Tüting T, Steitz J, Brück J, Gambotto A, Steinbrink K, DeLeo A B, Robbins P, Knop J, Enk A H
Department of Dermatology, J. Gutenberg-University, Mainz, Germany.
J Gene Med. 1999 Nov-Dec;1(6):400-6. doi: 10.1002/(SICI)1521-2254(199911/12)1:6<400::AID-JGM68>3.0.CO;2-D.
The induction of cellular immune responses to melanocyte-specific enzymes such as the tyrosinase family of proteins is the goal of various clinical studies for the immunotherapy of melanoma. Tyrosinase-related protein-2 (TRP2) is an attractive model antigen for preclinical studies in C57BL/6 mice because it is naturally expressed by the murine B16 melanoma and can be recognized by self-reactive cytolytic T lymphocytes (CTL). Here we describe efforts to develop genetic immunization with dendritic cells (DC) for the immunotherapy of melanoma in this clinically relevant system.
Recombinant adenoviruses encoding green fluorescent protein (Ad-EGFP) and murine TRP2 (Ad-mTRP2) were constructed using Cre-loxP-mediated recombination. DC were generated in vitro from precursors in bone marrow and transduced with Ad-EGFP or Ad-mTRP2. Mice were immunized by direct injection of adenovirus or by injection of Ad-transduced DC. Induction of tumor immunity was assessed by intravenous challenge with B16 melanoma cells and enumeration of experimentally induced lung metastases.
Flowcytometric analysis of DC transduced with Ad-EGFP demonstrated endogenous fluorescence due to cytoplasmatic expression of EGFP in 30-60% of cells. Ad-EGFP-transduced DC simultaneously displayed the DC-specific marker NLDC145 and high levels of MHC and costimulatory molecules on their cell surface. Transduction of DC with Ad-mTRP2 resulted in strong intracellular expression of TRP2 which could be readily detected by immunostaining. Importantly, immunization of mice with cultured Ad-mTRP2-transduced DC completely prevented the development of lung metastases following an intravenous challenge with B16 melanoma cells. This striking protective effect was observed with both the intravenous and the subcutaneous route of DC immunization. In vivo depletion of T-cell subsets suggested that the protective effect of an immunization with Ad-mTRP2-transduced DC involved both CD8+ and CD4+ T-cells.
Our results demonstrate that DC-based genetic immunization of mice with TRP2, a clinically relevant melanocyte-specific self-antigen, induces effective cellular immunity and prevents metastatic growth of B16 melanoma cells in vivo.
诱导针对黑素细胞特异性酶（如酪氨酸酶家族蛋白）的细胞免疫反应是黑色素瘤免疫治疗各种临床研究的目标。酪氨酸酶相关蛋白2（TRP2）是C57BL/6小鼠临床前研究中一种有吸引力的模型抗原，因为它在鼠B16黑色素瘤中自然表达，并且能被自身反应性细胞毒性T淋巴细胞（CTL）识别。在此，我们描述了在这个临床相关系统中为黑色素瘤免疫治疗开发树突状细胞（DC）基因免疫的工作。
使用Cre-loxP介导的重组构建编码绿色荧光蛋白（Ad-EGFP）和鼠TRP2（Ad-mTRP2）的重组腺病毒。DC在体外由骨髓前体细胞生成，并用Ad-EGFP或Ad-mTRP2转导。通过直接注射腺病毒或注射Ad转导的DC对小鼠进行免疫。通过静脉注射B16黑色素瘤细胞并计数实验诱导的肺转移来评估肿瘤免疫的诱导情况。
对用Ad-EGFP转导的DC进行流式细胞术分析显示，由于30%至60%的细胞中EGFP的细胞质表达而产生内源性荧光。用Ad-EGFP转导的DC同时在其细胞表面展示DC特异性标志物NLDC145以及高水平的MHC和共刺激分子。用Ad-mTRP2转导DC导致TRP2在细胞内大量表达，这可以通过免疫染色很容易地检测到。重要的是，用培养的Ad-mTRP2转导的DC免疫小鼠，在用B16黑色素瘤细胞静脉攻击后完全防止了肺转移的发生。在DC免疫的静脉和皮下途径中均观察到这种显著的保护作用。体内T细胞亚群的耗竭表明，用Ad-mTRP2转导的DC免疫的保护作用涉及CD8+和CD4+ T细胞。
我们的结果表明，用TRP2（一种临床相关的黑素细胞特异性自身抗原）对小鼠进行基于DC的基因免疫可诱导有效的细胞免疫，并在体内防止B16黑色素瘤细胞的转移生长。
