Single-cell and spatial profiling reveal tumor-immune features underlying pathologic complete response to neoadjuvant anti-PD-L1 and chemoradiotherapy in NSCLC

Background: Perioperative chemoimmunotherapy has become standard for resectable non-small cell lung cancer (NSCLC), yet how neoadjuvant anti-PD-L1 and chemoradiotherapy remodel the tumor microenvironment (TME) remains poorly defined. Methods: Tumors from patients with stage III NSCLC treated with neoadjuvant durvalumab plus concurrent chemoradiotherapy in the NCT03694236 trial were analyzed using single-cell RNA sequencing (scRNA-seq), T cell receptor sequencing (TCR-seq), and spatial transcriptomics to delineate cellular and molecular programs associated with pathologic response. Results: Neoadjuvant durvalumab combined with concurrent chemoradiotherapy was administered to 30 patients with resectable stage III NSCLC. All patients completed therapy without surgical delay, achieving a major pathologic response (MPR) rate of 74% and a pathologic complete response (pCR) rate of 41%, with R0 resection in all surgical cases. Integrated scRNA-seq, TCR-seq, and spatial analyses of 19 tumors revealed distinct immune architectures between pCR and non-pCR groups. pCR tumors exhibited expansion and spatial clustering of clonally enriched CD8⁺ effector-memory (T EM ) and progenitor-exhausted (T PEX ) cells forming a T EM →T PEX →T EX trajectory with cytotoxic and antigen-processing programs localized adjacent to tumor nests. In contrast, non-pCR tumors accumulated TNFalpha-producing OLR1⁺ monocytes and FOLR2⁺macrophages that promoted TNFR2⁺ regulatory T cell (Treg) differentiation through TNF-TNFR2 signaling, reinforcing immunosuppression. Chemokine-receptor mapping demonstrated divergent recruitment patterns: antigen-presenting CAFs (apCAFs) and myeloid cells in pCR expressed CCL5 and CCL3, recruiting T PEX cells via CCR5, whereas CCL20-CCR6/CCR4 signaling in non-pCR favored Treg attraction. apCAFs in pCR upregulated antigen-presentation and cytotoxicity-related genes while losing immunosuppressive traits, facilitating CD8⁺ T cell infiltration and spatial organization of an inflamed TME. Collectively, these findings delineate opposing immune circuits-cytotoxic CD8⁺ T PEX /T EX activation in pCR versus TNF-TNFR2-driven myeloid-Treg suppression in non-pCR tumors. Conclusion: Neoadjuvant durvalumab-based chemoradiotherapy reprograms the stage III NSCLC TME toward clonally expanded, cytotoxic CD8⁺ T cell immunity in pCR, while persistent TNF-TNFR2-Treg signaling sustains immune resistance in non-pCR tumors. Targeting TNFR2 signaling, modulating myeloid polarization, or enhancing CCL5-CCR5-mediated T cell recruitment may further potentiate antitumor efficacy. These findings provide a mechanistic rationale for integrating TNFR2 blockade or chemokine-axis modulation with neoadjuvant immunotherapy to improve pathologic response and long-term outcomes.