Re-activating Memory T Cells in the Microenvironment of Human Tumors

描述（由申请人提供）：具有效应记忆表型（Tem）的肿瘤相关CD4+和CD8+ T细胞存在于人类非小细胞肺肿瘤和卵巢癌的微环境中，但不能控制肿瘤进展。我们的数据已经确定，这些细胞对肿瘤的无反应性部分是由于它们未能响应通过T细胞受体（TCR）的激活信号。通过阐明TCR通路中阻滞的位置，并通过定义启动这种阻滞的分子和细胞事件，应该可以设计和测试原位重新激活Tem的策略。在治疗后的肿瘤微环境中，Tem的局部再激活预计会导致(a) T细胞介导的原位肿瘤细胞杀伤，(b)肿瘤抗原释放到循环中，(c)产生全身抗肿瘤免疫，(d) T细胞识别并根除最初治疗肿瘤部位附近或远处的现有肿瘤。结合多光谱成像流式细胞术、共聚焦显微镜、western blot和rt - pcr，我们的第一个目标是确定TCR通路中转导信号被阻断的位置。在相关的第二个目标中，与触发TCR阻滞有因果关系的细胞和分子是通过细胞消耗和添加回协议以及通过监测选定分子对该调节信号检查点启动的影响来确定的。我们的初步研究已经将信号检查点的位置定位在PLC-3的上游某处，TCR信号阻滞与TGF-21有因果关系，从而证明了我们的实验方案的可行性和可行性。从这些机制研究中获得的结果将用于目标2和目标3中，以确定直接或间接作用于T细胞以逆转其无反应性的生物活性因子，并开发和测试旨在以局部和持续的方式在体内递送这些因子的脂质体配方。在最后的目的，每个因子的治疗效果重新激活Tem原位和诱导局部和全身抗肿瘤反应进行评估。后者是通过一种已建立的异种移植模型来完成的，在这种模型中，未被破坏的人类肿瘤块通过手术植入SCID或NOD-SCID/ il - 2受体？链空鼠标。在这些异种移植物中，肿瘤微环境得以保存，肿瘤相关白细胞保持活力，并可预见地对细胞因子信号长时间作出反应。在将载因子脂质体接种到异种移植物中后，监测肿瘤杀伤和Tem反应模式，以确定每种脂质体制剂的治疗效果。这些研究有望为设计可用于提高我们目前癌症疫苗临床试验有效性的策略奠定基础。

DESCRIPTION (provided by applicant): Tumor-associated CD4+ and CD8+ T cells with an effector memory phenotype (Tem) are present within the microenvironment of human non-small cell lung tumors and ovarian carcinomas, but fail to control tumor progression. Our data have established that the non-responsiveness of these cells to the tumor is due in part to their failure to respond to activation signals via the T cell receptor (TCR). By elucidating the site of the arrest in the TCR pathway and by defining the molecular and cellular events that initiate this arrest it should be possible to design and test strategies to re-activate the Tem in situ. The local re-activation of the Tem within the treated tumor microenvironment is expected to result in, (a) T cell mediated killing of tumor cells in situ, (b) release of tumor antigens into the circulation, (c) generation of a systemic anti-tumor immunity, and (d) T cell recognition and eradication of existing tumors at sites that are adjacent to or distant from the initially treated tumor site. Using a combination of multispectral imaging flow cytometry, confocal microscopy, western blot and rtPCR our first aim is to determine where in the TCR pathway the transduction signal is blocked. In a related second aim the cells and molecules that are causally linked to triggering the TCR arrest are determined by cell depletion and add back protocols and by monitoring the effects of selected molecules on the initiation of this regulatory signaling checkpoint. Our preliminary studies have localized the site of the signaling checkpoint to occur somewhere upstream of PLC-3 and the TCR signal arrest has been causally linked to TGF-21 thereby demonstrating the feasibility and viability of our experimental protocols. The results obtained from these mechanistic studies will be utilized in aims 2 and 3 to identify biologically active factors that act directly or indirectly on the T cells to reverse their non-responsiveness, and to develop and test liposome formulations that are designed to deliver these factors in a local and sustained fashion in vivo. In the final aim the therapeutic efficacy of each factor for re-activating Tem in situ and for inducing a local and systemic anti-tumor response is evaluated. The latter is to be accomplished using an established xenograft model in which non- disrupted pieces of human tumor are surgically implanted into SCID or NOD-SCID/IL2 receptor ? chain null mice. In these xenografts the tumor microenvironment is preserved and the tumor- associated leukocytes remain viable and predictably responsive to cytokine signals for prolonged periods. Following the inoculation of the factor loaded liposomes into the xenografts tumor killing and Tem response patterns are monitored to determine the therapeutic efficacy of each liposomal preparation. These studies are expected to lay the foundation for the design of strategies that can be used to enhance the efficacy of our current cancer vaccination clinical trials. Memory T cells that are present within and surrounding cancer patients' tumors have the potential to recognize and kill tumor cells, but they often fail to control the growth and spread of the cancer cells in these patients. This failure is due in part to a non-responsive or quiescent state of the memory cells. We are investigating the reason for this quiescent state, designing strategies to reverse this non-responsiveness, and testing the therapeutic benefits of re-activating the tumor-associated memory T cells.