## 3 Results
### DHT Increases the Cytotoxicity of PBMCs  
The role of T cells is as important as that of NK cells in tumor clearance [24]. We cannot exclude the possibility that DHT may enhance the cytotoxicity of CD8\({}^{+}\) T cells against SNU719 cells as much as NK cells. To confirm this, PBMCs were isolated from donor blood, and the HLA-A type of these PBMCs was determined by the sequencing of HLA-A exon 2 (Supplementary Figure S3A) and _HLA-A_ exon 4 (Supplementary Figure S3B). In addition, EBVaGC cell lines with HLA-A types such as SNU719, YCCEL1, and NCC24 cells were identified. The PBMCs from donor blood, SNU719 cells and YCCEL1 cells were identified as HLA-A*24:02 type. However, NCC24 cells were identified as HLA-A*11:01:01 type (Supplementary Figure S3). Therefore, SNU719 and YCCEL1 cells were used to investigate the cytotoxicity of the aforementioned PBMCs by LDH release-based cytotoxicity assay. The PBMCs were then treated with EBBA1 and LMP2A peptides for 14 days. As a result, the PBMCs were stimulated, increasing the proportion of CD 8\({}^{+}\) T-cells in the PBMCs. Depending on the EBV peptide used, EBNA1 PBMCs and LMP2A PBMCs were constructed. Flow cytometry was used to examine the expression levels of CD8\({}^{+}\) and CD3\({}^{+}\) in EBNA1 and LMP2A PBMCs to measure the proportion of CD8\({}^{+}\) T-cells in the PBMCs (Figure 4A). While unstimulated PBMCs had 22.43% CD8\({}^{+}\) T-cells, EBNA1 PBMCs had 58.58% CD8\({}^{+}\) T-cells and LMP2A PBMCs had 66.45% CD8\({}^{+}\) T-cells. We also measured the amount of IFN-\(\gamma\)+ secreted by EBNA1 and LMP2A PBMCs using intracellular flow cytometry and determined the proportion of IFN-\(\gamma\)+ cells (Figure 4B). While unstimulated PBMCs had 0.22% IFN-\(\gamma\)+ cells, EBNA1 PBMCs had 99.02% IFN-\(\gamma\)+ cells and LMP2A PBMCs had 97.16% IFN-\(\gamma\)+ cells. The DHT-treated EBNA1 PBMCs showed significantly higher cytotoxicity against SNU719 cells compared to the EtOH-treated control (Figure 4C). However, there was no significant difference in the cytotoxicity of DHT-treated LMP2A PBMCs against SNU719 cells compared to the control (Figure 4D). Additionally, the DHT-treated EBNA1 PBMCs exhibited significantly higher cytotoxicity against YCCEL1 cells than the EtOH-treated control (Figure 4E). All these results suggested that PBMCs stimulated with EBNA1 peptide exhibited increased cytotoxicity against SNU719 cells by DHT treatment, and that this increased cytotoxicity was due to increased antigen presentation resulting from increased _EBNA1_ transcription in SNU719 cells. In the above results, DHT increased the EBNA1 PBMC killing of SNU719 cells. This prompted us to investigate how DHT regulates the transcript expression of T-cell-activating receptors in the PBMCs. Therefore, we analyzed the changes in transcription patterns of T-cell-activation receptor genes in EBNA1 PBMCs treated with 100 nM DHT for 24 h by RT-qPCR assay. Among the four activated T-cell receptor genes tested, DHT treatment statistically significantly increased the transcript levels of _FASL_ and _NKG2D_ (Figure 4F). Next, DHT-induced transcriptional changes in SNU719 cell ligand genes were analyzed using RT-qPCR assay. Among the 11 ligand genes tested, DHT significantly increased the expression of _MICA_ and _RAETIG_, both ligands for NKG2D (Figure 4G). These results suggest that the increased cytotoxicity of PBMCs against SNU719 cells by DHT is mainly due to the increased expression of NKG2D on CD8\({}^{+}\) T-cells and MICA on SNU719 cells. We hypothesized that EBNA1 PBMC cytotoxicity against SNU719 cells requires the interaction of NKG2D and MICA, similar to NK-92 cell cytotoxicity, and tested this hypothesis. The cytotoxicity of EBNA1 PBMCs against SNU719_pLKO.1, SNU719_MICA(\(-\)), and SNU719_HLA-A(\(-\)) cells was assessed using an LDH release-based cytotoxicity assay. DHT significantly increased the cytotoxicity of EBNA1 PBMCs against SNU719_pLKO.1 cells at T:E = 1:15 compared to EtOH (control) (Figure 4H).