Abstract 5816: TCR clonal tracking maps tumor-draining lymph nodes as reservoirs fueling neoadjuvant PD-1 blockade in resectable NSCLC

Introduction: PD-1 blockade with platinum chemotherapy (chemo/IO) has transformed treatment for resectable non-small cell lung cancer (NSCLC). However, gaps in understanding the adaptive T cell response impede progress, resulting in a lack of predictive biomarkers and mechanism-informed combination therapies. We hypothesize that novel T cell clones activated in the tumor draining lymph node (tdLN) in response to PD-1 blockade are critical drivers of therapeutic outcomes. Methods: Tumor, tdLNs (stations 11-12), and peripheral blood (PB) were collected from 16 NSCLC patients (stage I-IIIB). Nine patients received neoadjuvant chemo/IO, while seven were treatment naïve. Samples underwent pathological evaluation and stratified by pathological response based on residual viable tumor. Clonal dynamics were assessed across tissues using single-cell TCR barcoding integrated with transcriptional profiling (scRNASeq) by using machine learning (ML) approaches. Results: Paired scRNA/TCR sequencing of 670,000 T cells and 110,000 clones from 47 tissue samples revealed deep clonal dynamics across tumor, tdLN, and PB. Tumor matched and expanded (n≥ 10) shared TCRs between tumor and tdLN (TM clones) accounted for 5% of clones, 75% of which were found in the PB (3.7% of clonal population). Chemo/IO-treated patients exhibited an increase in expanded TM clonal diversity (69 vs. 47 clones/1000 expanded T cells, p = 0.07) and a higher proportion of TM T cells within expanded T cells across all three tissues (83% vs. 68%, p < 0.05). Notably, 12 of 111,000 unique TCRs were shared across at least two patients. Clonal fate mapping using TCR sequences and UMAP clustering revealed distinct phenotypic trajectories. TM tdLN-resident clones were enriched in stem-cell quiescence genes (SELL, TCF7, CCR7) and signatures (p < 0.001), relative to the cytotoxic and exhausted tumor-infiltrating T cells (TILs) (p < 0.001). Chemo/IO patients’ tdLN and TILs exhibited T cell activation via AP-1 pathway (p<0.05), while untreated patients’ TILs were exhaustion. Patients with pathologic response had a less differentiated TM TILs compared to non-responders. ML further identified a 280-gene activation signature indicative of tdLN-linked responses to PD-1 blockade (p < 0.05) led by CXCL13 and CXCR6, validated in a resectable murine model. Persisting TM clones circulating in PB 6 months post-resection retained effector-memory phenotypes (CXCR3, TCF7, CD44). Conclusion: Our findings highlight the distinct role of tdLNs in contributing to treatment responses to PD-1/PD-L1 blockade. This alternative mechanism appears driven by tdLN-mediated T cell activation and tumor infiltration, rather than the reactivation of pre-existing TILs. These insights strongly support the development of pre-lymph node resection treatment strategies, and future approaches targeting lymph node-driven immunity in resectable NSCLC. Citation Format: Rachel L. Honigsberg, Tatiana Cruz, Geoffrey Markowitz, Liron Yoffe, Stephanie Tang, Marissa Micheal, Ozge Dicle, Olivier Elemento, Nasser Altorki, Jonathan Villena-Vargas. TCR clonal tracking maps tumor-draining lymph nodes as reservoirs fueling neoadjuvant PD-1 blockade in resectable NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5816.