Assess NK Cell Function and Phenotype

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.

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. 

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.