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

肿瘤来源的热休克蛋白gp96含有肿瘤抗原肽，通过产生不受MHC限制的CD8CTL诱导对亲本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。本研究分析了卵蛋白转导的小鼠骨髓来源的树突状细胞(DC)对免疫活性C57BL/6小鼠建立的LLC肿瘤的治疗效果。分别于第3、7、10、14天将1×105的LLC-OVA注射到C57BL/6小鼠右侧背部皮下，将LLC-OVA来源的gp96、DC或负载LLC-OVA gp96的DC注入左侧背部皮下。LLC-OVA来源的gp96或DC对已建立的LLC-OVA肿瘤生长几乎没有影响。用3μg的LLC来源的gp96冲击DC后，其抗肿瘤作用明显增强。与转T细胞受体基因CD8细胞(OT-1)共孵育后，未见OVA特异性干扰素γ的产生。然而，经LLC-OVA来源的gp96冲击的DC治疗后，局部淋巴结中OVA-四聚体阳性的CD8 T细胞数量略有增加，这表明OVA多肽可能部分地诱导了抗肿瘤作用。强制激光显微镜显示gp96被DC摄取，进入内小体，并将其多肽转移到内小体中的MHC I类分子。我们的数据表明，用肿瘤来源的gp96包裹的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.