Generation of HLA-A2 restricted MAGE-A10 specific T lymphocytes in vitro from PBMC of healthy donors and their function evaluation

OBJECTIVE: To explore the method of generating HLA-A2 restricted MAGE-A10(254-262) specific T lymphocytes in vitro from PBMC of healthy donors.METHODS: Bulk Culture from PBMC and autologous DC loaded with MAGE-A-10(254-262) peptide to stimulate CD3+CD8+ T lymphocytes culture were used respectively,with the stimulation for three times to proliferate tetramer-MAGE-A10(254-262) positive T lymphocytes,and human flu virus peptide flu-matrix(58-66)(GILGFVFTV) was set as the positive control.An intracellular staining assay was used for their MAGE-A10(254-262) specific Perforin/Granzyme B secretion capacities.RESULTS:Both the generating methods for flu-matrix(58-66) obtained tetramer-positive T lymphocytes with the concentration over 10%,and the concentration of obtained positive T lymphocytes from DC stimulation culture was obviously higher than that from Bulk Culture,t=9.79,P0.01.By Bulk Culture,0.01% and 0.02% tetramer positive T lymphocytes were harvested from the two samples while 0.05% and 0.12% tetramer positive T lymphocytes by DC stimulation culture from the same samples.By the functional assays,the sorted tetra-mer-MAGE-A10(254-262) positive T lymphocytes showed 46% specific killing effects at effector:target/10∶1,and 39%/87% of Perforin/Granzyme B secretion capacities targeted to MAGE-A10(254-262) positive tumor cells.CONCLUSION: By autologous DC loaded with peptide to stimulate CD3+CD8+ T lymphocytes culture is more efficient than by Bulk Culture to generate tetramer positive T lymphocytes from healthy donor blood.