An alternative explanation for T cell activation by antibodies and soluble pMHC was proposed by Minguet and Schamel (84); multivalent binding of antibodies or pMHC tetramers can induce changes in the orientation of pMHC molecules in the plasma membrane due to the limited distances between antibody or pMHC binding sites, thereby providing weak mechanical forces around the engaged TCRs. dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of Vortioxetine topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs. protein tethers with different dimensions. Ovals represent Ig-like domains. The tethers are derived from BGP-1 (one Ig domain name), a monomeric IgG1 Fc domain name (two Ig domains), CD66 (three Ig domains), CD43 (extended rod-like structure), PTK-7 (seven Ig domains), or CD44 (unknown structure). Materials and Methods Cell Lines and Animals OKT3 hybridoma cells were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). BALB/c 3T3 cells, HT29 human colon cancer cells, BC3 hybridoma cells, HB65 hybridoma cells, and Jurkat human T cells were from the American Type Culture Collection (Manassas, VA, USA). 2B4 mouse T cells were kindly provided by Dr. Ming-Zong Lai, Institute of Molecular Biology, Academia Sinica. The B3Z mouse T cell hybridoma was kindly provided by Dr. Ya-Wun Yang, School of Pharmacy, College of Medicine, National Taiwan University. Rabbit Polyclonal to MAP2K3 (phospho-Thr222) OT-I (15) and OT-II (16) transgenic mice were a kind gift from Dr. Nan-Shih Liao, Institute of Molecular Biology, Academia Sinica. Mouse splenic T cells were obtained from BALB/c, C57BL/6, OT-I, or OT-II mice purification over Vortioxetine nylon wool. Human whole blood, obtained from healthy donors by the Taipei City Blood Bank mentioned already in ethics statement. Antibodies and Reagents Rat anti-HA (clone 3F10) was purchased from Roche (Mannheim, Germany). The 25D-1.16 antibody, which recognizes Kb-SINFEKL complexes, was generously Vortioxetine provided by Dr. Ron Germain, National Institutes of Health (Bethesda, MD). Mouse anti-HA (clone 16B12) was from Covance (Berkeley, CA, USA). FITC-labeled anti-CD8, PE-labeled CD44, Alexa Fluor 647-labeled anti-CD62L, PE-labeled AF6-88.5 (anti-H-2Kb), and FITC-labeled AF6-120.1 (anti-I-Ab) antibodies were from BD Biosciences (East Rutherford, NJ, USA). Rat anti-CD66acd antibody was from AbD Serotec (Kidlington, UK). Rabbit anti-6xHis Tag antibody was from Bioman Scientific (Jhonghe, Taiwan). HRP-conjugated affinipure donkey anti-mouse IgG, HRP-conjugated affinipure goat anti-rabbit IgG, HRP-conjugated affinipure goat anti-rat IgG, HRP-conjugated streptavidin, goat anti-mouse Ig(A?+?G?+?M), and goat anti-human Ig(A?+?G?+?M) were from Jackson ImmunoResearch (West Grove, PA, USA). FITC-conjugated goat F(ab)2 anti-mouse IgG Fc was from ICN Pharmaceuticals (Aurora, OH, USA). Mouse anti–actin (Clone AC-74) was from Sigma-Aldrich (St. Louis, MO, USA). Biotin-conjugated mouse anti-phosphotyrosine (clone 4G10) and rabbit polyclonal anti-ZAP-70 were from Millipore (Temecula, CA, USA). 2C11, BC3, and OKT3 antibodies were purified from ascites produced in BALB/c mice by affinity chromatography using Protein G Sepharose (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Recombinant DNA We used the murine B7.1 transmembrane and cytoplasmic domains to express and tether ligands on the surface of 3T3 APCs. The construction of p2C11-B7, p2C11-BGP-B7 (2C11-1), p2C11-1-B7 (2C11-2d), p2C11-CD44-B7 (2C11-CD44), and p2C11-CD43-B7 (2C11-CD43) have been described (14, 17, 18). We used PCR to amplify the CH2-CH3 domains from human IgG1 excluding the hinge region using p2C11-1-B7 Vortioxetine as a template, the modified 1 (m1) was used to replace BGP in p2C11-BGP-B7 to generate p2C11-m1-B7 (2C11-2). DNA fragments encompassing the ectodomains of human CD66 and PTK-7 with flanking sites were amplified from HT29 cells by RT-PCR. These DNA fragments were inserted in place of the BGP fragment in p2C11-BGP-B7 to generate p2C11-CD66-B7 (2C11-3) and p2C11-PTK-B7 (2C11-7), respectively. The 1 fragment in pLNCX-phOx-1-B7 (18) was replaced with the BGP fragment in p2C11-BGP-B7 to generate the control scFv construct pLNCX-phOx-BGP-B7 (phOx-1). The constructs encoding for BC3 and OKT3.