Lymphocyte Stimulation and Cytokine Analysis

Single cell suspensions from aseptically removed the head and neck lymph nodes (HNLNs), mesenteric LNs (MLNs), and spleens were prepared as previously described [35 (link)]. Lymphocytes were cultured in a complete medium: RPMI 1640 with 2 mM l-glutamine (Genesee Scientific, El Cajon, CA) containing 10% fetal bovine serum (Atlanta Biologicals, Oakwood, Georgia), plus supplements (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. Lymphocytes were cultured at 10⁶ cells/well in 96-well, round-bottomed tissue culture plates (Millipore, Billerica, MA) coated with 5 μg/mL anti-CD3 mAb (clone 17A2; Invitrogen, Carlsbad, CA, USA) plus 2.5 μg/mL of soluble anti-CD28 mAb (clone 37.51; Invitrogen) was stimulated for 48 h at 37 °C. Lymphocytes were stimulated in triplicate for 2 days for flow cytometry analysis or for 4 days for collection of cell culture supernatants, which were then stored at − 20 °C until assayed by cytokine-specific ELISAs.