
4. Yao Z, Zheng Z, Wu K1, Junhua Z. Immune environment modulation in pneumonia patients caused by coronavirus: SARS-CoV, MERS-CoV and SARS-CoV-2. Aging (Albany NY). 2020 May 2;12. doi: 10.18632/aging.103101
Representative Incucyte® and iQue® Publications
NK in Tumor Cell Suppression and Resistance
Sarhan D, et al. Adaptive NK Cells Resist Regulatory T-cell Suppression Driven by IL37. Cancer Immunol Res. 2018 Jul;6(7):766-775. doi: 10.1158/2326-6066.CIR-17-0498. Epub 2018 May 21
Kamiya T, Seow SV, Wong D, Robinson M, Campana D. Blocking expression of inhibitory receptor NKG2A overcomes tumor resistance to NK cells. J Clin Invest. 2019 Mar 12;129(5):2094-2106. doi: 10.1172/JCI123955. Print 2019 May 1
Li Y, Hermanson DL, Moriarity BS, Kaufman DS. Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell. 2018 Aug 2;23(2):181-192.e5. doi: 10.1016/j.stem.2018.06.002. Epub 2018 Jun 28
High Throughput ADCC Assay Development
Camacho-Sandoval R, et al. Taking advantage of a high-throughput flow cytometer for the implementation of an ADCC assay for regulatory compliance. Biotechnol Rep (Amst). 2020 Apr 19;26:e00456. doi: 10.1016/j.btre.2020.e00456. eCollection 2020 Jun
NK Cells and Vaccine Development
Boudreau CM, Yu WH, Suscovich TJ, Talbot HK, Edwards KM, Alter G. Selective induction of antibody effector functional responses using MF59-adjuvanted vaccination. J Clin Invest. 2020 Feb 3;130(2):662-672. doi: 10.1172/JCI129520