## 4 Discussion  
The role of male hormones, particularly dihydrotestosterone (DHT), in the progression and treatment of Epstein-Barr virus-associated gastric carcinoma (EBVAGC) has been underexplored compared to other cancers. Our study provides new insights into how DHT modulates the immune response against EBVAGC, highlighting its potential therapeutic implications. We observed that DHT significantly increases androgen receptor (_AR_) expression in EBVAGC cells. The DHT-AR complex activates various signaling pathways, including the upregulation of NF-kB, which is crucial for immune response modulation. This upregulation was associated with an increased expression of MICA protein, a crit  
Figure 6: **DHT does not regulate cytokine expression. ****(A)** Cytokine array analysis to identify cytokines secreted from SNU719 cells treated with 100 nM DHT for 24 h. (**B)** Cytokine array analysis to identify cytokines secreted from NK-92 cells treated with 100 nM DHT for 24 h. (**C)** Cytokine array analysis to identify cytokines secreted from EBNA1 PBMCs treated with 100 nM DHT for 24 h. (**D)** Analysis of transcriptional changes of _MIF1_ and _IL-18_ genes in SNU719 cells treated with 100 nM DHT for 24 h by RT-qPCR assay. (**E**) Analysis of transcriptional changes of _CXCL12_ gene in NK-92 cells treated with 100 nM DHT for 24 h by RT-qPCR assay. (**F**) Analysis of transcriptional changes of _MIF1_ and _IL-16_ genes in EBNA1 PBMCs treated with 100 nM DHT for 24 h by RT-qPCR assay. 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. ical ligand for the NKG2D receptor on natural killer (NK) and T cells. Increased MICA levels enhance interaction with the NKG2D receptor, boosting NK- and T-cell cytotoxicity against SNU719 cells--an EBVaGC cell line. This immune activation occurs without a concomitant increase in proinflammatory cytokine gene expression, suggesting a targeted and potentially less-inflammatory immune response. Previous studies have shown that DHT, along with DHEA and E2, reduces IL-6 expression in human bone-marrow cells and that testosterone replacement in androgen-deficient males decreases IL-10 expression [26; 27]. These findings suggest that male hormones modulate immune responses by regulating inflammatory cytokines in normal cells, but this modulation does not seem effective in cancer cells. In EBVaGC, no significant changes in cytokine expression were noted after 24 h treatment with 100 nM DHT, despite the sufficient induction of gene expression via NF-\(\kappa\)B. This suggests that DHT-AR signaling may contribute more to direct NK- and T-cell activation rather than relying on NF-\(\kappa\)B to upregulate inflammatory cytokine genes. NKG2D, expressed on T and NK cells, transmits activation signals by recognizing NKG2D ligands (NKG2 DLs) on tumor cells. Among these ligands, MICA and MICB are well characterized, with MICA more abundantly expressed in tumor cells [28; 29]. Our analysis revealed that DHT treatment upregulates _MICA_ and _RAETIG_ expression in EBVaGC, with a more substantial increase in _MICA_. The upregulation of _MICA_ by DHT counteracts the reduction caused by MICA/B shedding, enhancing NK-cell recognition and suppressing immune escape [30; 31]. We generated Epstein-Barr virus (EBV)-specific peripheral blood mononuclear cells (PBMCs) targeting EBNA-1 (EBNA1 PBMCs) and LMP2A (LMP2A PBMCs), proteins expressed during the EBV latent phase in EBVaGC [32]. Notably, DHT treatment only enhanced the cytotoxicity of EBNA1 PBMCs. Given that EBNA-1 is expressed in nearly all EBV latent phases, EBNA-1 peptides could serve as target antigens in most EBV-associated malignancies. Although EBNA-1 interferes with HLA-A presentation [28], DHT treatment did not upregulate _HLA-A_ expression in SNU719 cells.