Cellular immunotherapy and immune-gene therapy by heat shock protein gp96 and dendritic cells

肿瘤热休克蛋白gp 96具有肿瘤抗原肽，通过产生不依赖于MHC限制的CD 8 CTL来增强针对亲本肿瘤的特异性保护性免疫。然而，肿瘤衍生的gp 96对已建立的肿瘤的治疗效果并不乐观。本研究分析了免疫活性C57 BL/6小鼠中用卵清蛋白（LLC-OVA）转导的骨髓来源的树突状细胞（DC）对已建立的LLC（刘易斯肺癌）肿瘤的治疗作用。在第3、7、10和14天，将1×10^5的LLC-OVA皮下注射到C57 BL/6小鼠的右弗兰克中，并将LLC-OVA衍生的gp 96、DC或用LLC-OVA衍生的gp 96脉冲的DC皮下注射到左弗兰克中。用LLC-OVA衍生的gp 96或DC治疗几乎不影响已建立的LLC-OVA肿瘤生长。当给予用3μg LLC衍生的gp 96脉冲的DC时，抗肿瘤效果显著增强。当用LLC-OVA衍生的gp 96脉冲的DC与特异性T细胞受体（TCR）转基因CD 8+细胞（OT-1）共孵育时，未证实OVA特异性IFN-γ产生。然而，用LLC-OVA衍生的gp 96脉冲的DC治疗诱导局部淋巴结中OVA-四聚体阳性CD 8 T细胞的数量轻微增加，这表明抗肿瘤作用部分由OVA肽诱导。激光共聚焦显微镜观察显示，gp 96被DC摄取，进入内体，并将其肽段转移到内体的MHC I类分子上。我们的数据表明，用肿瘤衍生的gp 96包裹的DC治疗可能用作有效的抗肿瘤疫苗。

Heat shock protein gp96 derived from tumors holds tumor antigen peptides and elicits specific protective immunity against parental tumors through the generation of CD8 CTL independent of MHC restriction. However, the therapeutic effects of tumor-derived gp96 on established tumors have not been promising. The present study analyzes the therapeutic effects on established LLC (Lewis Lung Cancer) tumors transduced with ovalbumine (LLC-OVA) of bone marrow-derived dendritic cells (DC) pulsed with LLC-OVA-derived gp96 in immunocompetent C57BL/6 mice. 1×10^5 of LLC-OVA was subcutaneously injected into right frank in C57BL/6 mice and LLC-OVA-derived gp96, DC or DC pulsed with LLC-OVA-derived gp96 was subcutaneously injected into left frank on day 3,7,10 and 14. Therapy with either LLC-OVA-derived gp96 or DC barely affected established LLC-OVA tumor growth. The antitumor effect was significantly enhanced when DC pulsed with 3μg of LLC-derived gp96 was administered. When DC pulsed with LLC-OVA-derived gp96 was co-incubated with specific T-cell receptor (TCR) transgenic CD8+ cells (OT-1), OVA-specific IFN-γ production was not demonstrated. However, therapy with DC pulsed with LLC-OVA-derived gp96 induced slight increase in the numbers of OVA-tetramer positive CD8 T cells in the regional lymph nodes, which revealed that antitumor effect was induced partly by OVA peptides. Conforcal laser microscope showed that gp96 was taken up by DC, entered endosome and transfered their peptides to MHC class I molecules in the endosome. Our data suggest that therapy with DC pursed with tumor-derived gp96 may be useful as potent anti-tumor vaccines.