目的：探讨穿膜肽（cell penetrating peptide, CPP）Tat49-57携带NY-ESO-1155-163抗原肽致敏DC后诱导获得抗原特异性细胞毒性T淋巴细胞（cytotoxic lymphocyte, CTL)，并体外检测其对黑素瘤细胞株A-375的杀伤效果。 方法：采集健康志愿者外周血50 ml，用淋巴细胞分离液分离外周血单个核细胞，经细胞因子诱导，获得DC和T淋巴细胞。实验组用Tat49-57-NY-ESO-1155-163致敏DC，待DC成熟后与T淋巴细胞混合诱导产生CTL，并设PBS组和NY-ESO-1155-163组作为对照。流式细胞术检测致敏前后DC的表型，乳酸脱氢酶（LDH）释放法检测抗原肽疫苗致敏DC后诱导的CTL对黑素瘤细胞株A-375的体外杀伤活性，同时与对人肺癌A549细胞株、人白血病K562细胞的杀伤作用进行比较。结果：Tat显著提升NY-ESO-1155-163进入DC的穿膜能力。与NY-ESO-1155-163致敏的DC相比，Tat49-57-NY-ESO-1155-163致敏后DC的CD80/CD86\[(54.9 ±3.3 )% vs (43.8±5.7)%，P<0.05\]和CD40\[(42.1±1.9)% vs (23.7±2.8)%，P<0.05\]的表达率显著升高。Tat49-57-NY-ESO-1155-163刺激DC后诱导培养的T细胞亚群主要以MHC-Ⅰ类分子介导的CD3+CD8+细胞为主。Tat49-57-NY-ESO-1155-163组CTL对A-375细胞的杀伤能力显著高于NY-ESO-1155-163组，且随着效靶比的增加，杀伤活性逐渐增强（P<0.05）。A-375细胞高表达NY-ESO-1，Tat49-57-NY-ESO-1155-163组CTL对A-375细胞具有特异性杀伤作用，杀伤作用显著强于A549和K562细胞（均P<0.05）。结论：Tat49-57可以增强NY-ESO-1155-163抗原多肽的免疫原性，Tat49-57-NY-ESO-1155-163多肽致敏DC能有效诱导CTL抗黑素瘤细胞A-375的特异性免疫应答。

Objective:To investigate the cytotoxic effect of cytotoxic lymphocytes (CTLs) activated by Tat49-57-NY-ESO-1155-163 sensitized dendritic cells on melanoma A-375 cells. Methods:We collected the peripheral blood (about 50 ml) from healthy volunteers and isolated mononuclear cells by using lymphocyte separation medium. The cells were treated with cytokines to produce dendritic cells and T lymphocytes. After sensitized with the Tat49-57-NY-ESO-1155-163 peptide, the dendritic cells were co-cultured with the T lymphocyte cells to generate antigen-specific CTLs. Phenotypes of the dendritic cells were examined by flow cytometry. The antigen-specific cytotoxic activity of the CTLs against A-375 melanoma cells in vitro was assessed by the lactate dehydrogenase (LDH) method. Human lung cancer A549 cells and leukemia K562 cells were used as controls. Results: The expression rate of CD80/CD86 in dendritic cells sensitized with Tat49-57-NY-ESO-1155-163 was (54.9±3.3)% significantly higher than these sensitized with NY-ESO-1155-163 (\[43.8±5.7\]%，P<0.05). There were also significant increase of CD40 expression (\[42.1±1.9\]% vs \[23.7±2.8\]%，P<0.05) in Tat49-57-NY-ESO-1155-163 sensitized dendritic cells. These results indicated that the Tat fragment (49-57) significantly improved the cell penetrating ability of NY-ESO-1155-163. The T lymphocytes activated by the Tat49-57-NY-ESO-1155-163 sensitized DC were mainly CD3+CD8+cells. The cytotoxic activity of the CTLs induced by Tat49-57-NY-ESO-1155-163-sensitized dendritic cells toward A-375 melanoma cells was significantly higher than these induced with NY-ESO-1155-163-sensitized dendritic cells (P<0.05). The CTLs were also specific as they killed NY-ESO-1-expressing A375 more efficient than A549 cells and K562 cells (P<0.05). Conclusion: Tat49-57 cell penetrating peptide can enhance the immunogenicity of the peptide NY-ESO-1155-163. Tat49-57-NY-ESO-1155-163 polypeptide-sensitized dendritic cells can effectively induce the specific immune response against melanoma A-375 cells.

山西省生物治疗示范平台项目资助（No.2014091105-0101）