Cytokine release by CD4+ cell lines generated from C57BL/6 mice immunized with hTRP-2 DNA. Five days after the last immunization, CD4+ T lymphocytes were isolated from the spleen and draining inguinal lymph nodes by magnetic bead cell sorting (MACS) and co-cultured with naive irradiated splenocytes with the addition of hTRP-2 237-256 peptide. Following three weekly rounds of in vitro stimulation, cells were stimulated with plate-bound anti-CD3 and anti-CD28 for 6 hours, in the presence of Brefeldin A for the last 2 hours. Flow cytometry was then performed on the cells in order to analyze intracellular cytokines.