T-Cell Proliferation Assay

Draining lymph nodes (maxillary, mandibular, axillary, and inguinal) were harvested from EAM animals at day 21 after immunization. Single-cell suspensions were prepared by lysing the erythrocytes using 1× ammonium chloride potassium buffer (Lonza, Walkersville, MD). After washing, cell pellets were suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 4 mmol/L l-glutamine, 1× each of nonessential amino acids and vitamin mixture, and 100 U/mL penicillin-streptomycin (Lonza; hereafter called growth medium). Lymph node cells (LNCs) were stimulated at a density of 5 × 10⁶ cells/mL in triplicate with the indicated peptides (0 to 100 μg/mL) in growth medium; cells cultured with no peptides were used as medium controls. After 48 hours, cells were pulsed with tritiated ³[H]-thymidine (1 μCi/well; MP Biomedicals, Santa Ana, CA) for 16 hours, and proliferative responses were then measured as counts per minute using a Wallac liquid scintillation counter (Perkin Elmer, Waltham, MA). In some experiments, recall responses were tested in animals immunized with a single dose of peptide/CFA emulsion (100 μg per mouse).