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

来源于肿瘤的热休克蛋白gp96持有肿瘤抗原肽，并通过产生CD8 CTL而不受MHC限制，引发针对亲代肿瘤的特异性保护性免疫。然而，肿瘤来源的gp96对已建立的肿瘤的治疗效果并不乐观。本研究分析了骨髓源性树突状细胞（DC）在免疫功能正常的C57BL/6小鼠中以卵清胺（LLC- ova）转导的LLC （Lewis Lung Cancer）肿瘤的治疗效果。分别于第3、7、10、14天向C57BL/6小鼠右心房皮下注射1×10^5的LLC-OVA，左心房皮下注射LLC-OVA衍生的gp96、DC或以LLC-OVA衍生的gp96脉冲的DC。用lc - ova衍生的gp96或DC治疗几乎不影响已建立的lc - ova肿瘤生长。以3μg的llc源性gp96作为DC脉冲给药，其抗肿瘤作用明显增强。当lc - ova衍生的gp96脉冲DC与特异性t细胞受体（TCR）转基因CD8+细胞（OT-1）共孵育时，ova特异性IFN-γ的产生未被证实。然而，用llc -OVA衍生的gp96脉冲DC治疗可诱导局部淋巴结中OVA-四聚体阳性CD8 T细胞的数量略有增加，这表明抗肿瘤作用部分是由OVA肽诱导的。共焦激光显微镜显示gp96被DC携带进入核内体，并将其肽转移到核内体的MHC I类分子上。我们的数据表明，用肿瘤来源的gp96进行DC治疗可能是有效的抗肿瘤疫苗。

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.