Natural killer (NK) cells represent crucial players of the innate immune system and fulfil their main function in first line of defense by recognizing and eliminating tumor degenerated and virus infected cells. To analyze and influence NK cell behavior it is necessary to be able to specifically activate NK cells. In this work, known NK cell-specific stimulants were used in whole blood cultures (TruCulture) to investigate the specificity of the NK cell-activating stimuli in the high complexity of this culture system and to determine whether and to what extent co-activation of further immune cells of the peripheral blood occurs. For this purpose, it was necessary to generate appropriate test systems. Thus, two bead-based multiplex Luminex immunoassays, IMAP 1 and IMAP 2, were developed for the detection of nine (IL-4, -6, -8, 10, GM CSF, IFN-γ, MCP-1, MIP-1β, TNF-α) and six (IL-1β, -1Ra, -12p70, -13, VEGF, M-CSF) analytes respectively. Additionally, highly sensitive single-molecule arrays (Simoa) were established for IL 4 and IL 12p70 as single-plex assays and for IL-6 and TNF-α as 2-plex assays as these four analytes required higher sensitivities than those provided by the Luminex technology. During assay development, parameters such as the basic buffer system and detector antibody concentration were optimized for optimal performance and sensitivity, with cross-reactivity between multiplexed analytes evaluated and reduced. Developed assays were then validated to confirm their potential as an analytical method for the TruCulture system and to confirm their reproducibility and validity. Method suitability was confirmed for the majority of analytes. Only for VEGF the pre-defined acceptance criteria for precision were not met, while for IL 4, IL-12p70, GM-CSF, VEGF and M-CSF, as part of the IMAPs, parallelism could not be demonstrated. A method comparison (Luminex vs. Simoa) using Passing-Bablok regression and Blant-Altman plots was performed for IL-6 and TNF-α assays to be able to use data from both assays for analysis. This showed comparability for both technologies.
The developed and validated assays were then used to assess NK cell activation in whole blood cultures, supplemented by flow cytometric analyses. Synergistic effects of the stimulant combinations IL-12 + IL-18, R848 + IL-2 and K562 cells + IL 2 were shown to induce the strongest activation states of NK cells. It was observed that R848 + IL-2 stimulated not only cytokine production but also the degranulation process as NK cell effector functions and led to a broad activation of all immune cell populations of the peripheral blood. In contrast, the combination IL-12 + IL-18 showed NK cell stimulation only in the direction of cytokine production and moderately activated other immune cells. Although it is not yet possible to store these cells at -20 °C, which is a prerequisite for their use as default stimulant in the TruCulture application, the most specific NK cell activation was observed by stimulation with K562 cells in combination with IL-2
