Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest cancers, features a highly immunosuppressive environment, with expansion of cancer-associated fibroblasts (CAFs), and absence/inactivation of antitumor immune cells. Therefore, ways to manipulate this environment, favoring the antitumor cells, will be critical for effective therapies for PDAC. Recently, we identified the neuronal protein Netrin G1 Ligand (NGL-1) to be overexpressed in PDAC tissue, including its novel expression in immune cells and CAFs. However, the role that NGL-1 plays in the tumor microenivronment (TME) is unknown and warrented further investigation. Using wild-type and NGL-1 full body knockout mice orthotopically injected or not with pancreatic tumor cells, we assessed tumorigenesis and secretion of immunosuppresive factors. Moreover, using our in vitro 3D system we explored the ability of NGL-1+ CAFs to rescue PDAC cell survival under nutrient deprivation, their immunosuppressive profile and the ability of a peptide targeting NGL-1 to reverse these features. Finally, we assessed the overall survival of 140 PDAC patients according to NGL-1 expression in the TME. Comparing immune cells from naïve and tumor-bearing mice we observed that myeloid, T and NK cells from tumor-bearing mice tend to overexpress NGL-1. Moreover, CD8+ and CD4+ T cells from NGL-1 KO mice proliferated more when stimulated in vitro, suggesting that NGL-1 could represent a functional brake for T cell activation and proliferation. Interestingly, the absence of NGL-1 in bone marrow-derived macrophages stimulated in vitro led to decreased production of pro-inflammatory cytokines, further suggesting a functional role for NGL-1 in myeloid cells. Importantly, NGL-1 KO mice orthotopically injected with PDAC cells developed smaller tumors and these produced less immunosuppressive factors. In accordance, CAFs lacking NGL-1 were not supportive of PDAC cell survival in vitro and produced less immunosuppressive cytokines, which was phenocopied by the treatment with the peptide targeting NGL-1. Finally, data from PDAC patients showed that low expression of NGL-1 in CAFs and immune cells correlated with better survival of these patients, therefore highlighting NGL-1 as a potential new target that could be manipulated in different compartments in pancreatic cancer (cancer cells, CAFs, immune cells). This represents an innovative perspective for such a complex disease. Citation Format: Debora Barbosa Vendramini-Costa, Ralph Francescone, Tiffany Luong, Janusz Franco-Barraza, Igor Astsaturov, Kathy Q. Cai, Andres J. Klein-Szanto, Huamin Wang, Kerry Campbell, Edna Cukierman. The synaptic protein netrin G1 ligand (NGL-1) modulates the immunosuppressive environment in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT019.