转化生长因子β不敏感的树突状细胞疫苗对肾细胞癌荷瘤小鼠细胞毒性T淋巴细胞的影响

Cytotoxic effect of TGF-β insensitive dendritic cell vaccine on cytotoxic T lymphocytes in mice with renal cell carcinoma

目的树突状细胞(dendritic cell,DC)是一种最强的抗原提呈细胞,转化生长因子β(transforming growth factor-β,TGF-β)能降低其递呈抗原的效率并促进其凋亡,文中观察TGF-β不敏感肿瘤特异性的DC疫苗免疫后的荷瘤Balb/c小鼠的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)对小鼠肾细胞癌Renca细胞的免疫治疗作用。方法利用修饰的TGF-βⅡ型受体(TβRⅡ)质粒构建逆转录病毒载体,转染肾细胞癌Renca细胞裂解物负载的DC,获得表达显性负相TβRⅡ(TβRⅡDN)的DC,流式细胞仪测定细胞表型变化,Western blot检测Smad-2的磷酸化情况,观察TGF-β体外增殖抑制效果。TβRⅡDN的DC疫苗接种荷瘤Balb/c小鼠模型,获取CTL与肿瘤细胞共同孵育51Cr释放实验测定细胞毒作用,同时行酶联免疫吸附(ELISA)测定细胞因子IFN-γ和IL-2变化情况。结果 TβRⅡDN-DC组细胞与DC细胞表型相比无显著变化(P>0.05);对TGF-β增殖抑制不敏感同时检测不到磷酸化的Smad-2,能显著提高免疫小鼠CTL的特异性杀伤作用(P<0.01);IFN-γ和IL-2水平也显著升高(P<0.01)。结论 TGF-β不敏感的DC疫苗能显著提高肾细胞癌荷瘤Balb/c小鼠CTL对相关肿瘤细胞的细胞毒性作用。

Objective Dendritic ceils （DC） are the most potent antigen-presenting ceils, and transforming growth factor beta （TGF-β） can reduce their efficiency of antigen presentation and promote their apoptosis. This study was to observe the immunotherapeutic effect of cytotoxie T lymphocytes （CTL） isolated from tumor bearing Balb/c mice vaccinated with TGF-β insensitive tumor specific DC vaccine on Renca cells in mice with renal cell carcinoma （RCC）. Methods DCs were pulsed with freeze-thaw tumor lysates and rendered insensitive to TGF-β by infecting them with a retrovirus containing dominant-negative TGF-β type Ⅱ receptor （TβR Ⅱ DN）. The immunophenotype of TβR Ⅱ DN DCs was analyzed by flow cytometry, and the antiproliferative reaction of TGF-β and the level of phosphorylated Smad-2 detected by Western blot. The Balb/c tumor-bearing mice were challenged with TβR Ⅱ DN DC vaccine. Then CTLs were harvested and cultured with RCC Renca cells, their cytotoxic effect measured by 51Cr releasing test, and the levels of serum IFN-γ and IL-2 determined by ELISA. Results Statistic analyses showed that the expression of TβR Ⅱ DN DCs did not affect the phenotype of transduced DCs （ P 〉 0.05 ）. The TβR Ⅱ DN DCs insensitive to the antiproliferative effect of TGF-β and devoid of phosphorylated Smad-2 markedly enhanced the specific lethal effect of CTLs （P 〈 0. 01 ）. The levels of serum IFN-γ and IL-2 were both elevated remarkably （P 〈 0. 01 ）. Conclusion TGF-β insensitive DC vaccine can significantly increase the cytotoxic effect of CTLs on RCC Renca cells in tumor bearing Balb/c mice.