J Immunol 2014 193, 96-101
Vassiliki MagafaTg was purified from thyroid glands of female CD-1 mice (Charles River, Quebec, QC, Canada) after centrifugation of thyroid extract at 16,000 3 g, followed by passage through a Sepharose CL-4B column (Amersham Biosciences, Uppsala, Sweden), as previously described (10). F-moc L-thyroxine was produced as previously described (18, 19). The 12-mer Tg peptide T4p2553 (aa 2549-2560), STDD(T4)ASFSRAL, was synthesized at the University of Patras, Greece, and at Biosynthesis (Lewisville, TX) at .90% purity. The 19-mer OVA peptide (322-340), CISQAVHAAHAEI-NEAGRY, was synthesized at the Alberta Peptide Institute (Edmonton, AB, Canada) at .80% purity. Both peptides were blocked with an acetyl group at the N terminus and with an amide group at the C terminus. OVA was purchased from Sigma. CFA (with Mycobacterium butyricum) and IFA were purchased from Difco (Detroit, MI). Culture media and lymphocyte proliferation assays Inguinal, brachial, and axillary LNCs from Ag-primed mice were cultured in DMEM supplemented with 10% FCS (PAA Laboratories, Etobicoke, ON, Canada). In ISAT, thyroid-draining cLNCs or spleen cells were cultured in HL-1 medium (Lonza, Walkersville, MD). Media were supplemented with 100 U/ml penicillin/streptomycin, 2 mM glutamine (Life Technologies, Invitrogen, Grand Island, NY), and 5 3 10 25 M 2-ME (Sigma-Aldrich, St. Louis, MO). RPMI 1640 (Life Technologies, Invitrogen) with 10% FCS was used in activation assays of T cell hybridomas. Lymphocyte proliferation assays were performed by culturing 4 3 10 5 cells per well with Ag in 96-well flatbottom plates for 3-4 d at 37˚C. During the last 18 h, 1 mCi [ 3 H]thymidine (PerkinElmer, Boston, MA) was added to each well. Cells were harvested in a Classic Cell Harvester (Skatron Instruments), and radioactivity was measured in a LS6500 MultiPurpose Scintillation Counter. Stimulation index (S.I.) was defined as cpm in the presence of Ag/cpm in the absence of Ag. Generation and characterization of the KC1 hybridoma clone LNCs from T4p2553-primed NOD.H2 h4 mice were activated in vitro with 20 mM T4p2553 for 4 d and were subsequently fused with BW5147 a 2 b 2 cells (20), kindly provided by P. Marrack (National Jewish Medical and Research Center, Denver, CO), using polyethylene glycol 1500 (Roche Diagnostics, Mannheim, Germany). Screening for IL-2-secreting, peptidespecific hybrids was done using TA3 cells as APCs and the IL-2-dependent CTLL line, as previously described (21). Cloning was performed by limiting dilution at 0.3 cell per well. The IgG2a mAbs specific for A k (TIB 92) or influenza A nucleoprotein (NP) (HB 65) (American Type Culture Collection, Manassas, VA) were purified from culture supernatants, as previously described (22). Percent inhibition of KC1 activation by blocking mAbs was calculated as follows: [1 2 (cpm in the presence of mAb)/(cpm in the absence of mAb)] 3 100.
