热休克瘤细胞裂解物冲击的异基因树突状细胞促进抗肿瘤免疫

目的探讨热休克瘤细胞裂解物冲击的异基因树突状细胞（DC）抗肿瘤免疫效应。方法应用热休克（43℃,1h）小鼠Lewis肺癌细胞（LLC）裂解物（LhTCL）冲击的小鼠骨髓细胞衍生的树突状细胞（DC）作为瘤苗（DC／LhTCL）。应用Western印迹检测可诱导HSP70表达。用FITC-标记的LhTCL和CFSE-标记的异基因DC分别进行体外吞噬和体内示踪实验,藉酶联免疫吸附（ELISA）、流式细胞术（FCM）和共聚焦荧光显微镜观察分析不成熟异基因DC体外摄取LhTCL对其表型和IL-12产生的影响以及受鼠接种局部引流淋巴结DC（LD-DC）对异基因DC的摄取。然后,通过免疫接种的小鼠脾细胞体外诱生CTL的杀瘤活性（LDH法）和产生的Th-1相关细胞因子检测,LLC移植瘤的免疫预防和早期带瘤鼠的治疗实验以评价C57BL小鼠接种异基因或同系DC／LhTCL瘤苗的产生的抗肿瘤免疫效应。结果（1）热休克增加LLC胞浆内可诱导HSP70表达。（2）小鼠不成熟异基因DC能充分摄取LhTCL并促进其DC成熟（增加DC表面共刺激分子表达和IL-12分泌）。（3）皮下注射LhTCL冲击的异基因DC观察到有促进受鼠DC摄取异基因DC的效应。（4）LhTCL冲击的异基因DC免疫的C57BL小鼠脾细胞能特异性杀伤活LLC,分泌Th-1相关的细胞因子（高水平IFN-γ和低水平IL-4和IL-10）。（5）C57BL小鼠LLC移植瘤免疫保护实验和治疗实验均证实,免疫接种异基因DC／LhTCL瘤苗能够阻止或延缓LLC肿瘤的生长,其产生的免疫保护效应要比同系DC／LhTCL瘤苗强。结论接种热休克处理的肿瘤裂解物冲击的异基因树突状细胞瘤苗有促进抗肿瘤免疫的效应。

Allogeneic dendritic cell vaccine pulsed with heat shocked tumor cell lysate can enhance antitumor immunity

OBJECTIVE: To explore whether the allogeneic dendritic cells-pulsed with heat shock protein (HSP)-70-rich tumor cell lysate can enhance the antitumor immunity. METHODS: Mouse Lewis lung cancer cells were dipped into water of the temperature of 43 degrees C for 1 h so as to undergo heat shock. C57BLK(b)) to elicit increased hsp70 expression, The LLCs were lysed by freeze thawing and the supernatant was collected (LhTCL). Dendritic cells (DCs) were isolated from allogeneic inbred mice (615Kk or BALB/CKd) bone marrow (Bm-DCs), and were pulsed with the LhTCL that underwent heart shock. The phenotypes of the Bm-DCs were analyzed by flow cytometry (FCM). The level of IL-12 was detected by ELISA. The Bm-Dcs of Balb/c mice that were pulsed by LhTCL or not, were injected into the foot pads of C57BL mice as vaccine, 3 d later the lymph nodes in the drainage area of popliteal fossa were taken out to make single cell suspension to undergo immunohistochemistry. Inducible hsp70 in LhTCL was assayed by Western Blotting. The ability of taking up LhTCL by immature DC was evaluated by phagocytosis experiment in vitro and the uptake of CFSE labeled allogeneic DC by recipient DC was evaluated by tracing experiment in vivo. The supernatant cytokine release was determined by sandwich ELISA. Double stained DC was confirmed by FCM and confocal microscopy. Cytotoxic activity was assessed by LDH release assay. The immuno-effect of vaccine was valued by immunoprophylaxis in LLC transplanted model and therapy experiment of LLC-bearing mice of early stage. RESULT: 43 degrees C heating significantly increased the expression of inducible hsp70 in the LLCs. The allogeneic immature DC s showed full ability to uptake LhTCL and enhanced the expression of CD86 molecules on DC surface and IL-12 secretion, showing that they promoted the maturation of DCs. Subcutaneous injection of allogeneic DC/LhTCL promoted the uptake of allogeneic DC by recipient DC. The spleen cells from the C57BL mice immunized with allogeneic DC/LhTCL vaccine specifically killed the viable LLCs, and secreted Th-1 related cytokine (i.e. high level of INF-gamma and low level of IL-4/IL-10). The experiment of immunoprophylaxis of LLC transplant and therapy of LLC-bearing C57BL mice both confirmed that immunization with allogeneic DC/LhTCL prevented or delayed the growth of LLCs, which elicited higher immunological effect than that with syngeneic DCs/HLTCL. CONCLUSION: Immunization with vaccine of allogeneic DC pulsed with heat shocked hsp70-rich tumor cell lysate is able to enhance antitumor immunity.