IMMUNOTHERAPY OF MELANOMA WITH ACTIVATED LYMPHOCYTES

这项建议的广泛和长期目标是：(1)开发有效的针对人类黑色素瘤的免疫治疗策略；(2)鉴定强大的抗肿瘤效应T细胞。我们的假设是，通过主动的特异性免疫治疗，可以在引流的淋巴结中诱导出针对自体黑色素瘤的前效应细胞。然而，这些前效应细胞可能并不活跃，除非它们在体外被激活成为效应细胞。本研究旨在通过主动免疫治疗在体内诱导特定的前效细胞，并在体外激活和扩增这些前效细胞，使其成为过继免疫治疗的抗肿瘤效应细胞。其具体目的是：(1)研究T细胞免疫治疗患者的免疫反应；(2)研究GM-CSF作为免疫佐剂在体内增敏淋巴T细胞的机制，特别是抗原提呈细胞的作用；(3)研究淋巴T细胞的免疫学特性和体外反应与其体内抗肿瘤效果的关系。IV期黑色素瘤患者将用照射后的自体肿瘤细胞与GM-CSF混合作为佐剂进行免疫接种，以增强免疫反应。7天后手术采集的引流淋巴结的肿瘤疫苗将被细菌超抗原激活并在体外扩增，随后是抗CD3。大量被激活的T细胞将被静脉输注给患者进行过继免疫治疗。在这项以研究为导向的临床试验中，将使用细胞因子产生的酶联免疫吸附试验和ELISPOT、T细胞毒性的51/铬释放试验、T细胞标志的流式细胞术和免疫组织化学染色以及T细胞受体的聚合酶链式反应，详细研究这些激活的T细胞的免疫学特性。将进行皮肤测试并进行分析。这些细胞的体外反应性将与它们在体内的抗肿瘤效果和皮试反应相关。希望这项研究将使我们能够更好地了解宿主-肿瘤的相互作用，并改进产生具有治疗能力的效应性T细胞的方法。如果能够建立产生针对自体黑色素瘤的特定T细胞的标准，该项目的健康相关性对于转移性黑色素瘤患者来说是一个重大的治疗突破，因为到目前为止还没有针对这些患者的有效治疗方法。

The broad and long-term objectives of this proposal are: (1) the development of effective immunotherapeutic strategies against human melanoma; and (2) the identification of potent anti-tumor effector T cells. Our hypothesis is that pre-effector cells against autologous melanoma may be induced in the draining lymph nodes by active specific immunotherapy. However, these pre-effector cells may not be active unless they become activated in vitro to become effector cells. This study is meant to induce specific pre-effector cells in vivo by active specific immunotherapy and to activate and expand these pre-effector cells in vitro to become anti-tumor effector cells for adoptive immunotherapy. The specific aims are: (1) to study the immune response of patients undergoing T cell immunotherapy; (2) to study mechanisms of GM-CSF as an immune adjuvant sensitizing lymph node T cells in vivo with special attention to the role of antigen-presenting cells; and (3) to correlate between immunologic characteristics and in vitro reactivities of lymph node T cells with their in vivo anti-tumor efficacy. Stage IV melanoma patients will be immunized with irradiated autologous tumor ells mixed with GM-CSF as an adjuvant to boost the immune response. Tumor vaccine draining lymph nodes harvested surgically 7 days later will be activated and expanded in vitro with a bacterial super- antigen followed by anti-CD3. A large number of these activated T cells will be infused intravenously to the patient for adoptive immunotherapy. In this research-driven clinical trial, immunological characteristics of these activated T cells will be studied in detail using ELISA and ELISPOT for cytokine production, 51/chromium release assay for T cell cytotoxicity, flow cytometry and immunohistochemical staining for T cell markers and polymerase chain reactions for T cell receptor usage. Skin tests will be performed and analyzed. The in vitro reactivities of these cells will be correlated with their in vivo anti-tumor efficacy and skin test reactivity. It is hoped that this study will allow us to have a better understanding of host-tumor interactions and to improve methods for the generation of therapeutically potent effector T cells. If the criteria to generate specific T cells against autologous melanoma can be established, the health relatedness of the project is a significant therapeutic breakthrough for patients with metastatic melanoma as there is no effective treatment for these patients to date.