HLA-A2型人外周血MAGE-A10特异性T细胞的体外激发及功能检测

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

目的:探讨体外激发HLA-A2型人外周血单个核细胞(PBMC)中的MAG-E-A10(254-262)特异性T淋巴细胞及其功能检测方法。方法:分别采用肽段MAGE-A-10(254-262)直接刺激PBMC的Bulk Culture法和自体DC负载肽段MAGE-A10(254-262)刺激CD3+CD8+法,均经3次刺激进行体外扩增tetramer-MAGE-A10(254-262)阳性T细胞。以人流感病毒肽段flu-matrix(58-66)为阳性对照。Intracellular staining法检测其针对MAGE-A10(254-262)的穿孔素、颗粒酶B的分泌能力。结果:HLA-A2型PBMC用BuckCulture或DC负载肽段刺激CD3+CD8+法培养,激发的tetramer-flu阳性T细胞率均>10%,DC法激发的tetramer-flu阳性T细胞率显著高于Buck Culture,t=9.79,P<0.01。Bulk Culture法得到的阳性T细胞率分别为0.01%和0.02%,DC法则分别是0.05%和0.12%。Sorting得到的tetram-er-MAGE-A10(254-262)阳性T细胞,效靶比为10∶1时其特异性杀伤为46%,细胞针对MAGE-A10(254-262)分泌穿孔素、颗粒酶B的百分率分别为39%和87%。结论:从捐献的人血细胞中用自体DC负载肽段刺激CD3+CD8+法激发tetramer阳性T细胞的效率优于Bulk Culture法。

OBJECTIVE： To explore the method of generating HLA-A2 restricted MAGE-A10（254-262） specific T lympho cytes 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 pro liferate 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, P〈0.01. 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 tetramer-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 pep tide to stimulate CD3^＋ CD8^＋ T lymphocytes culture is more efficient than by Bulk Culture to generate tetramer positive T lymphocytes from healthy donor blood.