CX3CR1⁺ CD8⁺ cytotoxic T cells drive tumor killing in esophageal squamous cell carcinoma during neoadjuvant therapy

Abstract

Immune checkpoint inhibitor (ICI) responses in esophageal squamous cell carcinoma (ESCC) are highly variable, and the immune mechanisms underlying therapeutic sensitivity remain unclear. Here we show, using single-cell RNA, T-cell receptor and whole-exome sequencing of 52 ESCC patients from a phase 3 neoadjuvant trial, that treatment response is associated with coordinated tumor microenvironment remodeling. Responders exhibit increased abundance and clonal enrichment of cytotoxic CX3CR1⁺CD8⁺ T-cell clonotypes, accompanied by reduced frequency and clonal dominance of exhausted CXCL13⁺CD8⁺ T-cell states after treatment. CX3CR1⁺CD8⁺ T-cell accumulation is associated with CX3CL1–CX3CR1 signaling and supported by functional, mouse-model and peripheral blood validation. Genomic analyses link higher cancer cell fraction mutation burden and neoantigen abundance to response, consistent with post-treatment immune editing. Tumor epithelial differentiation states and stromal remodeling are also associated with therapeutic outcome. These findings identify a response-associated CX3CR1⁺CD8⁺ cytotoxic effector state that may inform neoadjuvant ICI-based therapy in ESCC. Responses to immune checkpoint inhibitors (ICI) in esophageal squamous cell carcinoma (ESCC) are highly heterogeneous, and the mechanisms underlying therapeutic sensitivity remain incompletely understood. Here the authors present multi-omics profiling of ESCC patients receiving neoadjuvant therapy and identify a CX3CR1⁺CD8⁺ T-cell cytotoxic effector state linked to response.