CTL-based Immunotherapy in an Organotypic Me

原发性黑色素瘤与T淋巴细胞的浸润性与良好的临床结局有关。这表明基于T细胞的免疫疗法可能对黑色素瘤患者有益。然而，针对黑色素瘤患者的过继细胞溶解T淋巴细胞(CTL)免疫治疗和以CTL为基础的抗原/多肽疫苗的主动免疫治疗并不是很有希望。这些局限性可能在于体外CTL免疫治疗的选择不能反映原位CTL的抗原特异性。传统上，CTL是在二维混合淋巴细胞-肿瘤培养(MLTC)中直接在塑料表面上选择的。我们已经建立了三维人类黑色素瘤器官典型培养模型(黑色素瘤重建)。 其中CTL是在类组织条件下培养的。我们的初步研究表明，与MLTC中的T细胞受体(TCR)库相比，肿瘤重建中的T细胞受体(TCR)库更接近于原位T细胞的库。然而，这两种文化体系是相辅相成的。因此，与单独培养相比，当两种培养系统中的T细胞结合在一起时，产生类似于原位(组织)TCR的T细胞的机会更大。我们的主要目标是发展，包括黑色素瘤的重建作为培养系统，改进的基于CTL的过继和主动免疫治疗黑色素瘤。具体地说，我们将：1.比较表型(CD标志物、TCR、黏附和凋亡相关 来自重建或MLTC新鲜组织来源的CTL的功能特性(增殖和裂解活性；细胞因子产生)。2.细胞毒性T淋巴细胞识别的克隆抗原(S)具有高度的细胞毒性，并表达原位发现的原始TcR。这些AGS具有黑色素瘤患者的疫苗潜力，并可能增强先前存在的免疫反应。3.确定参与CTL向肿瘤细胞迁移的趋化因子和趋化因子受体。这包括：1)选择移行性CTL；2)阻断趋化因子及其受体以确定它们在CTL移行中的作用。重建过程中与CTL迁移相关的趋化因子可用于增强过继和主动免疫治疗的抗肿瘤效果。拟议的研究开启了新的 以CTL为基础的过继和主动免疫疗法的方法，可能在孢子的第三年启动。

Infiltration of primary melanomas with T lymphocytes is associated with a favorable clinical outcome. This suggests that T cell-based immunotherapies may be beneficial for melanoma patients. However, adoptive cytolytic T lymphocyte (CTL) immunotherapy of melanoma patients and active immunotherapy with CTL-based antigen (Ag)/peptide vaccines have not held promise. These limitations may rest in the selection for immunotherapy of CTL in vitro that do not reflect the Ag specificity of the CTL in situ. CTL traditionally are selected in two-dimensional mixed lymphocyte-tumor culture (MLTC) directly on plastic surfaces. We have developed the three-dimensional human melanoma organolypic culture model (melanoma reconstruct) in which CTL are cultured under tissue-like condition. Our preliminary studies suggest that the T cell receptor (TCR) repertoire in tumor reconstructs more closely resembles the repertoire of the T cells in situ as compared to the TCR repertoire in MLTC. However, the two culture systems are complementary. Thus, the chances of generating T cells with in situ (tissue)-Iike TCR are enhanced when T cells from both culture systems are cornbined, compared to either culture alone. Our major goal is to develop, including the melanoma reconstruct as a culture system, improved CTL-based adoptive and active immunotherapies against melanoma. Specifically we will: 1. Compare the phenotypic (CD markers, TCR, adhesion and apoptosis-related molecules, HLA, chemokines, chemokine receptors) and functional characteristics (proliferative and lytic activity; cytokine production) of CTL derived from fresh tissues in the reconstruct or MLTC. 2. Clone Ag(s) recognized by CTL that are highly cytolytic and express the original TCR found in situ. These Ags have vaccine potential for melanoma patients and may boost pre-existing immune responses. 3. Identify chemokines and chemokine receptors that are involved in CTL migration toward tumor cells in the reconstruct. This involves: i) Selection of migrating CTL and ii) blocking of chemokines and their receptors to determine their role in CTL migration. Chemokines relevant in CTL migration in the reconstruct may be used to enhance anti-tumor effects of adoptive and active immunotherapy. The proposed studies open new approaches to CTL-based adoptive and active immunotherapies, which may be initiated during the third year of the SPORE.