Regulation of DC Activity by Memory and Effector CD8+ T Cells

在该项目的前一个（第一个）资助期，我们证明了记忆和效应CD 8 + T细胞 发挥相互的DC-杀伤（“抑制”）与DC-激活（“辅助”）活动，并不同地影响 癌症疫苗的免疫学和抗肿瘤活性。我们的数据表明，效应CD 8 + T细胞 以穿孔素（Pfn）和粒酶B（GrB）依赖性机制杀死携带抗原（Ag）的DC。与此相反， 记忆性CD 8 + T细胞起“辅助”作用，诱导DC表达丝氨酸蛋白酶抑制剂PI 9（CAP 3/B 9/SPI 6）， 内源性GrB抑制剂和保护DC免受细胞毒性T细胞（CTL）介导的杀伤。他们还诱导 DC的1型极化，表现为IL-12 p70的产生增加， Th 1和CTL应答，并介导抗肿瘤作用。此外，我们已经证明， 名义上的“抑制性”效应细胞毒性T淋巴细胞在药理学阻断后可转化为“辅助细胞”， 它们的细胞溶解机制，或在用IFNα加IL-18非TCR依赖性活化后。 基于这些数据，我们假设清除携带肿瘤相关抗原（TAA）的DC TAA特异性CTL的抑制是限制癌症治疗的长期有效性的重要因素 疫苗。此外，我们假设记忆性CD 8 + T细胞激活DC和保护DC的能力可能与其免疫功能有关。 它们来自效应细胞诱导的细胞凋亡，可用于增加 预防针在下一个拨款期内，我们建议测试上述假设，并制订方法， 抵消预先存在的肿瘤特异性CD 8 + T细胞的抑制作用，并利用它们的“辅助” 在以下具体目标中的潜力： 1.鉴定人CD 8+效应子杀伤DC和DC保护/极化的分子机制 （Teff）与记忆（Teff）细胞，作为免疫干预的潜在靶点。 2.探讨了小鼠CD 8 + Teff和Tcb细胞体外调控DC的关键机制。 tehse研究的成功将使我们能够优化设计前瞻性的体内小鼠研究， 个体调节机制对Teff和Tmem介导的免疫应答的相对贡献 调节，并开发策略来抵消CTL介导的DC杀伤消除， 在治疗性癌症疫苗接种的小鼠模型中利用CD 8 + T细胞依赖性帮助。 该项目的积极成果及其后续研究将有助于我们了解 免疫记忆和调节功能的Teff和Teff细胞，并将使我们能够开发新的现成的 治疗性癌症疫苗和利用保护原理的联合癌症疗法， 极化癌症患者的内源性DC，以实现持续的免疫和 接种疫苗对已确定的癌症的治疗效果。

In the previous (first) period of funding of this project we demonstrated that memory and effector CD8+ T cells exert reciprocal, DC-killing (“suppressive”) versus DC-activating (“helper”) activities and differentially affect the immunologic and anti-tumor activity of cancer vaccines. Our data demonstrate that effector CD8+ T cells kill antigen (Ag)-bearing DCs in a perforin (Pfn)- and Granzyme B (GrB)-dependent mechanism. In contrast, memory CD8+ T cells play a “helper” role, inducing the DC expression of the serpin PI9 (CAP3/B9/SPI6), an endogenous GrB inhibitor, and protecting DCs from cytotoxic T cell (CTL)-mediated killing. They also induce type-1 polarization of DCs, manifested by their enhanced production of IL-12p70, enhanced ability to support Th1- and CTL responses and to mediate antitumor effects. Moreover, we have demonstrated that the nominally “suppressive” effector CTLs can be converted to “helper cells” following pharmacologic blockade of their cytolytic machinery, or following their TCR-independent activation with IFNα plus IL-18. Based on these data, we hypothesize that the elimination of tumor-associated antigen (TAA)-harboring DCs by the TAA-specific CTLs is a significant factor limiting the prolonged effectiveness of therapeutic cancer vaccines. Furthermore, we hypothesize that the ability of memory CD8+ T cells to activate DCs and protect them from the effector cell-induced apoptosis can be utilized to increase the continued effectiveness of vaccination. In the next period of funding, we propose to test the above hypotheses and to develop means to counteract the suppressive impact of pre-existing tumor-specific CD8+ T cells and to utilize their “helper” potential in the following Specific Aims: 1. Identify the molecular mechanisms of DC-killing and DC protection/polarization by human CD8+ effector (Teff) versus memory (Tmem) cells, as potential targets of immunointervention. 2. Validate the key mechanisms of DC modulation by mouse CD8+ Teff and Tmem cells in vitro. Success of tehse studies will allow us to optimally design prospective in vivo mouse studies testing the relative contribution of the individual regulatory mechanisms to the Teff -and Tmem-mediated immune regulation in vivo and to develop strategies to counteract the CTL-mediated DC killing elimination and to utilize the CD8+ T cell-dependent help in mouse models of therapeutic cancer vaccination. The positive outcome of this project and it follow-up studies, will help us to understand basic principles of immune memory and regulatory functions of Teff and Tmem cells, and will allow us to develop new off-the-shelf therapeutic cancer vaccines and combined cancer therapies utilizing the principles of protection and polarization of endogenous DCs of cancer patients, in order to achieve continued immunologic and therapeutic effects of vaccination against established cancer.