Abstract

The existence of intra-tumoral tertiary lymphoid structures (TLSs) has been reported to be correlated with reduced recurrence of hepatocellular carcinoma (HCC). However, the cellular characteristics and driving mechanism of TLSs in HCC remain largely unknown. In this study, we compared clinical outcomes of TLSs in HCC using whole exome sequencing, bulk RNA sequencing, and single-cell RNA sequencing on a cohort of 339 HCC patients belonging to different TLS groups. Intra-tumoral TLSs were significantly associated with improved recurrence-free survival in HCC (p = 0.00013), with higher maturity of TLSs correlating with better prognosis (p = 0.00033). A B cell-related seven-gene signature effectively predicted TLS presence (AUC = 0.78) and patient prognosis, outperforming previously reported signatures, which was validated in situ by spatial transcriptomic data. Bulk and single-cell transcriptomic analyses revealed that TLS-positive (TLS+) tumors were immunologically active and strongly associated with immunotherapy response signatures. IgG-producing plasma cells, identified as key effector subsets enriched in TLS+ tumors, exhibited clonal expansion, somatic hypermutation, and high-affinity antibody production. Among potential tumor-enriched TLS-associated genes, HAPLN3 was overexpressed in TLS+ HCC and induced high serum antibody titers (p = 0.0032). Spatial transcriptomics and in vivo experiments confirmed that HAPLN3 promotes B cell activation, leading to suppressed tumor growth. Administration of Hapln3 protein displayed immunostimulatory and antitumor effect in orthotopic mouse model. These findings reveal that targeting TLS-associated B cell responses or leveraging HAPLN3-specific immunity may offer therapeutic avenues for improving immunotherapy outcomes in hepatocellular carcinoma.