IMMUNOTHERAPY OF MELANOMA WITH ACTIVATED LYMPHOCYTES

该提案的广泛和长期目标是：(1)开发针对人类黑色素瘤的有效免疫治疗策略；(2)强效抗肿瘤效应T细胞的鉴定。我们的假设是，主动特异性免疫治疗可能会在引流淋巴结中诱导抗自体黑色素瘤的前效应细胞。然而，这些前效应细胞可能没有活性，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特异性免疫治疗在体内诱导特异性前效应细胞，并在体外激活和扩增这些前效应细胞，使其成为抗肿瘤效应细胞，用于过继免疫治疗。具体目的是：(1)研究T细胞免疫治疗患者的免疫反应；(2)在体内研究GM-CSF作为免疫佐剂致敏淋巴结T细胞的机制，特别关注抗原呈递细胞的作用；(3)淋巴结T细胞的免疫特性、体外反应性与其体内抗肿瘤效果之间的关系。IV期黑色素瘤患者将使用放射的自体肿瘤细胞混合GM-CSF作为佐剂进行免疫接种，以增强免疫反应。肿瘤疫苗引流7天后手术切除的淋巴结，在体外用细菌超抗原和抗cd3进行激活和扩增。大量这些活化的T细胞将被静脉注射到患者体内进行过继免疫治疗。在这项研究驱动的临床试验中，这些活化的T细胞的免疫学特性将通过ELISA和ELISPOT来详细研究细胞因子的产生，51/铬释放法来研究T细胞的细胞毒性，流式细胞术和免疫组织化学染色来研究T细胞标志物，以及聚合酶链反应来研究T细胞受体的使用。将进行皮肤测试并进行分析。这些细胞的体外反应性将与其体内抗肿瘤效果和皮肤试验反应性相关。希望这项研究能让我们更好地了解宿主与肿瘤的相互作用，并改进产生治疗有效效应T细胞的方法。如果能够建立针对自体黑色素瘤产生特异性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，因为迄今为止这些患者还没有有效的治疗方法。

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.