## 4 Discussion  
Our aim was to identify activation signatures in murine immune cells derived from spleen that had been co-cultured with plasma-treated melanoma cells. Although many responses observed in immune cells were rather subtle, their sum point to a principle recognition of plasma-treated compared to control melanoma cells. Direct plasma treatment induced pro-inflammatory cytokine release of IL-1\(\beta\) or TNF-\(\alpha\) in B16F10 melanoma cells. IL-1\(\beta\) is processed and released under caspase-8 regulated apoptotic cell death [41]. Apoptotic cells release uric acid, activating systemic inflammation and resulting in release of IL-1\(\beta\)[42]. Interleukin-1\(\beta\) is a potent activator molecule in T and B cell responses and supports survival of naive and memory T cells [43; 44; 45], potentially forming anti-tumor immunity. Tumor necrosis factor alpha (TNF\(\alpha\)) leads to pyrogenic activation, inducing apoptotic cell death via the extrinsic pathway [46]. Plasma treatment of melanoma cells in mono and co-culture also increased release of IL-10 and CCL, both positively regulating T cell function [47]. Hence, plasma-derived oxidative cell stress and/or death of melanoma cells profoundly affected inflammatory conditioning of cells of the immune system. Presence of FCS during plasma treatment was of minor importance corroborating previous results that excess protein has negligible effects on plasma-derived long-lived oxidants [36]. Plasma-induced tumor cell death supposedly was of an immunogenic nature, as release of ATP and CXCL1 suggests [48], which is in line with earlier reports [34; 35; 49; 50]. In this study, immune cells derived from spleens were exposed to plasma. Similar to studies with human immune cells [51; 52; 53], splenocytes' viability was affected by direct exposure to plasma, while their mitogenic activation (with PMA) reduced plasma-mediated toxic effects. As the conditions (cell number, treatment time, volume of media, etc.) were the same in both non-activated and activated regimens but with different outcomes, our results point to differences in intracellular signal translation upon expose to reactive species, as these responses ultimately determines the amplitude of toxicity. At lower concentrations, reactive species can also serve in cell signaling [54]. Several studies manifested the role of ROS, in particular H\({}_{2}\)O\({}_{2}\), as a stimulant and second messenger in lymphocytes [55]. An increase in intracellular calcium was found in plasma-treated T cells in our study, which points to their activation [56; 57]. Elevated intracellular calcium levels need to be maintained for more than 30 min for T cell activation [57; 58], which was the case with our results. Moreover, we determined a significant increase in Erk1/2 activation in T cells, which is associated with activation [59; 60]. In general, we do no propose a specific but rather non-specific stimulus of lymphocytes with plasma as this is highly unlikely without proper T cell receptor stimulation as previously described [22] and suggested experimentally [61; 62]. Co-culture of splenocytes with plasma-treated melanoma cells provoked calcium influx neither in T cells nor in macrophages. For T cells, this suggests a lack of specific antigen being presented either on tumor (MHC I) or antigen presenting cells (MHC II). For macrophages, calcium signaling is often related to aberrant stress of the endoplasmic reticulum and cell death [63] or sensing of microorganisms [64]. Both CD4\({}^{+}\) T helper and CD8\({}^{+}\) cytotoxic T cells contribute to antitumor immunity in tumor patients [65; 66; 67]. CD4\({}^{+}\) and CD8\({}^{+}\) T cells co-cultured with plasma-treated compared to untreated melanoma cells showed an increased expression of CD28. When binding to its ligands (B7 receptors, e.g., CD80/86) triggering IL-2 release [68], the CD28 cost stimulatory receptor is required for efficient and optimal immune response in T lymphocytes [69]. CD28-signaling through Bcl-2 and Bcl-xL promotes T cell survival [70; 71; 72].