Hypofractionated radiotherapy-induced tertiary lymphoid structures potentiate the efficacy of PD-1 inhibitors in lung cancer brain metastases

Radiotherapy (RT) has emerged as an effective synergistic therapy to enhance PD-1 inhibitor efficacy in Lung cancer brain metastases (LCBM). Nonetheless, not all patients benefit from this radioimmunotherapy strategy. Further research is imperative to determine the optimal RT fractionation regimen for enhancing PD-1 inhibitor efficacy in LCBM. An intracranial LCBM model was established by intracranial injection of LLC-Luc-BrM cells. Mice received PD-1 inhibitors combined with RT delivered at the same biologically effective dose (BED) using three RT fractionation regimens: conventionally fractionated radiotherapy (CFRT, 2 Gy × 8f), hypofractionated radiotherapy (HFRT, 3 Gy × 5f), and single-fraction high-dose radiotherapy (SF-HDRT, 10 Gy × 1f). Intracranial tumor growth was monitored by IVIS imaging, and survival was assessed using Kaplan–Meier analysis. T cell infiltration and function were evaluated using mIHC and ELISA. Cell depletion experiments were performed to clarify the essential cell populations involved in this synergistic efficacy. To investigate endothelial adhesion molecule expression and tertiary lymphoid structure (TLS) formation-related programs, RNA sequencing, IHC, and mIHC were employed. Among the three regimens, HFRT combined with PD-1 inhibitors most effectively inhibited tumor growth and prolonged survival. This combination significantly enhanced infiltration of CD4 + CD3 + and CD8 + CD3 + T cells and increased secretion of GZMB, IFN-γ, and TNF-α. T cell depletion experiments confirmed that this antitumor efficacy depended on both CD8 + T cells and CD4 + T cells. Mechanistically, HFRT upregulated ICAM-1 and VCAM-1 on CD31 + endothelial cells and promoted perivascular accumulation of CD3 + T cells, consistent with enhanced vascular permissiveness. HFRT also induced TLS-like aggregates and enriched TLS-associated gene signatures, with a nonclassical phenotype. HFRT was identified as an optimal RT fractionation regimen for enhancing PD-1 inhibitor efficacy in LCBM. The synergistic efficacy requires both CD4 + and CD8 + T cells and is associated with vascular remodeling and nonclassical TLS formation induced by HFRT.