## 4 Discussion
Importantly, basal CD28 expression in healthy volunteers, as well as in patients, correlates with a T cell phenotype more sensitive to stimulation, and low CD28 expression is a marker of disease [73]. Moreover, plasma treatment of splenocytes significantly increased levels of IL-4 and IL-12. The former induces T\({}_{\rm H}\)2 and the latter T\({}_{\rm H}\)1 responses, having opposite roles in health and disease [74]. Both cytokines are derived from myeloid cells such as basophils and monocytes/macrophages [75]. Although calcium increase was not observed in immune cells upon co-cultured with plasma-treated melanoma cells, an upregulation of surface marker was observed in monocytes in that condition. CD86 expression is induced upon stimulation in monocytes, leading to an inflammatory milieu [76] and proper T cell co-stimulation [77]. Increase of IgE receptor (Fc\(\varepsilon\)R) dependent activation of monocytes is associated with tissue inflammation and cytotoxicity allergic diseases and asthma [78; 79]. However, significantly regulated was only colony-stimulating factor 1 receptor (CSF-1R or CD115). CD115 contributes to macrophage maturation [80] and is crucial for the development of certain myeloid subsets as CD115 blockage shortens the life span of monocytes [81]. Several studies by others and us have observed plasma-induced tumor cell death before [82; 83; 84; 85; 86; 87; 88]. [PERSON] and colleagues observed in several pancreatic cancer cell lines an increase in apoptosis concomitant with downregulation of MAPK7, BCL2, and CHK1 [99]. In line with our results, they have observed an increase in H\({}_{2}\)O\({}_{2}\) with plasma treatment using the kINPen, and moreover found apoptosis-inducing effects of plasma treatment in lung cancer cells based on increase in plasma-mediated ROS formation and intracellular oxidation signaled via ATM and p53 machinery [100], which is supported by our recent data in HaCaT keratinocytes [101]. Figure 4: CD28 expression in T lymphocyte splenocytes subpopulations co-cultured with control or plasma-treated melanoma cells. (**a**) Representative flow cytometric dot plots used to gate for two T lymphocyte subpopulations, namely CD4+ T helper and CD8+ cytotoxic T cells; (**b**,**c**) representative overlay histograms for CD28 marker in expression in CD4+ and CD8+ cells, respectively; (**d**) fold change quantification of CD28 expression in T cells co-cultured for 24 h with either control or plasma-treated melanoma cells; (**e**) CD4/CD8 of T cells co-cultured for 24 h with either control or plasma-treated melanoma cells. Data are mean + S.E. of several spleens, statistical analysis was performed using t test. Figure 5: Activation of monocyte and macrophage splenocyte subpopulations after co-culture with melanoma cells. (**a**) Representative gating strategy to identify monocytes and macrophages within splenocytes; (**b**) of change expression of activation marker expression in monocytes/macrophages 24 h after co-culture with either control (C) or plasma-treated (P) melanoma cells; (**c**) cytokine profile supernatants retrieved from transwell co-cultures of control or plasma-treated melanoma cells with splenocytes. Data are presented as mean + S.E. of several spleens; statistical analysis was performed using t test.