Expansion of Anti Tumor T Cells from Tumor Bearing Hosts

描述(由申请人提供)：我们已经证明了抗原特异性 已建立的小鼠肿瘤的消退和对挑战的抵抗力 过继免疫疗法(AIT)体外激活肿瘤致敏T细胞 用Bryostatin 1和离子霉素(B/I)。B/I选择性地激活 效应/记忆细胞，表达低水平的L选择(CD62L)。我们 提出实验来描绘细胞和分子机制 解释了这些新奇的观察结果。具体目标将涉及两个方面 问题：1.我们将检验可能解释为什么B/I选择性的假设 激活抗原致敏的CD62L T细胞。我们将确定是否 蛋白激酶C(PKC)亚型的差异表达或激活 其他信号事件解释了这种选择性激活。肿瘤致敏 CD62L表达的T细胞和致敏的T细胞 TCR转基因小鼠将用于这些研究。2.使用特定于多肽的 探查和回应，我们将检验以下假设： B/I体外激活抗原致敏T细胞增强免疫功能 通过增加抗原反应性的数量来对抗肿瘤抗原的反应 T细胞，与直接接种相比。我们还将检验这一假设 这种扩增可以增加多肽疫苗的抗肿瘤效果。 用于已建立的肿瘤和自发转移的治疗。我们已经这么做了 研究表明，AIT与来自肿瘤的药物激活的T细胞 细胞免疫的小鼠比直接接种更有效。这些研究 将使用4T07/4T1小鼠乳腺癌模型和 白血病病毒源性AH1多肽(SPSYVYHQF)最近被描述为一种 这种肿瘤的主要T细胞表位。AH1特异性T细胞的数量将 通过ELISpot分析和商业使用的流式细胞术进行计数 提供LD-Ig调光剂和合成肽。我们还将确定是否 环磷酰胺预处理对AIT抗肿瘤作用的增强作用 过继转移的T细胞的增殖增加。我们还将测试 B/I激活的T细胞在领养后体内增殖的概念 通过使用一种重要的荧光染料来“跟踪”细胞的数量和 从过继转移的细胞中获得的细胞分裂数。 最后，我们将测试ait对肿瘤细胞或多肽致敏的dln细胞的作用。 用B/I激活将在佐剂设置中有效 已确定4T1肿瘤的自发转移。这些研究将会增加 我们对如何操纵对肿瘤抗原的免疫反应和 基础T淋巴细胞生物学。

DESCRIPTION (provided by applicant): We have demonstrated antigen-specific regression of established murine tumors and resistance to challenge with adoptive immunotherapy (AIT) sing tumor-sensitized T cells activated in vitro with bryostatin 1 and ionomycin (B/I). B/I selectively activates effector/memory cells, which express low levels of L-selection (CD62L). We propose experiments to delineate the cellular and molecular mechanisms that account for these novel observations. The specific aims will address two sets of questions: 1. We will test hypotheses that may explain why B/I selectively activates antigen-sensitized CD62L T cells. We will determine whether differential expression or activation of protein kinase C (PKC) isoforms or other signaling events accounts for this selective activation. Tumor-sensitized T cells separately by CD62L expression and sensitized versus naive T cells from TcR transgenic mice will be used for these studies. 2. Using peptide-specific probes and responses, we will test the hypothesis that adoptive transfer of antigen-sensitized T cells activated in vitro with B/I amplifies immune responses against tumor antigen by increasing the number of antigen-responsive T cells, compared to direct vaccination. We will also test the hypothesis that this amplification can increase the antitumor efficacy of peptide vaccination for treatment of established tumors and spontaneous metastases. We have already shown that AIT with pharmacologically activated T cells from tumor cell-vaccinated mice is more effective than direct vaccination. These studies will be carried out using the 4T07/4T1 murine mammary carcinoma model and the leukemia virus-derived AH1 peptide (SPSYVYHQF) recently described to be a dominant T cell epitope for this tumor. The number of AH1-specific T cells will be enumerated by ELISpot assays and by flow cytometry using commercially available Ld-Ig dimmers and synthetic peptides. We will also determine whether enhancement of AIT antitumor effects by cyclophosphamide pre-treatment reflects increased proliferation of adoptively transferred T cells. We will also test the concept that B/I-activated T cells proliferate in vivo after adoptive transfer by using a vital fluorescent dye to "track" the number of cells and the number of cell division derived from the adoptively transferred cells. Finally, we will test whether AIT with tumor or peptide-sensitized DLN cells activated with B/I will be effective in the adjuvant setting against established spontaneous metastases from 4T1 tumors. These studies will increase our understanding of how to manipulate immune responses to tumor antigens and of basic T lymphocyte biology.