## 3 Results
### DHT Increases the Cytotoxicity of NK Cells
1e2 means 1 \(\times\) 10\({}^{2}\), which equals 100. 1e3 means 1 \(\times\) 10\({}^{3}\), which equals 1000. **B**) The proportion of cells secreting INF-\(\gamma\) in each EBNA1 PBMCs and LMP2A PBMCs was examined by FACs analysis. **C**) Investigation of cytotoxicity of EBNA1 PBMCs against SNU719 cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of EBNA1  
\(1\) PBMCs against SNU719 cells treated with EtOH for 24 h was used as a control. **(D)** Investigation of cytotoxicity of LMP2A PBMCs against SNU719 cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of LMP2A cells against SNU719 cells treated with EtOH for 24 h was used as a control. **(E)** Investigation of cytotoxicity of EBNA1 PBMCs against YCCEL1 cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of EBNA1 PBMCs against YCCEL1 cells treated with EtOH for 24 h was used as a control. **(F)** Analysis of transcriptional patterns of DHT-regulated T-cell-activation receptor genes. The transcription of T-cell-activation receptor genes in EBNA1 PBMCs treated with 100 nM DHT for 24 h was investigated by RT-qPCR assay. One-way ANOVA and [PERSON]'s multiple comparisons test were used for statistical processing. **(G)** Analysis of transcriptional patterns of T-cell-activating receptor ligand genes regulated by DHT. The transcription of ligand genes of T-cell-activating receptor in SNU719 cells treated with 100 nM DHT for 24 h was investigated by RT-qPCR assay. One-way ANOVA and [PERSON]'s multiple comparisons test were used for statistical processing. **(H)** Investigation of cytotoxicity of EBNA1 PBMCs against SNU719_pLKO.1 cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of EBNA1 PBMCs against SNU719_pLKO.1 cells treated with EtOH for 24 h was used as a control. **(I)** Investigation of cytotoxicity of EBNA1 PBMCs against SNU719_MICA(\(-\)) cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of EBNA1 PBMCs against SNU719_MICA(\(-\)) cells treated with EtOH for 24 h was used as a control. **(J)** Investigation of cytotoxicity of EBNA1 PBMCs against SNU719_HLA-A(\(-\)) cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of EBNA1 PBMCs against SNU719_HLA-A(\(-\)) cells treated with EtOH for 24 h was used as a control. E(effector cells) and T (target cells) refer to PBMCs and SNU719 cells, respectively. The E:T ratio represents the ratio of the number of effector cells to target cells. An E:T ratio of 1:1 corresponds to a combination of 5 \(\times\) 10\({}^{3}\) PBMCs and 5 \(\times\) 10\({}^{3}\) SNU719 cells. We performed a one-way ANOVA test for comparisons involving more than three groups, followed by [PERSON]'s multiple comparison test if the results were significant. The results were presented using a compact letter display, with uppercase letters used to denote significant differences. Statistically significant differences identified by the two-tailed Student's \(t\)-test are indicated by asterisks (*). The DHT-mediated cytotoxicity of NK cells against SNU719_MICA(\(-\)) and SNU719_HLA-A(\(-\)) cells was then assessed by LDH release-based cytotoxicity assay. The DHT-mediated cytotoxicity of NK-92 cells against SNU719_pLKO.1, SNU719_MICA(\(-\)) and SNU719_HLA-A(\(-\)) cells was assessed. As expected, compared to EtOH treatment (control), DHT significantly increased NK-cell cytotoxicity against SNU719_pLKO.1 cells at T:E = 1:15 (Figure 3F). However, compared to the control, DHT significantly decreased NK-cell cytotoxicity against SNU719_MICA(\(-\)) cells at T:E = 1:15 (Figure 3G). Interestingly, compared to the control, DHT did not significantly change NK-cell cytotoxicity against SNU719_HLA-A(\(-\)) cells at T:E = 1:15 (Figure 3H). Based on these results, it is proposed that the DHT-mediated increase in NK-cell cytotoxicity against SNU719 cells is based on the interaction of NKG2D and MICA.