Abstract Background: We and others have reported the major role of mature tertiary lymphoid structures (TLS) in the efficacy of PD-L1 blockade in NSCLC. The objective response rate (ORR) to inhibition of PD1/PDL1 used as single agents observed in TLS-positive NSCLC is similar to ORRs observed in all-comers NSCLC treated with a combination of immune checkpoint inhibitor (ICI) and chemotherapy. However, a substantial proportion of patients with TLS-positive NSCLC did not display clinical benefit. The aim of our study was to identify the determinants of response to ICI in TLS-positive NSCLC. Methods: We spatially profiled the expression of >18,000 protein-coding genes across six patients with mature TLS-positive NSCLC (three with objective response and three with progressive disease as the best response) using the GeoMx whole-transcriptome atlas (WTA) assay. Using two consecutive slides stained with CD3/CD20 and CD45/PanCK markers, a first set of regions of interest (ROIs) selected the TLS (CD3+/CD20+) as an entire area of illumination (AOIs). A second set of ROIs was selected outside the TLS region and further segmented into ‘tumor’ (PanCK+/CD45-) versus ‘stroma’ (PanCK-/CD45+) AOIs. To further validate our findings, we performed multiplex immunofluorescence (mIF) on 70 mature TLS-positive NSCLC tumors. Results: Comparative transcriptomic analysis of TLS between non-responders and responders revealed upregulation in responders of CCL21 which plays a crucial role in T cell recruitment, and of IGHM indicating the presence of unswitched B cells. Outside of TLS, in the stromal compartment, the most significantly differentially expressed genes between responders and non-responders were those belonging to a subset of cancer associated fibroblasts (CAFs) expressing both FAP and αSMA markers, and recently described as linked to more inflammatory activated immune cells and involved in T cell exclusion in NSCLC. We then used FFPE tissue mIF to histologically profile CAF subset composition and spatial distribution of T cells in a cohort of 70 mature TLS-positive NSCLC. In tumor lesions enriched in FAP+αSMA+ CAFs, the tumor-to-stroma ratio of infiltrating CD8+ cells was significantly lower, consistent with a decreased infiltrating CD8+ T cell density in the tumor. Strikingly, ORR and survival of patients with mature TLS NSCCLC enriched in FAP+αSMA+ CAFs were significantly lower than those of patients with low abundance of CAFs. Conclusions: We report here the first study investigating the mechanisms of resistance to immunotherapy in TLS-positive tumors. Our analysis of human tumor samples suggests a crucial role of FAP+αSMA+ CAFs in excluding T cells and in driving resistance to immune checkpoint inhibition in mature TLS-positive NSCLC. The development of novel CAF-targeting strategies may be particularly relevant in this subset of tumors. Citation Format: Florent Peyraud, Jean-Philippe Guegan, Christophe Rey, Oren Ngouala, Ophélie Odin, Emma Clot, Lucile Vanhersecke, Maxime Brunet, Thomas Grellety, Marie Del Castillo, Sylvestre Le Moulec, François Le Loarer, Alban Bessede, Antoine Italiano. Cancer-associated fibroblasts promote T-cell exclusion and resistance to immunotherapy in non-small cell lung cancer with tertiary lymphoid structures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6767.