Intratumoral CXCL13 + T follicular helper cells are associated with the formation and maturation of tertiary lymphoid structures in unresectable hepatocellular carcinoma treated with triple therapy

Hepatocellular carcinoma (HCC) is a heterogeneous, lethal cancer with a poor prognosis. Transarterial chemoembolization (TACE) combined with lenvatinib and anti-programmed death-1 antibodies (triple therapy) treats unresectable HCC (uHCC) without severe adverse events. However, mechanisms underlying this triple therapy remain unclear. Patients with uHCC who underwent salvage resection after triple therapy, with a partial pathological response, were enrolled. Because intratumoral tertiary lymphoid structures (iTLSs) cannot be reliably assessed in pre-therapy fine-needle aspiration biopsies, no true pre-treatment tissue was available; a comparator cohort of specimens from patients treated by upfront surgery without prior therapy was matched 1:1 by propensity score on age, sex, etiology, BCLC stage, and maximum tumor diameter ( n = 62 per cohort). The comparison was cross-sectional and between-patient, not longitudinal within-patient; balance was assessed using standardized mean differences. Patients with a pathological complete response were ineligible because extensive necrosis precluded microenvironment assessment. Immunohistochemistry, multiplex immunofluorescence, and single-cell RNA sequencing (10 tumor samples) characterized the tumor microenvironment. TLSs were identified in 93.5% (58/62) of post-treatment specimens and 59.6% (37/62) of comparator specimens. iTLS density was higher in the post-treatment cohort (0.216 ± 0.125 versus 0.028 ± 0.046 TLS/mm², P < 0.001), whereas peritumoral TLS (pTLS) density did not differ (0.020 ± 0.020 versus 0.022 ± 0.019 TLS/mm², P = 0.63), and iTLS density was positively correlated with maturation. Overall, 41 of 62 patients (66%) achieved a major pathological response (MPR), and higher iTLS density and maturity were associated with a pathological response and longer relapse-free survival (RFS) and overall survival (OS). Single-cell analysis showed higher proportions of CXCL13 + T follicular helper (TFH) cells and CXCR5 + germinal center (GC) B cells after triple therapy, and ligand-receptor analysis indicated stronger CXCL13-CXCR5 signaling between these populations. Triple therapy was associated with denser, more mature iTLSs and enrichment of CXCL13 + TFH and CXCR5 + GC B cells. We propose a model wherein triple therapy is associated with increased TFH-derived CXCL13 signaling, which may promote the recruitment of CXCR5 + GC B cells and thereby support iTLS assembly and maturation. iTLS density and maturity are candidate biomarkers for stratifying patients after salvage resection.