Spatial multi-omics implicate the interaction between Tpex and B cells in tertiary lymphoid structures after neoadjuvant therapy.

Tertiary lymphoid structures (TLSs) are associated with the efficacy of various oncological therapies. However, the comprehensive spatial TLS pharmacodynamics are largely unclear. Here, we performed multifaceted spatial transcriptomic analysis with whole-transcriptome coverage and single-cell resolution, complemented by the high-throughput spatial proteomics, to thoroughly characterize TLSs in clinical breast cancer samples after neoadjuvant therapy. Notably, spatial multi-omics data identified that precursors of exhausted T cells (Tpex cells) preferentially reside within TLSs. Spatial transcriptomics with TCR-seq revealed the presence of tumor-specific Tpex cells inside TLSs and their clonally related terminally differentiated effector T cells outside TLSs. B cells are nearest neighbors of Tpex cells in TLSs and B cells promote invigoration of Tpex cells via ICOSL-ICOS and CD86-CD28 interactions within TLSs. These findings extend the current understanding of TLS spatial architecture and highlight a therapy-induced evolution of anti-tumor immune responses driven by the interaction between Tpex cells and B cells within TLSs.