Anti igg1 clone mopc 21

Cells were stimulated with plate-bound anti-CD3 (R&D, clone UCHL1, various concentrations), anti-IgG1 (clone MOPC-21) or anti-CD161 (clone HP-3G10) (Biolegend) (HPV16-specific CD4+ T cell clones: 5 µg/mL, OPSCC TIL: 10 µg/mL). Alternatively, cells were preincubated with biotinylated anti-IgG1 (clone MOPC-21) or anti-CD161 (clone HP-3G10) (Biolegend, 5 µg/mL) for 30 min, washed, and crosslinked with anti-biotin antibody (Stemcell Technologies, 5 µg/mL) during stimulation with plate-bound anti-CD3 (1 µg/mL) and soluble anti-CD28 (2 µg/mL, BD, clone CD28.2). 2 days before coculture, autologous B lymphoblastoid cell lines (B-LCL) were cultured (0.25×10⁶ cells/mL) and after 1 day loaded overnight with indicated amounts of 22-mer E6 and E7 peptides (pool or specific peptide). T cells were preincubated with plate-bound anti-IgG1 or anti-CD161 (10 µg/mL) for 1 hour before coculture with irradiated (7500 RAD) peptide-loaded B-LCL at indicated ratio’s. Cells were cultured for 4 hours in presence of Brefeldin A (10 µg/mL) or 24 hours with Brefeldin A added after 1 hour of culture and analyzed by intracellular cytokine staining, or 3 days and analyzed for interferon γ (IFNγ) and GM-CSF secretion using ELISA (Mabtech).