细胞周期抑制因子p27对肿瘤免疫反应的影响及作用机制

Although immune responses directed towards various tumor antigens can be detected in rodent models and cancer patients, these anti-tumor responses are incapable of eliminating cancer in vivo. Three major mechanisms appear to be responsible for the poor anti-tumor immune responses in vivo. First, the inability of tumors to initiate, amplify and maintain CTL responses because they lack costimulatory molecules, thereby inducing T cell anergy instead of priming in tumor infiltrating T lymphocytes. Second, the expression of PD-L1 by tumor cells via which they initiate PD-1-mediated negative signals to tumor infiltrating T lymphocytes, leading to their inactivation. Third, the production of soluble factors (predominantly TGF-?) by tumor cells inhibits T cell activation and expansion. p27 is a critical inhibitor of cell cycle progression. Recently, we observed that p27 deficient T cells are resistant to tolerance induction by blockade of costimulation. Consistently, p27 deficient recipients reject cardiac allografts under conditions that induce long-term allograft survival in wild-type recipients. We have also observed that p27 deficient CTL express lower levels of PD-1 than their wild-type counterparts. Finally, compared to their wild-type counterparts, p27 deficient CD8+ cells are less susceptible to the inhibitory effects of TGF-? and highly sensitive to IL-2 receptor mediated signals. These properties of p27 deficient lymphocytes suggest that elimination of p27 may overcome three major mechanisms via which tumor cells escape immune surveillance in vivo. In this proposal we will explore the hypothesis that p27 deficient CD8+ T cells may be capable of mounting effective anti-tumor responses and potentially suppress tumor growth. We will undertake the following specific aims to: 1) Examine whether p27 deficient CD8+ T cells can differentiate into efficient tumor-specific CTL under tolerogenic conditions. To address this question we shall use OT-I TCR transgenic T cells (which express a TCR specific for OVA257-264 peptide), OT-I /p27 deficient T cells (OT-I/p27?), and EG7 tumor, a syngeneic thymoma that expresses Ovalbumin. We shall examine: 1) CD8+ T cell proliferation and production of IL-2, IFN-? and TNF??in response to tumor cell stimulation; 2) generation of functional CTL, as determined by expression of perforin and granzyme B and assessment of effector function by tumor-specific killing assay in vitro. 2) Examine whether p27 deficient CTL can efficiently suppress tumor growth. We shall use the same experimental system as in Aim 1. We will examine: 1) whether adoptive transfer of OT-I /p27 deficient CD8+ T cells into tumor-bearing mice prevents the growth of tumor, and 2) the properties of adoptive transferred OT-I cells in tumor -bearing host. 3) Examine whether knockdown of p27 in OT-I cells from tumor-bearing mice enhances their cytolytic activity against EG7 tumor in vitro.

肿瘤免疫逃逸的主要机制之一是肿瘤细胞在缺少协同激活因子表达的同时却表达高水平的协同抑制因子从而导致免疫耐受。如何调节T细胞使它们在肿瘤微环境下抵制免疫耐受，增强抗肿瘤免疫反应.是当前肿瘤免疫领域所要解决的关键问题，也是本项目拟解决的问题。细胞周期调控因子p27蛋白阻断细胞增殖反应，是T细胞的免疫耐受过程中的重要环节。p27蛋白缺失的CD4+T细胞对CD28协同激活信号的需求降低，能够抵抗由阻断CD28信号通路引起的免疫耐受。我们最近的研究表明p27基因缺陷的CD8+T细胞在受到同种异体抗原刺激时，活化和增殖较野生型细胞显著增加。我们推测p27基因缺陷的CD8+T细胞在体内能有效地抵抗肿瘤免疫耐受环境的影响，杀伤肿瘤细胞。我们拟利用体外和动物体内实验检测p27基因缺陷对CD8+Ｔ细胞介导的抗肿瘤免疫反应，并对p27蛋白调节免疫反应的分子机制加以研究，为肿瘤免疫治疗提供新方案及理论依据。

项目背景：T细胞受到抗原刺激后的细胞增殖反应是T细胞功能成熟和形成记忆细胞所需要的过程。p27抑制CDK的活性从而阻断T细胞的增殖反应。我们提出的科学问题是能否通过基因调控p27在T细胞中的表达从而在一定程度上影响T细胞的抗肿瘤免疫作用。经典评估抗肿瘤免疫反应至少包括细胞增殖和产生效应分子IFN-g这两个功能，两者在抗肿瘤中起到的作用是否有差异。..研究内容：..1. 肿瘤细胞能否诱导肿瘤抗原特异性p27基因缺陷的CD8+ T细胞分化为杀伤细胞。.2. 过继转移肿瘤抗原特异性p27基因缺陷的CD8+Ｔ细胞能否抑制肿瘤生长。.3. 肿瘤浸润T细胞增殖和分泌IFN-g功能之间的相互关系及其临床意义。.4. IFN-g对肺癌细胞生物学特征的影响及其作用机制。..主要结果：..1. 肿瘤细胞激活p27基因缺陷CD8+Ｔ细胞，表现为诱导这些T细胞表达CD25和发生细胞增殖反应。但是肿瘤细胞不能有效刺激p27基因缺陷CD8+Ｔ细胞产生IFN-g并分化为杀伤性T细胞。.2. 过继转移肿瘤抗原特异性p27基因缺陷的CD8+Ｔ细胞到肿瘤宿主体内显著抑制肿瘤生长，提示T细胞增殖能力在抗肿瘤免疫应答中起到重要作用。.3. 肺癌组织浸润T细胞表型和细胞功能密切相关。肿瘤浸润CD103+T细胞增殖能力强于CD103-T细胞，但是产生IFN-g的能力低下。CD103+T细胞在肿瘤组织中密度高的患者预后较好。PD-1抑制剂有效提高CD103+ T 细胞产生IFN-g的能力。另一类肿瘤浸润T细胞表达CD57, 产生IFN-g的能力高于CD57-T细胞，但两类细胞增殖功能没有显著差别。肿瘤组织高密度CD57+T细胞和不良预后相关。CD57+T细胞对PD-1抑制剂不敏感。综上所述，肿瘤浸润T细胞的细胞增殖而不是产生IFN-g的功能是决定患者预后，甚至免疫检查点抑制剂治疗疗效的关键因素。.4. IFN-g抑制肿瘤细胞增殖，但也引发适应性肿瘤免疫逃逸。IFN-g激活的STAT1和AKT都参与调控PD-L 1的表达。抑制PI3K-AKT通路可能会提高IFN-g的抗肿瘤作用。..科学意义：调控p27在T细胞中的表达可以影响T细胞的抗肿瘤作用。肿瘤浸润T细胞的细胞增殖而不是产生IFN-g的功能是决定患者预后，甚至PD-1抑制剂治疗疗效的关键因素。我们的研究结果为免疫治疗提供了新思路。