Shared tumor-reactive T cell clonotypes from tumor-infiltrating lymphocytes and tumor-associated lymph nodes defined by single-cell sequencing and tumor organoid models in non-small cell lung cancer.

275 Background: Tumor-infiltrating lymphocytes (TILs) are a critical subset of immune cells in solid tumors, particularly in non-small cell lung cancer (NSCLC), and serve as a cornerstone in cancer immunotherapy. While TILs can be enriched for tumor-specific reactivity, the challenge remains in the precise identification and isolation of the potent tumor-reactive T cells. This study investigates TILs and tumor-associated lymph nodes (TALNs) as alternative sources of tumor-specific T cells and leverages single-cell sequencing to delineate their molecular signatures under stimulation with tumor antigens derived from tumor cells or patient-derived tumor organoids (PDTOs). Methods: TILs, TALN-derived T cells and PDTOs were isolated from paired surgically resected NSCLC tumor tissues to model tumor-immune interactions ex vivo. Functional assays, along with single-cell RNA sequencing were employed to identify and characterize putative tumor-reactive T cells. Additionally, T-cell receptor (TCR) sequencing was utilized to track tumor-reactive TCR clonotypes within three types of T cells. Results: CD137+ TILs from NSCLC tumors harbor a significantly higher proportion of tumor-specific T cells and enhanced activation potency and effector functions than T cells from PBMC. PDTOs closely mimic the primary tumors. TILs activated by PDTOs or autologous tumors have similar TCR clonotypes and tumor-specific expression. T cells derived from TALNs share similar TCR clonotypes with TILs. Conclusions: This study underscores the superior tumor specificity of TILs with CD137 expression and supports the utility of PDTOs as a robust platform for evaluating immune function in NSCLC. Additionally, TALNs demonstrate a distribution pattern that aligns with the tumor's lymphatic drainage and metastatic behavior.