Abstract 4075: LAG3 and PD1 co-inhibitory molecules directly interact to limit CD8+ T cell signaling

Abstract

Abstract Increasing evidence indicate that dual blockade of Lymphocyte Activation Gene-3 (LAG3) and programmed cell death 1 (PD1) synergistically enhances autoimmunity and anti-tumor immunity. Despite the fact that these co-inhibitory receptors individually attenuate T cell receptor signaling, it is not clear whether they interact at the physical, biochemical and/or molecular levels, to regulate the functional fate of T cells. To answer these questions, we first simulated possible direct interaction of LAG3 and PD1 using in-silico molecular docking techniques. Using confocal microscopy and ImageStream analyses, we demonstrated that LAG3 and PD1 molecules co-localized in a subset of activated murine CD8+ T cells. The co-localization occurred at the trans-Golgi vesicles, early/recycling endosomal compartments, lysosomes, and microtubule organizing center (MTOC). Importantly, LAG3 and PD1 cluster with pLck at the immunological synapse of CD8+ T cell and dendritic cell conjugates. Reciprocal immunoprecipitation of the T cell extracts with specific antibodies for LAG3 and PD1 revealed a physical association between LAG3 and PD1 and this interaction was absent in T cells isolated from LAG3 and PD1 knockout mice. Finally, LAG3 protein was also associated with Src family tyrosine phosphatase-2 (SHP2) that is recruited by PD1 during T cell signaling. The recruitment of phosphatases is additively reduced in CD8+ T cells deficient in both LAG3 and PD1. Taken together, our data suggest that LAG3 interacts and collaborates with PD1, following T cell activation, for rapid translocation from early/recycling endosomal compartments to the immunological synapse, leading to a synergistic inhibitory effect on T cell signaling. Citation Format: Ruea-Yea Huang, Cheryl Eppolito, Ahmed Fadiel, Adel Hamza, Shashikant Lele, Junko Mastuzaki, Kunle Odunsi. LAG3 and PD1 co-inhibitory molecules directly interact to limit CD8+ T cell signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2014-4075