Abstract 4005: Understanding the mechanism of 2FF-induced immune modulation

Abstract

Abstract Fucosylation is the process of adding a fucose sugar to a glycan chain. The fucose analog, 2-fluorofucose (2FF), has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate, GDP-fucose, as well as direct inhibition of fucosyltransferases. 2FF has shown promising activity in a number of tumor models. For example 2FF enhanced the protective effect of a lymphoma vaccine (Okeley et al, PNAS 110, 2013), and this protection was determined to be immune dependent since depletion of CD4 and CD8 T-Cells reduced the 2FF/vaccine activity. Due to the complex nature of immune interactions in vivo we established an experimental system using human PBMCs to assess how 2FF-mediated immune changes may contribute to 2FF anti-tumor activity. Human PBMCs were matured in the presence of 2FF for 10 days and phenotypic analysis was performed. Using lectin binding, we found that 2FF dose-dependently decreased cell surface fucosylation which correlated with decreased levels of GDP-fucose and formation of GDP-2FF. 2FF-treatment alone had minimal effect on immune cell phenotype and no significant changes in activation or lineage markers were identified. Recently receptor fucosylation has been shown to be critical for BCR antigen recognition and antibody production (Li et al, J Immunol 194, 2015) as well as TLR recognition and signaling through the scavenger receptor DC-SIGN (Gringhuis et al, Nat Commun 5, 2014). As the TCR is reported to be fucosylated and basal signaling and activation of this receptor is regulated by a galectin-glycoprotein lattice we hypothesized that afucosylation following 2FF treatment may affect TCR functionality. During TCR engagement and activation the galectin-glycoprotein lattice is disrupted. 2FF-treated T cells show decreased galectin-3 levels and when activated have increased and more sustained TCR signaling, shown by increased levels of phosphorylated TCR associated proteins. Additionally, 2FF-treated T cells show increased tetramer specific binding. These data indicate that alteration in surface fucosylation on T cells impacts the regulatory glycoprotein lattice that negatively influences T cell action. Following up on this observation, in co-cultures 2FF-treated T cells activate dendritic cells in a contact dependent and antigen specific manner, shown by upregulation of maturation markers MHCII, CD83, CD86 and CD40. Overall we hypothesize that 2FF-treated T cells are more easily activated due to decreased avidity of galectin-3 binding to glycoproteins which lowers the threshold of lattice disruption by peptide-MHC resulting in easier TCR engagement, increased TCR signaling, and dendritic cell maturation. Citation Format: Jessica J. Field, Nicole M. Okeley, Weiping Zeng, Che-Leung Law, Peter D. Senter, Shyra J. Gardai. Understanding the mechanism of 2FF-induced immune modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4005.