PD-L1(+) tumor-associated macrophages induce CD8(+) T Cell exhaustion in hepatocellular carcinoma.

The therapeutic efficacy of immune checkpoint inhibitors (ICIs) in patients with hepatocellular carcinoma (HCC) is profoundly influenced by the tumor immune microenvironment (TIME), where tumor-associated macrophages (TAMs) expressing programmed death-ligand 1 (PD-L1) serve as key modulators of immune suppression and tumor progression. Although PD-L1(+) TAMs have attracted increasing attention, their precise immunological functions in patients with HCC remain incompletely understood. In this study, we conducted an integrated analysis combining single-cell transcriptomics, spatial profiling, in vitro functional assays, and in vivo therapeutic modeling to clarify the role of PD-L1(+) TAMs in HCC. Single-cell RNA sequencing of tumor samples from patients with HCC (GSE189903) revealed that intratumoral PD-L1(+) TAMs were enriched for immune-related signaling pathways and expressed chemokines including CXCL9, CXCL10, and CXCL11. In vitro, GM-CSF-induced PD-L1(+) macrophages promoted CD8(+) T cell exhaustion, characterized by increased expression of TIM3 and suppression of cytotoxic molecules such as GZMB. Spatial analysis using multiplex immunofluorescence staining of surgical specimens from 113 patients with HCC demonstrated that close proximity between PD-L1(+) TAMs and CD8(+) T cells within tumors was an independent predictor of early postoperative recurrence and poor outcome. Moreover, in a murine orthotopic liver cancer model, the combination of anti-GM-CSF and anti-PD-L1 antibodies inhibited the differentiation of PD-L1(+) TAMs, reduced their contact with CD8(+) T cells, alleviated T cell exhaustion, and potentiated antitumor immunity. These findings highlight the critical contribution of PD-L1(+) TAMs to immune evasion in patients with HCC and support their therapeutic targeting as a strategy to enhance ICI responses.