Chapter 4 │ Page 137 Figure 6: Exposure to NTP results in moderate expressional changes of the MHC-I complexes HLA-C and HLA-E in several HNSCC cell lines. Quantification of (a) HLA-C and (b) HLA-E expression after exposure to di erent regimes of NTP. Results are depicted as mean fluorescence intensity (MFI) minus FMO control, normalized to untreated controls (normalized (Norm) ΔMFI), immediate (0h) and one day (24h) post-treatment for all three HNSCC cell lines. Data are represented as mean ± SEM, with individual values shown (n = 5). Statistical significance between untreated cells and the treated conditions was determined using the generalized linear mixed model with post hoc Dunnett’s test (* p ≤ 0.05; ** p ≤ 0.01). Outliers were calculated with the Grubbs’ Test. 3.4. Key Targets In The TIGIT Axis Are Downregulated By NTP-Induced Ligand Oxidation As NK functionality is determined by the integration of complex activation and inhibition signals rather than a single interaction impulse [61-63], we subsequently investigated a panel of key tumor ligands of the TIGIT receptor, a major inhibitory pathway for NK functionality [61, 64]. CD155 and CD112 are inhibition signals for this highly immunosuppressive axis, though CD115 is the dominant antigen while the binding with CD112 is weaker [64, 65]. As recent studies indicated that targeting of CD155 and CD73 works synergistically and surpasses the failure in overcoming NK cell dysfunction by CD155 monotherapy [64], we also included this immune checkpoint in the analysis (Figure 7). Immediate NTP-induced oxidation of the target ligand resulted in significantly reduced expression of both CD155 and CD112, in the Cal27 and SCC61 cell line with the higher treatment regime (0h; 500Hz), as shown in Figure 7a,b. Both ligands were diminished with 21.9% and 24.8%, respectively for the Cal27 cell line, while 12-15% reduction was noted for SCC61 (p ≤ 0.0304). These e ects became even more pronounced for the immune checkpoint CD73 with a strong decline in ΔMFI, around 36%, for both cell lines (Figure 7c). It needs to be stated that these oxidative e ects are transient, as 24h later, expression levels stabilized again to baseline. It is apparent that both cell lines responded very similar to therapeutic oxidation, while the SCC22B cell line exploits a more robust, oxidation-resistant phenotype. Although a trend in decreased CD155-ΔMFI could be observed
RkJQdWJsaXNoZXIy MTk4NDMw