Liu Wujiang, O'Donnell Michael A, Chen Xiaohong, Han Ruifa, Luo Yi
Department of Urology, University of Iowa, 3202 MERF, 375 Newton Road, Iowa, IA 52242, USA.
Cancer Immunol Immunother. 2009 Oct;58(10):1647-55. doi: 10.1007/s00262-009-0673-z. Epub 2009 Feb 13.
The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-alpha 2B secreting recombinant (r) BCG (rBCG-IFN-alpha). We demonstrated that rBCG-IFN-alpha augmented T helper type 1 (Th1) cytokine IFN-gamma production by PBMC. In this study, we further investigated whether rBCG-IFN-alpha could also enhance PBMC cytotoxicity toward bladder cancer cells.
PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-alpha or control MV261 BCG, and used as effector cells in (51)Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-alpha as well as endogenously expressed IFN-gamma and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-alpha in the presence of neutralizing antibodies to IFN-alpha, IFN-gamma or IL-2. To determine the role of natural killer (NK) and CD8(+) T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in (51)Cr-release assays.
Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-alpha further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-gamma and IL-2 by PBMC was observed after rBCG-IFN-alpha stimulation. Blockage of IFN-alpha, IFN-gamma or IL-2 by neutralizing antibodies during rBCG-IFN-alpha stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8(+) T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-alpha with the former cell type being more predominant.
rBCG-IFN-alpha is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.
有效的卡介苗（BCG）膀胱癌免疫疗法需要适当诱导细胞免疫。已知BCG刺激人外周血单个核细胞（PBMC）可在体外产生对膀胱癌细胞具有细胞毒性的效应细胞。为了改进BCG疗法，我们之前开发了分泌人干扰素（IFN）-α 2B的重组（r）BCG（rBCG-IFN-α）。我们证明rBCG-IFN-α可增强PBMC产生1型辅助性T细胞（Th1）细胞因子IFN-γ。在本研究中，我们进一步研究rBCG-IFN-α是否也能增强PBMC对膀胱癌细胞的细胞毒性。
从健康个体中制备PBMC，不进行处理或用rBCG-IFN-α或对照MV261 BCG刺激，然后用作铬（51）释放试验中的效应细胞。人膀胱癌细胞系T24、J82、5637、TCCSUP和UMUC-3用作靶细胞。为了确定分泌的rIFN-α以及内源性表达的IFN-γ和IL-2在诱导细胞毒性中的作用，在存在针对IFN-α、IFN-γ或IL-2的中和抗体的情况下，用rBCG-IFN-α刺激PBMC。为了确定自然杀伤（NK）细胞和CD8（+）T细胞在诱导细胞毒性中的作用，在PBMC受到BCG刺激后分离这两种细胞类型，并用作铬（51）释放试验中的效应细胞。
未刺激的PBMC对所有测试的靶细胞系显示出基础水平的细胞毒性。MV261 BCG使PBMC细胞毒性增加了1.8至4.2倍。rBCG-IFN-α使PBMC细胞毒性进一步增加了高达2倍。在rBCG-IFN-α刺激后，观察到PBMC中IFN-γ和IL-2的产生增加。在rBCG-IFN-α刺激期间，用中和抗体阻断IFN-α、IFN-γ或IL-2可降低或消除PBMC细胞毒性的诱导。发现NK细胞和CD8（+）T细胞均对rBCG-IFN-α诱导的PBMC细胞毒性增强负责，其中前者细胞类型更为主要。
rBCG-IFN-α是一种改良的BCG制剂，可在体外诱导PBMC对膀胱癌细胞的细胞毒性增强。这种rBCG菌株可作为治疗浅表性膀胱癌的BCG替代品。
