The PTBP1-RORA axis is a central mediator of the cytotoxic T-cell response to multimodal thermal therapy in hepatocellular carcinom

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality, necessitating therapies that bridge local tumor control with systemic anti-tumor immunity. While multimodal thermal therapy (MTT) can induce immunogenic responses, the precise molecular programs governing antigen-driven cytotoxic CD8⁺ T-cell immunity following ablation remain undefined. This study aims to delineate the transcriptional mechanisms driven by MTT that govern CD8⁺ T-cell mediated anti-tumor immunity. Integrated single-cell transcriptomic profiling and proteomic analyses were performed on clinical samples obtained from HCC patients treated with MTT. Functional assays, including cytokine production, cytotoxicity analyses, and molecular interaction studies, were used to characterize CD8⁺ T-cell effector programs. Genetic manipulation and conditional knockout models were employed to interrogate the mechanistic role of key regulatory factors in vivo using murine HCC models. Statistical analyses were conducted using appropriate parametric or nonparametric tests as indicated, with significance determined based on standard criteria. Single-cell RNA sequencing revealed that MTT drives a distinct differentiation trajectory of CD8+ T cells toward a highly cytotoxic state, which was strongly associated with elevated expression of the nuclear receptor RORA. CD8 + T cells isolated from MTT-treated tumor-bearing mice exhibited a robust anti-tumor effector program characterized by increased production of IFN-γ, Perforin, Granzyme-B, and the chemokines CXCL9 and CXCL10, resulting in enhanced tumor cell killing. Mechanistically, the RNA-binding protein PTBP1 was identified as a critical co-activator of RORA. MTT treatment upregulated PTBP1, which directly interacted with RORA, facilitated its nuclear import, and promoted its recruitment to the promoters of cytotoxic effector genes. In murine HCC models, elevated RORA expression in CD8+ T cells was associated with enhanced anti-tumor responses following MTT. Notably, conditional deletion of Rora in CD8+ T cells completely abrogated the therapeutic efficacy of MTT. This study identifies the PTBP1–RORA axis as a central transcriptional regulator of CD8+ T-cell mediated anti-tumor immunity induced by multimodal thermal therapy. Our findings provide mechanistic insight into how MTT initiates systemic anti-tumor immune remodeling and reshapes cytotoxic T-cell function highlight PTBP1–RORA signaling as a promising biomarker and therapeutic target for enhancing cancer immunotherapy in HCC.