CD4+ Memory T-Cells in Human Tumor Microenvironment

描述（由申请人提供）：在卵巢肿瘤相关抗原患者接种疫苗后，在外周血中发现肿瘤特异性T细胞通过其T细胞受体（TCR）完全响应激活。然而，来自卵巢肿瘤微环境的T细胞（CD4+和CD8+）对与外周血T细胞相同的TCR刺激反应迟钝或无反应。肿瘤相关T细胞的低反应性部分是由于TCR信号级联的阻滞，并且在体外和体内都可以通过IL-12逆转。虽然人类T细胞在进入肿瘤微环境后的命运尚未确定，但我们预测并将测试功能性T细胞在进入肿瘤后引起TCR信号级联阻滞的可能性，从而使这些细胞无能。由于间质成纤维细胞是肿瘤微环境中的主要细胞类型，并且它们表达具有生物活性的细胞因子和共抑制表面结合分子，因此我们的重点将放在这些细胞在调节淋巴细胞功能和存活中的作用上。首先在体外通过将来自外周血的肿瘤特异性T细胞与来自肿瘤的自体成纤维细胞共同培养来解决这个问题，并比较T细胞存活率、T细胞活化电位以及在TCR刺激下不培养成纤维细胞的T细胞后信号分子表达和磷酸化模式的变化。在目标1和目标2中，将确定由成纤维细胞、淋巴细胞、树突状细胞或肿瘤细胞产生或表达的可溶性和膜结合生物活性因子，这些因子有助于TCR信号阻滞，并延长肿瘤相关记忆T细胞的存活时间。鉴于公认的成纤维细胞的异质性及其对T细胞的多性作用，在Aim 3中讨论了存在表型不同的成纤维细胞亚群的可能性，这些亚群负责增强T细胞的存活，阻止T细胞信号传导，促进肿瘤生长。在最后的目标中，测试了旨在预防或逆转抑制性TCR信号阻滞的策略的治疗效果，以增强肿瘤相关记忆T细胞的存活，并延缓肿瘤进展。最后一个目标是通过在异种移植模型中使用功能阻断抗体来实现的，在异种移植模型中，肿瘤细胞、T淋巴细胞和成纤维细胞被植入NOD-scid/IL-2受体？链null （NSG）小鼠。该模型首次在体内长时间监测人成纤维细胞和其他基质细胞对肿瘤生长和T细胞功能的影响。使用该模型，将确定提高肿瘤相关T细胞的存活和预防或逆转TCR阻滞对肿瘤进展的控制可能产生的有益影响。预计这些研究将为设计可用于提高当前癌症疫苗治疗效果的新策略提供基础。公共卫生相关性：癌症疫苗诱导肿瘤特异性T细胞在外周血中循环，但当这些T细胞离开循环进入肿瘤时，它们不能攻击和杀死肿瘤细胞。我们打算确定是什么原因导致肿瘤特异性T细胞的明显功能阻滞，并设计和测试方法，以防止和/或逆转这些细胞到达肿瘤后的关闭。

DESCRIPTION (provided by applicant): Following vaccination of patients with an ovarian tumor-associated-antigen, tumor-specific T cells are found in the peripheral blood that are fully responsive to activation via their T cell receptor (TCR). However, T cells (both CD4+ and CD8+) derived from the microenvironment of the ovarian tumors are either hyporesponsive or nonresponsive to the same TCR stimulation used for peripheral blood T cells. The hyporesponsiveness of the tumor-associated T cells is due in part to an arrest in the TCR signaling cascade, and is reversible both in vitro and in vivo by IL-12. While the fate of human T cells following their entry into the tumor microenvironment has not yet been determined we predict and will test the possibility that functional T cells, upon entry into the tumor, incur an arrest in the TCR signaling cascade rendering these cells anergic. Because stromal fibroblasts represent such a major cell type within the tumor microenvironment, and because they express biologically active cytokines and co-inhibitory surface bound molecules, our focus will be upon the role of these cells in modulating lymphocyte function and survival. This is to be addressed first in vitro by co-cultivating tumor-specific T cells derived from the peripheral blood with autologous fibroblasts derived from the tumor and comparing T cell survival, T cell activation potential, and changes in the expression and phosphorylation patterns of the signaling molecules following a TCR stimulation to T cells cultivated without fibroblasts. In Aims 1 and 2, the soluble and membrane bound biologically active factors produced or expressed by fibroblasts, lymphocytes, dendritic cells or tumor cells that contribute to the TCR signaling arrest, and to the prolonged survival of the tumor-associated memory T cells are to be identified. In view of the recognized heterogeneity of fibroblasts and their demonstrated pleiotropic effects on T cells, the possibility that phenotypically distinct subsets of fibroblasts exist, and are responsible for the enhanced survival of T cells, the arrest of T cell signaling, and the enhancement of tumor growth is to be addressed in Aim 3. In the final Aim, a test is made of the therapeutic efficacy of strategies designed to prevent or reverse the inhibitory TCR signaling arrest, to enhance the survival of tumor-associated memory T cells, and to retard tumor progression. This last aim is achieved using function blocking antibodies in a xenograft model in which tumor cells, T lymphocytes and fibroblasts are implanted into NOD-scid/IL-2 receptor ? chainnull (NSG) mice. This model has made it possible for the first time to monitor the effect of human fibroblasts and other stromal cells on tumor growth, and T cell function for prolonged periods in vivo. Using this model the possible beneficial effects of enhancing the survival and preventing or reversing the TCR arrest in tumor-associated T cells upon the control of tumor progression will be determined. These studies are expected to provide a basis for the design of new strategies that could be used to enhance the therapeutic effectiveness of current cancer vaccines. PUBLIC HEALTH RELEVANCE: Cancer vaccines induce tumor-specific T cells that circulate in the peripheral blood, but when these T cells leave the circulation and enter the tumor they fail to attack and kill tumor cells. We intend to determine what is responsible for the apparent functional arrest of the tumor-specific T cells and to design and test ways to prevent and/or reverse the shutdown of these cells once they reach the tumor.