Abstract Agonist anti-CD137(4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs covering four distinct epitopes on the CD137 glycoprotein co-stimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in antigen and antibody internalization towards a vesicular compartment. Internalization was observed in activated T cells from humans and mice, not only in culture but also in antibody-injected living animals. These in vivo experiments were carried out upon systemic intravenous injections with anti-CD137 mAbs and showed internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to mice. CD137-internalization required K63-polyubiquitination and endocytosed CD137-containing deposits were decorated with K63-polyubiquitins. CD137 stimulation activates NF-kB through a K63-linked polyubiquitination-dependent route and CD137-associated TRAF2 becomes K63-polyubiquitinated. Consistent with a role for TRAF2 in CD137 signalling, transgenic mice functionally deficient in TRAF2 show a delayed immunotherapeutic activity of anti-CD137 mAbs Citation Format: Ivan Martinez-Forero, Arantza Azpilicueta, Elixabet Bolaños-Mateo, Estnislao Nistal-Villan, Asis Palazon, Alvaro Teijeira, Gema Perez-Chacon, Aizea Morales-Kastresana, Oihana Murillo, Maria Jure-Kunkel, Juan Manuel Zapata, Ignacio Melero. T cell costimulation in cancer immunotherapy with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B2.