CD4+ Memory T-Cells in Human Tumor Microenvironment

描述（由申请方提供）：在患者接种卵巢肿瘤相关抗原后，在外周血中发现肿瘤特异性T细胞，其通过其T细胞受体（TCR）对活化完全应答。然而，来自卵巢肿瘤微环境的T细胞（CD 4+和CD 8+）对用于外周血T细胞的相同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信号级联中的停滞，并且在体外和体内都可通过IL-12逆转。虽然人类T细胞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信号级联阻滞从而使这些细胞无反应性的可能性。因为基质成纤维细胞代表了肿瘤微环境中的主要细胞类型，并且因为它们表达生物活性细胞因子和共抑制表面结合分子，所以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。这将首先在体外通过共培养源自外周血的肿瘤特异性T细胞与源自肿瘤的自体成纤维细胞并将TCR刺激后的T细胞存活、T细胞活化潜力以及信号分子的表达和磷酸化模式的变化与无成纤维细胞培养的T细胞进行比较来解决。在目的1和2中，将鉴定由成纤维细胞、淋巴细胞、树突细胞或肿瘤细胞产生或表达的可溶性和膜结合的生物活性因子，其有助于TCR信号传导阻滞，并有助于肿瘤相关记忆T细胞的延长存活。鉴于成纤维细胞的公认异质性及其对T细胞的多效性效应，目的3中将阐述存在成纤维细胞的表型不同亚群的可能性，并负责T细胞存活增强、T细胞信号传导阻滞和肿瘤生长增强。在最后的目的中，测试了设计用于预防或逆转抑制性TCR信号传导停滞、增强肿瘤相关记忆T细胞的存活和延缓肿瘤进展的策略的治疗功效。这最后一个目的是实现使用功能阻断抗体在异种移植模型中，肿瘤细胞，T淋巴细胞和成纤维细胞植入NOD-scid/IL-2受体？链缺失（NSG）小鼠。该模型首次能够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，将确定在控制肿瘤进展时增强肿瘤相关T细胞中的存活和预防或逆转TCR停滞的可能有益效果。这些研究有望为设计可用于增强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细胞离开循环并进入肿瘤时，它们无法攻击和杀死肿瘤细胞。我们打算确定是什么导致了肿瘤特异性T细胞的明显功能停滞，并设计和测试方法来防止和/或逆转这些细胞一旦到达肿瘤就关闭。

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.