Abstract Background: While being neglected for decades, tumor immune escape is now considered as a new paradigm that promotes tumor growth and evasion. Besides the prototypical PD1 / PDL1 axis, several mechanisms has been identified as important actor in immune editing process. Indoleamine 2,3 dioxygenase (IDO1), a first rate-limiting enzyme of the Kynurenine pathway, has been described to favor tolerance and immune escape through the production of a series of metabolites including L-Kynurenine. However, functional evidences have not yet been clearly demonstrated. Methods: We reconstituted in vitro a human immune - tumor cell synapse where ability of primary immune cells to promote tumor cell death has been kinetically followed through fluorescent-based live cell imaging in the presence and absence of L-Kynurenine. Two different donors as source of Peripheral Blood Mononuclear Cells (PBMCs) and three different tumor cell lines - namely IB115 and IB136 as sarcoma models & H1299 as reference lung adenocarcinoma model - have been used in this study. Finally, in order to determine cancer subtypes that might be highly exposed to Kynurenine pathway dysregulation and associated immune dysfunction, a comprehensive analysis of Kynurenine and Tryptophan levels was done in a series of 691 patients with advanced solid tumors Results: L-Kynurenine demonstrated a clear propensity to limit the immune cell-mediated tumor apoptosis elicited by TCR ligation - an event that was consistent within the two different donors and the three different cell lines. Interestingly, this immune suppressive feature of L-Kynurenine was associated with a limited amount of gamma-Interferon (IFNγ) in the cell culture supernatant while no effect was observed on Tumor Necrosis Factor alpha (TNFα). It's worth to note that L-Kynurenine had no effect on tumor cells without immune cells thus suggesting that Kynurenine has direct action on immune cells through which it limits their anti-tumor function -The Kyn/Trp ratio in cancer patients was significantly higher than that reported in healthy subjects (median: 71.2, range: 5.8-2825.2). Median value was the highest in breast cancer (126) and the lowest in colorectal (54) and pancreatic cancer (51) patients. No correlation with IDO1 tumor expression neither with systemic markers of inflammation (C-reactive protein) was observed. In conclusion, these results represent the first large description of the Kynurenine/Tryptophan ratio levels in cancer patients and the first functional evidence of a direct inhibition by Kynurenine of lymphocytes's effector functions. Citation Format: Alban Bessede, Antoine Italiano, Assia Chaïbi, Christophe Rey, Imane Nafia, Sylvestre le Moulec, Sophie Cousin, Maud Toulmonde, Céline Auzanneau, Marina Pulido. Functional evidence for an immunosuppressive role of kynurenine in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5716.