Cellular and molecular mechanisms of NK cell suppression

描述（由申请人提供）：自然杀伤（NK）细胞是抵御感染的第一道防线。在它们的一生中，NK细胞经历了一个动态的过程：随着它们的发育，NK细胞承诺，增殖，分化，然后停止。一旦被激活，它们立即获得细胞毒性，并变得非常贪婪地杀死目标细胞。NK细胞倾向于杀戮，因为它们在发育过程中就被设定了这种功能。我们的长期目标是了解控制NK细胞动力学的调节机制，这是成功开发基于NK细胞的过继免疫疗法的先决条件。尽管在过去20年的共同努力下，我们发现了NK细胞的关键正调节因子，但目前的知识还未能识别或阐明NK细胞负调节的机制。我们搜索了潜在的候选者，并成功地鉴定出TGF？作为NK细胞的有效负调节因子1。迄今为止，TGF？仍然是唯一确定的NK细胞数量负调节因子。因此，迫切需要研究TGF？在NK细胞中起作用我们提出的研究的具体假设是：TGF？在病毒感染的反应中，限制NK细胞的产生和细胞毒性获得。这一假设基于三个初步数据：第一，TGF？R信号导致骨髓中NK细胞在终末分化时的扩增。二是TGF？R信号显示了先前未描述的Granzyme Blow NK细胞群，否则在正常小鼠中被抑制。第三，TGF？NK细胞对小鼠巨细胞病毒具有耐药性。结合体外和体内方法，我们将在两个特定目标中验证我们的假设：在Aim 1中，我们将确定TGF？控制NK细胞的产生。两项研究旨在探讨1)TGF如何？调节骨髓NK细胞产生过程中的分化、存活和增殖过程，以及i)-为什么NK细胞在分化末期特别容易受到TGF的影响？信号。未来成功发展NK细胞免疫疗法的一个先决条件是确定影响NK细胞生存、扩增和自我更新的因素。TGF缺乏的可能性？R信号可以延长NK细胞的存活，这支持了阻断TGF？在过继转移到患者体内之前，NK细胞中的R通路可能会改善NK细胞免疫治疗策略的设计。在Aim 2中，我们将确定TGF？在应答性感染中限制NK细胞的功能能力。三项研究旨在确定TGF？i)细胞毒性的获得，ii)响应病毒感染的克隆扩增和收缩，以及iii)对病毒配体的耐受性。我们认为了解TGF是如何负调控的？限制NK细胞的细胞毒性将提供一种策略，使NK细胞在面对不断进化的病毒时更强大。

DESCRIPTION (provided by applicant): Natural Killer (NK) cells are the first line of defense against infection. During their lifetime, NK cells undergo dynamic processes: as they develop, NK cells commit, proliferate, differentiate, and then arrest. Once activated, they immediately acquire cytotoxicity and become quite rapacious in killing target cells. NK cells are predisposed to killing because they are programmed for that function during their development. Our long-term goal is to understand the regulatory mechanisms controlling NK cell dynamics as a prerequisite to the development of successful NK-cell based adoptive immunotherapy. Although the collective efforts, accumulated for the last 20 years, resulted in the identification of key positive regulators of NK cells, the current knowledge failed to identify or elucidate the mechanisms of NK cell negative regulation. We searched for potential candidates and successfully identified TGF? as a potent negative regulator of NK cells1. To date, TGF? remains the only identified negative regulator of NK cell number. Therefore, it is imperative that the mechanisms by which TGF? operate in NK cells are understood. The specific hypothesis driving the proposed research is that TGF? limits the production and restricts the cytotoxicity acquisition of NK cells in response to viral infection. This hypothesis is based on three preliminary data: First, lack of TGF?R signaling results in augmented production of NK cells at terminal differentiation in the bone marrow. Second, lack of TGF?R signaling revealed a previously undescribed population of Granzyme Blow NK cells which is otherwise suppressed in normal mice. Third, mice having TGF?-resistant Granzyme Blow NK cells are resistant to murine cytomegalovirus. Using a combination of in vitro and in vivo approaches, we will test our hypothesis in two Specific Aims: In Aim 1, we will determine the mechanisms by which TGF? controls the production of NK cells. Two studies are designed to investigate i)-how TGF? regulates the processes of differentiation, survival, and proliferation during the production of NK from the bone marrow, and i)-why NK cells at their terminal differentiation are particularly susceptible to TGF? signaling. One prerequisite for the development of successful NK-cell-based immunotherapy in the future is to determine the factors that condition the survival, expansion, and self renewal of NK cells. The possibility that a lack of TGF?R signaling can prolong NK cell survival will support the notion that blocking the TGF?R pathway in NK cells prior to adoptive transfer into patients is a possibility for improving the design of NK-cell-based immunotherapeutic strategies. In Aim 2, we will determine the mechanism by which TGF? restricts functional competence of NK cells during response infection. Three studies were designed to determine direct outcomes of a lack of TGF?R signaling on i)- acquisition of cytotoxicity, ii)-clonal expansion and contraction in response to viral infection, and iii)-tolerance to viral ligands. We believe that understanding how negative regulation by TGF? restricts NK cell cytotoxicity will provide a strategy to make NK cells stronger in facing evolving viruses. PUBLIC HEALTH RELEVANCE: A type of white blood cells called Natural Killer (NK) cells may hold the key to combating infection. First discovered in the 1980's, NK cells are recognized as the immune system's front-line defense against infection. Circulating through the body by way of the blood and lymph systems, the majority of NK cells present in the body are in a resting state. Once activated, NK cells become quite rapacious in their search-and-destroy activities. Upon encountering a target cell (an infected cell or cancer cell), the activated NK cell attaches to the membrane of the target cell and injects cytoplasmic granules that quickly dissolve (lyse) the target cell. In less than five minutes, the infected or cancer cell is dead and the NK cell moves on to its next target. A single NK cell can destroy up to 27 target cells before it dies. The importance of NK cells in combating infection is undisputable but viruses are cleaver because they have evolved mechanisms for modulating NK cell activity to persist in spite of an active host immune system. That means we need to develop stronger NK cells to face the evolving viruses. In this research, we propose to study the function of "super"-NK cells developed to be stronger in killing infected cells. These super-NK cells were generated by genetic blockage of TGF? signaling pathway which renders them resistant to TGF? action. They will be called "TGF?-resistant NK cells". Studying the "super" killing functions of TGF?-resistant NK cells is the major goal of this project. Overall, this comprehensive study has a strong potential of clinical applicability. It is novel because it is one of a very few studies to directly assess in vivo the role of TGF? in NK cells, and it is innovative because it uses a unique in vivo model. Outcomes of the project are expected to generate new insight into the fundamental knowledge of NK cells and likely will provide a promising strategy to improve current NK cell- based immunotherapies.