Abstract Background: The presence of mature tertiary lymphoid structures (mTLS) within the tumor microenvironment (TME) has been linked to the effectiveness of PD-L1 blockade in non-small cell lung cancer (NSCLC). However, only a subset of patients experiencing clinical benefit. Our study aims to delve into the determinants of immunotherapy response in mTLS-positive NSCLC. Methods: We included 77 TLS-positive NSCLC from the BIP study (NCT02534649). First, we employed the GeoMx whole-transcriptome atlas (WTA) assay to spatially profiled the expression of >18,000 protein-coding in patients exhibiting extreme responses (3 responders [R] and 3 non-responders [NR]). To target regions of interest (ROIs), we selected areas that encompassed TLS (CD3+/CD20+) as entire areas of illumination (AOIs). Additionally, we segmented areas outside TLS into ‘tumor’ (PanCK+/CD45−) vs ‘stroma’ (PanCK−/CD45+) AOIs. To validate our findings, we conducted multiplex immunofluorescence (mIF) and complemented our results with bulk gene expression analysis using the HTG whole human transcriptome panel. Results: Spatial transcriptomic analysis pinpointed significant differences in gene expression within the stromal compartment between R and NR. Notably, a subset of cancer-associated fibroblasts (CAFs) expressing both FAP and αSMA markers emerged as the most differentially expressed genes. Subsequently, we used FFPE tissue mIF to delineate the histological profile CAF subsets and the spatial distribution of T cells. Notably, the stromal density of FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs were significantly higher in NR compared to R. Correspondingly, tumors enriched in FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs exhibited significantly lower overall response rates (ORR) and progression-free survival (PFS) than those with a low abundance of CAFs. Interestingly, tumors with elevated FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs were associated with a higher infiltration of CD8+ T cells. To delve deeper into the CD8+ T cell infiltrate, we conducted a comparative bulk transcriptomic analysis. Both FAP+αSMA+ CAFs High and MYH11+αSMA+ CAFs High demonstrated significant associations with regulatory T cells (Treg), exhaustion signatures, and inflammatory processes. To validate the CD8+ T cell phenotype, we performed complementary mIF. Consistently, the CD8+ T cell infiltrate in tumors featuring FAP+αSMA+ CAFs High and MYH11+αSMA+ CAFs High exhibited higher levels of exhaustion markers (PD1, LAG3, TIM3, TIGIT) and Treg infiltration, underscoring an immunosuppressive TME. Conclusions: FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs play a pivotal role in driving T cell exhaustion and Treg cell infiltration, contributing to the resistance to immune checkpoint inhibition. Targeting these specific CAF subsets may hold significant promise in overcoming resistance in mature TLS-positive NSCLC. Citation Format: Florent Peyraud, Jean-Philippe Guegan, Lucile Vanhersecke, Christophe Rey, Oren Lara, Ophélie Odin, Emma Clot, Maxime Brunet, Thomas Grellety, Marie Del Castillo, Sylvestre Le Moulec, François Le Loarer, Alban Bessede, Antoine Italiano. Cancer-associated fibroblasts drive T cell infiltration and resistance to immunotherapy in non-small cell lung cancer with mature tertiary lymphoid structures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7471.