IMMUNOTHERAPY OF MELANOMA WITH ACTIVATED LYMPHOCYTES

该提案的广泛和长期目标是：（1）开发针对人黑色素瘤的有效免疫策略;和（2）鉴定有效的抗肿瘤效应T细胞。我们的假设是，针对自体黑色素瘤的前效应细胞可以通过主动特异性免疫治疗在引流淋巴结中诱导。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，使其成为过继免疫治疗的抗肿瘤效应细胞。具体目标是：（1）研究接受T细胞免疫治疗的患者的免疫应答;（2）研究GM-CSF作为免疫佐剂在体内使淋巴结T细胞敏化的机制，特别关注抗原呈递细胞的作用;和（3）淋巴结T细胞的免疫学特性和体外反应性与其体内抗肿瘤功效之间的关联。IV期黑色素瘤患者将用与GM-CSF混合的经辐照的自体肿瘤细胞作为佐剂进行免疫以增强免疫应答。7天后手术收获的肿瘤疫苗引流淋巴结将在体外用细菌超抗原活化和扩增，然后用抗CD 3。大量的这些活化的T细胞将被静脉输注给患者用于过继免疫治疗。在这项研究驱动的临床试验中，将使用ELISA和ELISPOT（用于细胞因子产生）、51/铬释放试验（用于T细胞毒性）、流式细胞术和免疫组织化学染色（用于T细胞标志物）以及聚合酶链反应（用于T细胞受体使用）详细研究这些活化T细胞的免疫学特征。将进行皮肤试验并进行分析。这些细胞的体外反应性将与其体内抗肿瘤功效和皮肤试验反应性相关。希望这项研究将使我们能够更好地了解宿主-肿瘤相互作用，并改进产生治疗有效效应T细胞的方法。如果能够建立产生针对自体黑色素瘤的特异性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.