Td 139

Galectin inhibitors used in this study were β-lactose (Sigma Aldrich, St. Louis, Mo), thiodigalactoside (TDG) (Cayman Chemical, Ann Arbor, MI), TD-139 (Selleckchem, Houston, TX), GB1107 & G3-C12 (MedChemExpress, Monmouth Junction, NJ). Their structures and binding affinities to galectins were shown in Table 2. All are sugar-based inhibitors, except G3-C12 which is a peptide. Lactose binds to most structures of galectins with low affinity (dissociation constant [Kd] at micromolar to millimolar range). TDG binds to galectin-1,−2,−3,−8,−9 with slightly higher affinity than lactose (Kd at micromolar range). Inhibitors TD139 and GB1107 have similar targets as TDG but have a higher affinity to galectin-3 (Kd at nanomolar range) selectively. G3-C12 binds to galectin-3 specifically at high affinity. Anti-human monoclonal antibodies used for BAT assay were purified α-IgE (G7-18, BD Biosciences, Franklin Lakes, NJ), fluorescein isothiocyanate (FITC) labeled α-CD63 (H5C6; BioLegend, San Diego, CA), allophycocyanin (APC) labeled α-CD203c (NP4D6; BioLegend), phycoerythrin (PE) labeled α-IgE PE (Ige21; eBioscience, Carlsbad, CA). Vivinal^® GOS syrups were obtained from FrieslandCampina, The Netherlands. A fraction of GOS with enriched allergenic GOS DP4 structures and Blo t extract were prepared as previously described (6 (link)).

The 24-well plates were coated with CD3 antibody (OKT3, Invitrogen, Carlsbad, CA, USA) overnight. PBMCs were isolated from the peripheral blood of healthy donors using Human PBMC Isolate kit (TBD, Tianjin, China). CD8⁺ T cells were positive selected from PBMC using magnetic beads coated with CD8 antibodies (BD, Franklin Lakes, NJ, USA). These CD8⁺ T cells were placed into the Transwell lower chamber along with CD28 antibody (Invitrogen, USA) and IL-2 (Procell, Wuhan, China). A549 or A549-3a cells were seeded in Transwell inserts (Corning, Corning, NY, USA). For certain experiments, cells were treated with the galactin-3 inhibitor (TD-139) (Selleck, Houston, TX, USA). The co-culture system was incubated for 48 h at 37 °C and 5% CO₂.