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 已建立的鼠肿瘤的消退和对用 采用体外激活的肿瘤致敏T细胞的过继免疫治疗（AIT） 苔藓抑素1和离子霉素（B/I）。B/I选择性激活 效应/记忆细胞，其表达低水平的L-选择（CD 62 L）。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。具体目标将涉及两套 问题：1.我们将检验可能解释为什么选择性地选择B/I的假设 激活抗原致敏的CD 62 L T细胞。我们将决定 蛋白激酶C（PKC）同种型的差异表达或活化，或 其它信号事件解释了这种选择性激活。肿瘤致敏的 T细胞分别通过CD 62 L表达和致敏与幼稚T细胞， TcR转基因小鼠将用于这些研究。2.使用肽特异性 探针和反应，我们将测试的假设，过继转移的 体外用B/I活化的抗原致敏T细胞增强免疫 通过增加抗原应答细胞的数量， 与直接接种相比，T细胞。我们还将检验以下假设： 这种扩增可以增加肽疫苗接种的抗肿瘤功效 用于治疗已建立的肿瘤和自发转移。我们已经 显示AIT与来自肿瘤的活化T细胞 细胞接种的小鼠比直接接种更有效。这些研究 将使用4 T07/4 T1鼠乳腺癌模型和 白血病病毒衍生的AH 1肽（SPSYVYHQF）最近被描述为一种 这种肿瘤的显性T细胞表位。AH 1特异性T细胞的数量将 通过ELISpot测定和通过流式细胞术使用市售的 可用的Ld-Ig二聚体和合成肽。我们还将确定 环磷酰胺预处理增强AIT抗肿瘤作用反映了 过继转移的T细胞增殖增加。我们还将测试 B/I激活的T细胞在过继后体内增殖的概念 通过使用活体荧光染料来“跟踪”细胞数量， 来自过继转移细胞的细胞分裂数。 最后，我们将测试AIT是否与肿瘤或肽致敏的DLN细胞 用B/I激活的药物将在辅助治疗中有效， 4 T1肿瘤的自发转移。这些研究将增加 我们对如何操纵对肿瘤抗原的免疫反应的理解， 基本的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.