Chapter 4 │ Page 142 significantly after oxidation. Indeed, most of the solvent accessible amino acids prone to oxidation in the protein complex were found to be located in the α3 region of both HLA-Cw4 and HLA-E, and in β2M. Furthermore, of the amino acids likely oxidized by plasma treatment that are situated in the binding domain of the complex, none play a direct role in the actual interaction between the HLA proteins and their receptors. In accordance with our computational results, we experimentally observed only minor changes in the expression status of HLA-C and HLA-E by NTP oxidation. A significant decrease in the detection of HLA-C directly after treatment was solely observed in the SCC61 cell line (Figure 6). In contrast, NTP caused a rapid reduction of the two TIGIT ligands CD155 and CD112, and immune checkpoint CD73 in two out of three tested cell lines (Figure 7). It is clear that some immune checkpoints are more vulnerable to therapeutic oxidation, and linking this vulnerability to the structure of these proteins forms an interesting avenue for future computational analysis. Even so, it is essential to recognize some nuances in this context, because the experimental approach of binding a monoclonal antibody for fluorescence staining to the target ligand only partially approximates the complete ligand-receptor interaction in vivo. Indeed, the binding mechanism of the antibody can be [32], but is not necessarily, the same as that of the biological receptor. Still, far-leading structural changes due to (NTP-induced) oxidation would a ect the binding to both antibody and receptor. 24h after NTP application, the ∆MFI of HLA-C and HLA-E remained around baseline (Figure 6). The same is true for ligands CD155, CD112, and CD73, indicating that the e ects observed immediately after treatment were transient. However, NTP did stimulate the cellular upregulation of the activating NK ligands MICA/B (Figure 8). This aligns with previous reports, highlighting MICA/B as important stress proteins in response to cellular imbalance and DNA damage, which are key mechanisms of action of NTP [59, 73]. Moreover, we can conclude that the NTP investigated in this
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