CD4+ Memory T-Cells in Human Tumor Microenvironment

描述(申请人提供)：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，通过其T细胞受体(TCR)对激活完全反应。然而，来自卵巢肿瘤微环境的T细胞(包括CD4+和CD8+)对用于外周血T细胞的TCR刺激反应迟钝或无反应。肿瘤相关T细胞的低反应性在一定程度上是由于TCR信号级联中的停滞，并且在体外和体内都可以被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细胞进入肿瘤后，在TCR信号级联中发生停滞，使这些细胞失去能力的可能性。由于间质成纤维细胞在肿瘤微环境中代表着一种主要的细胞类型，而且它们表达生物活性细胞因子和共抑制表面结合分子，我们的重点将放在这些细胞在调节淋巴细胞功能和生存方面的作用。这首先要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，并比较T细胞存活率、T细胞激活潜力以及在TCR刺激后信号分子的表达和磷酸化模式的变化与没有成纤维细胞培养的T细胞。在目标1和目标2中，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的生物活性因子有助于TCR信号停滞和肿瘤相关记忆T细胞的延长生存。鉴于已知的成纤维细胞的异质性及其对T细胞的多效性作用，在目标3中将讨论存在表型不同的成纤维细胞亚群的可能性，并负责提高T细胞的存活率、阻止T细胞信号转导和促进肿瘤生长。在最终目的中，对旨在防止或逆转抑制性TCR信号停滞、提高肿瘤相关记忆T细胞的存活率和延缓肿瘤进展的策略的治疗效果进行测试。最后一个目的是在异种移植模型中使用功能阻断抗体来实现的，在该模型中，肿瘤细胞、T淋巴细胞和成纤维细胞被植入NOD-SCID/IL-2受体？链空(NSG)小鼠。这一模型首次使监测人成纤维细胞和其他基质细胞对肿瘤生长的影响以及T细胞在体内的长期功能成为可能。利用这一模型，将确定提高肿瘤相关T细胞存活率和防止或逆转肿瘤相关T细胞中TCR停滞在控制肿瘤进展方面的可能有益效果。这些研究有望为设计新的策略提供基础，这些策略可用于提高当前癌症疫苗的治疗效果。

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.