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

肿瘤相关的具有效应记忆表型(Tem)的CD4+和CD8+T细胞存在于 人非小细胞肺癌和卵巢癌的微环境，但未能控制肿瘤 进步。我们的数据证实，这些细胞对肿瘤的无反应性在一定程度上是由于 他们未能通过T细胞受体(TCR)对激活信号做出反应。通过澄清逮捕地点 在TCR途径中，通过定义启动这种停滞的分子和细胞事件，它应该是 可以设计和测试策略，以在现场重新激活TEM。Tem的本地重新激活在 治疗的肿瘤微环境有望导致：(A)T细胞介导的原位杀伤肿瘤细胞，(B) 释放肿瘤抗原进入循环，(C)产生系统性抗肿瘤免疫，和(D)T细胞 识别和根除与最初治疗对象相邻或相距较远的现有肿瘤 肿瘤部位。使用多光谱成像流式细胞术、共聚焦显微镜、蛋白质印迹 而RTPCR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。在一个 相关第二目的确定与触发TCR停滞有因果联系的细胞和分子 通过细胞耗尽和添加回方案，通过监测选定的分子对启动 这个监管信号检查站。我们的初步研究已经确定了信号检查站的位置 发生在PLC上游的某个地方，因此TCR信号停止与转化生长因子1有因果关系 论证了我们实验协议的可行性和可行性。从这些研究中获得的结果 在AIMS 2和AIMS 3中将利用机械学研究来确定直接作用于或 间接作用于T细胞以逆转其无反应性，并开发和测试脂质体制剂 旨在以局部和持续的方式在体内传递这些因子。在最终目标中， 各因子对原位再激活TM和诱导局部和全身抗肿瘤的疗效 对响应进行评估。后者是使用已建立的异种移植模型来完成的，在该模型中 将人类肿瘤的片断通过外科手术植入SCID或NOD-SCID/IL2受体缺失的小鼠体内。 在这些异种移植中，肿瘤微环境得以保留，肿瘤相关的白细胞得以保留。 对细胞因子信号有较长时间的可行和可预测的反应。在接种了 因子载脂体入异种移植瘤的杀伤和Tem反应模式的监测以确定 每种脂质体制剂的治疗效果。这些研究可望为 设计可用于提高我们当前癌症疫苗临床试验的有效性的策略。

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 immaging 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-γ and the TCR signal arrest has been causally linked to TGF-β1 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 nondisrupted pieces of human tumor are surgically implanted into SCID or NOD-SCID/IL2 receptor γ chainnull 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.