## 3 Results
### DHT Increases the Cytotoxicity of NK Cells
(**H**) Investigation of cytotoxicity of NK-92 cells against SNU719_HLA-A(\(-\)) cells treated with 100 nM DHT for 24 h by LDH release-based cytotoxicity assay. Cytotoxicity of NK-92 cells 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 NK-92 cells 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\({}^{3}\) NK-92 cells 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 both uppercase letters used to denote significant differences. The results indicated that DHT enhanced the cytotoxic activity of NK-92 cells against SNU719 and YCCEL1 cells, leading us to hypothesize that this effect was linked to the DHT-induced increase in the interaction between MICA and its corresponding receptors. Therefore, we analyzed the expression pattern of all NK-cell-activating receptors on NK-92 cells upon DHT treatment (Figure 3D). In details, we investigated changes in transcription of NK-cell-activating receptor genes in NK-92 cells treated with 100 nM DHT for 24 h. Among the nine activated NK-cell receptor genes tested, DHT slightly increased the receptor transcription of _NKp46_, _NTB-4_ and _NKG2D_, but this increase was not statistically significant. Next, the expression pattern of the above ligands of NK-cell-activating receptors was also extensively analyzed. In detail, we investigated changes in the transcription of ligands of NK-cell-activating receptor genes in SNU719 cells treated with 100 nM DHT for 24 h.  
Regarding the ligand genes for NK-cell receptors, DHT statistically significantly increased the transcription of _MICA_ (ligand of NKG2D), _RAETIG_ (ligand of NKG2D), and _NTB-A_ (ligand of NTB-A) among nine ligands (Figure 4E). The above results suggest that the increased NK-92 cytotoxicity against SNU719 cells by DHT is mainly due to the increased expression of NK-92 cell NKG2D ligands such as MICA and RAETIG. NKG2D is an activation receptor for NK and CD8\({}^{+}\) T cells, and MICA is a ligand for NKG2D [22]. Previous experiments have shown that DHT increases the transcription of _MICA_ and _RAETIG_. Therefore, we hypothesized that DHT-mediated increased NK-cell cytotoxicity is associated with the DHT-mediated upregulation of _MICA_. To test this hypothesis, we knocked down _MICA_ using an shRNA lentiviral system to generate SNU719_MICA(\(-\)) cells (Supplementary Figure S1C,D). As an internal control, SNU719_HLA-A(\(-\)) cells were generated by knockdown of _HLA-A_ (Supplementary Figure S1E,F). HLA-A is a ligand for the NK-cell-activating receptor KIR2 DS4 and the NK-cell inhibitory receptor KIR3 DL2 [22], and is one of the three major types of MHC class I transmembrane proteins [23]. As a negative control, negative control cells were constructed by transfecting SNU719 cells with the shRNA expression vector pLKO.1. These negative control cells were named SNU719_PLKO.1 cells. Figure 4: **DHT increases the cytotoxicity of PBMCs. ** (**A**) Fresh PBMCs were treated (stimulated) with EBV EBNA1 full peptide (5 g/mL) and LMP2A full peptide (5 g/mL) for 14 days to establish EBNA1 PBMCs and LMP2A PBMCs, respectively. The proportion of CD8\({}^{+}\) T cells in each of these two PBMCs was investigated by FACs analysis. The bright cyan letters and numbers in the dot plot represent the intensity and percentage of antibody binding to the antigen. Specifically, UL4 indicates the percentage of CD8-positive and CD3-negative cells, LL4 represents the percentage of CD8-negative and CD3-negative cells, UR4 corresponds to the percentage of CD8-positive and CD3-positive cells, and LR4 denotes the percentage of CD8-negative and CD3-positive cells. 1e1 means 1 \(\times\) 10\({}^{1}\), which equals 10.