The existence of intratumoral tertiary lymphoid structures (TLS) has been reported to be correlated with reduced recurrence of hepatocellular carcinoma (HCC). However, the cellular characteristics and driving mechanisms of TLSs in HCC remain largely unknown. In this study, we compared the clinical outcomes of TLSs in HCC using whole-exome sequencing, bulk RNA sequencing, and single-cell RNA sequencing on a cohort of 339 patients with HCC belonging to different TLS groups. Intratumoral 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 (area under the curve = 0.78) and patient prognosis, outperforming previously reported signatures, which were 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 effects in an 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 HCC. See related Spotlight, p. 716

Background: γδ T cells represent a heterogeneous family of innate-like lymphocytes with adaptive immunity features. Although abundant in barrier tissues such as skin and intestine, γδ T cells are rare in the cardiovascular system, which severely limits exploration of their roles in atherosclerosis. Consequently, the spatial localization, functional states, and antigen-driven responses of γδ T cells within atherosclerotic lesions remain poorly defined. Methods: To nevertheless examine γδ T cells in atherosclerosis, we employed an integrative, three-pronged strategy combining γδ T cell single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq), spatial transcriptomics, and large-scale multi-dataset scRNA-seq integration across multiple hyperlipidemic mouse models. Large-scale multi-dataset scRNA-seq integration was also applied to human atherosclerotic plaque datasets. Results: We found significant enrichment of γδ T cells within both atherosclerotic plaques and artery tertiary lymphoid organs (ATLOs), particularly of the proinflammatory IL17-producing γδ T17 subtype. Unexpectedly, γδ T17 cells containing paired Vγ6Vδ4 TCR chains with identical complementarity-determining region 3 (CDR3) sequences underwent clonal expansion in atherosclerotic plaques and ATLOs. Transcriptomic analysis revealed that plaques and ATLOs locally educate γδ T17 cells - here termed γδ eduT17 cells - towards an anti-apoptotic, tissue-resident, and apparent hypofunctional phenotype. Furthermore, γδ eduT17 cells underwent metabolic reprogramming within the atherosclerotic microenvironment. Spatial transcriptomics revealed that γδ T cells preferentially localize in ATLOs, particularly at the interface between T cell zones and B cell follicles. Multi-dataset integration confirmed the conservation of these features across multiple hyperlipidemic mouse models. In contrast, human atherosclerotic plaques harbored substantially fewer γδ T cells and the human phenotypes were dominated by an effector/cytolytic γδ T cell subtype, characterized by transcriptomes enriched in cytotoxic effector molecules. Conclusions: Our findings identify γδ T cells as a previously underappreciated T cell lineage population in atherosclerosis. Murine atherosclerosis is characterized by the enrichment, education, and clonal expansion of proinflammatory γδ eduT17 cells within plaques and ATLOs. In contrast, human plaques harbor γδ T cells with cytolytic features, suggesting divergent roles of γδ T cells between species. These results highlight the importance of local vascular microenvironments in shaping γδ T cell function and emphasize the need for caution when extrapolating mechanistic insights from mouse models to human atherosclerosis.

Supplementary Table S3 List of gene signatures for scoring.

Supplementary Table S1 Clinicopathological information of 314 patients from FAH-SYSU cohort.

Supplementary Table S4 Marker genes for B and Plasma cell subclusters.