弱酸洗脱提取的肿瘤抗原肽致敏的树突状细胞对CTL的体内外激活效应

In vivo and in vitro activation of CTL by murine dendritic cells pulsed with acid eluted tumor antigen peptides

目的 弱酸洗脱提取小鼠红白血病细胞膜表面肿瘤抗原肽 ,用于致敏树突状细胞(dendriticcells,DC) ,观察其对细胞毒性T淋巴细胞 (CTL)的体内外激活效应。方法 以pH3 3的枸橼酸 磷酸盐缓冲液室温下处理FBL 3小鼠红白血病细胞 5min,流式细胞术检测洗脱效率 ;弱酸洗脱的抗原肽经SepPakC18柱提取纯化 ,用于致敏DC ,检测其体外刺激CTL细胞株增殖的能力及体内激发特异性抗肿瘤免疫应答的能力。结果 弱酸可以洗脱肿瘤细胞膜表面绝大部分与MHCⅠ类分子结合的抗原肽 ;经此抗原肽致敏的DC ,体外可以激发特异性识别FBL 3细胞抗原九肽CCLCLTVFL的CD8+CTL细胞株增殖 ,回输小鼠 ,可以诱导体内生成高水平的特异性CTL活性 ,使小鼠获得抵抗后继FBL 3细胞攻击的免疫保护能力 ,并显著高于肿瘤冻融抗原致敏的DC回输组。结论 弱酸可以有效洗脱肿瘤细胞表面的Ⅰ类抗原肽 ,该类多肽致敏的DC ,在体内外均可激活CTL 。

Objective To observe the activation effect on cytotoxic T lymphocytes (CTL) by murine dendritic cells (DCs) pulsed with mild acid eluted tumor antigen peptides presented on erythroleukemia cell surface. Methods FBL 3 erythroleukemia cells were treated with citrate phosphate buffer (pH3.3) at room temperature for 5 min. Flow cytometry was used to analyze the elution effect. The acid eluted peptides were subsequently purified through SepPark C18 column. DCs pulsed with these peptides were used to stimulate the proliferation of the specific CTL line in vitro and to induce specific anti tumor response in vivo. Results Mild acid could elute tumor antigen peptides from tumor cells effectively. DCs pulsed with these peptides could stimulate the proliferation of the specific CD8 + CTL line which could recognize Friend murine leukemia viral nonapeptides CCLCLTVFL presented on the surface of FLB 3 cells. Vaccination with DCs pulsed with the peptides could also induce high level of specific CTL activity in vivo and make mice resistant to the subsequent challenge of parental tumor cells. The in vivo effects induced by DCs pulsed with thawed tumor antigens were also achieved, but were less effective than that induced by DCs pulsed with acid eluted antigen peptides. Conclusions Mild acid could elute class Ⅰ restricted peptides from tumor cells. DCs pulsed with acid eluted tumor antigen peptides could activate CTL and induce specific anti tumor immunity effectively.