The activation of Natural Killer (NK) cells depends on the stimulation of a broad range of receptors, which have overlapping and potentially redundant functions. Here, we aim to identify cause-effect relationships between the stimulation of distinct receptors and subsequent NK cell effector functions including cytokine secretion, degranulation, and cytotoxicity.

To stimulate human NK cells, we utilized NIH3T3 cells, an embryonic mouse fibroblast cell line that is not recognized by human NK cells. NIH3T3 cells were transfected to express defined ligands for activating NK cell receptors. To characterize different aspects of NK cell activity upon exposure to these targets, a combination of flow cytometry, chromium-release and xCelligence killing assays is employed.

We generated NIH3T3 cells stably expressing the NKp30 ligand B7H6, the 2B4 ligand CD48, the DNAM-1 ligands PVR (CD155) or Nectin-2 (CD112), the NKG2D ligand MICA, or the CD16 ligand CD20 (in combination with anti-CD20 antibody Obinutuzumab). In addition, the LFA-1 ligand ICAM-1 (CD54) was subsequently introduced into these cells to gain insights into the influence of adhesion. Initial findings suggested that target cells expressing B7H6, MICA, or CD20 are superior in triggering NK cell degranulation, cytokine release, and specific target cell killing. This activity could, to different extends, be further enhanced by addition of ICAM-1.

In the next steps of this ongoing project, combinations of B7H6, MICA, and CD20 with CD48 or PVR, respectively, will be tested to investigate the influence of co-stimulating ligand-receptor interactions on NK cell stimulation. Furthermore, a fluorescence localization reporter developed by our group will be employed to allow determination of which cytotoxic pathway is primarily triggered in NK cells upon stimulation of different receptors. Finally, the aspect of NK cell serial killing will be investigated in real-time microscopy-based assays.