Thymoquinone enhances efficacy of cervical Cancer therapeutic vaccines via modulating CD8⁺ T cells

Cervical cancer, primarily driven by persistent high-risk human papillomavirus (HPV) infection, remains a global health challenge. While therapeutic vaccines offer promising alternatives to conventional treatments, their clinical efficacy is often hindered by limitations in antigen presentation and the immunosuppressive tumor microenvironment (TME). In this study, we explore the potential of thymoquinone (TQ), a bioactive compound derived from Nigella sativa seeds, to enhance the efficacy of cervical cancer therapeutic vaccines. Utilizing a mouse cervical cancer xenograft model with TC-1 cells expressing HPV16 E6/E7 and ras genes, we observed substantial heterogeneity in vaccine-induced antitumor responses. Mice were stratified into hyporesponsiveness (Hypo) and hyperresponsiveness (Hyper) groups based on tumor progression. The Hyper group exhibited significantly reduced tumor growth, marked by increased CD4⁺ and CD8⁺ T cell infiltration, reduced regulatory T cells (Tregs), and altered systemic immune parameters. Metabolite profiling identified 63 differential metabolites, with TQ being notably upregulated in the Hyper group, correlating positively with CD4⁺ and CD8⁺ T cells and negatively with Tregs and tumor volume. Combining TQ with the vaccine potentiated antitumor responses, leading to smaller tumors without significant changes in body weight. Immunohistochemical and flow cytometry analyses revealed augmented CD4⁺ and CD8⁺ T cell infiltration and reduced Tregs, alongside increased IFN-γ production in the combination of vaccine with (Vax + TQ) group. RNA-Seq analysis of TQ-treated CD8⁺ T cells revealed upregulation of immune-related genes, including Spp1, Grem1, Cxcl10, Isg15, Lcn2, and Sema3c, highlighting enriched immune pathways. Furthermore, TQ treatment upregulated ISG15 expression and enhanced CD8⁺ T cell cytotoxicity and cytokine secretion both in vitro and in vivo. In conclusion, our findings suggest that TQ enhances the efficacy of cervical cancer therapeutic vaccines by modulating immune responses, particularly by upregulating ISG15 and boosting CD8⁺ T cell function. This study provides a theoretical foundation for TQ as a promising adjuvant in cancer immunotherapy.