Cellular and molecular mechanisms of NK cell suppression

描述(申请人提供)：自然杀伤(NK)细胞是抵御感染的第一道防线。在其一生中，NK细胞经历了动态的过程：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，它们就会立即获得细胞毒性，并变得非常贪婪地杀死目标细胞。NK细胞易于杀伤，因为它们在发育过程中被编程为具有这一功能。我们的长期目标是了解控制NK细胞动力学的调控机制，以此作为发展成功的基于NK细胞的过继免疫治疗的先决条件。尽管经过近20年的共同努力，人们已经发现了NK细胞的关键正调控因子，但目前的认识未能明确或阐明NK细胞负调控的机制。我们寻找了潜在的候选者，并成功地鉴定出了转化生长因子？作为NK细胞的有效负性调节因子1。到目前为止，是不是，转化生长因子？仍然是唯一已发现的NK细胞数量的负调节因子。因此，研究转化生长因子？在NK细胞中的作用已被了解。推动这项研究的具体假设是：转化生长因子？限制NK细胞的产生和细胞毒作用的获得，以应对病毒感染。这一假说基于三个初步数据：第一，缺乏转化生长因子受体信号导致骨髓在终末分化时NK细胞的产生增加。其次，缺乏转化生长因子受体信号，揭示了一个以前未被描述的颗粒酶Blow NK细胞群体，否则在正常小鼠中被抑制。第三，具有抗转化生长因子的颗粒酶BLOW NK细胞的小鼠对小鼠巨细胞病毒具有抵抗力。使用体外和体内相结合的方法，我们将在两个特定的目标上检验我们的假设：在目标1中，我们将确定转化生长因子？控制NK细胞的产生。两项研究旨在调查i)--转化生长因子如何？调节从骨髓产生NK过程中的分化、存活和增殖过程，以及i)为什么NK细胞在其终末分化时特别容易受到转化生长因子的影响？发信号。未来以NK细胞为基础的免疫治疗的成功发展的前提之一是确定影响NK细胞存活、扩增和自我更新的因素。缺乏转化生长因子受体信号可以延长NK细胞存活的可能性将支持这样一种观点，即在过继转移到患者体内之前阻断NK细胞中的转化生长因子受体通路是改进基于NK细胞的免疫治疗策略设计的可能性。在目标2中，我们将确定转化生长因子？在应答感染过程中限制NK细胞的功能。三项研究旨在确定缺乏转化生长因子受体信号的直接结果：i)获得细胞毒性，ii)对病毒感染的克隆性扩张和收缩，以及iii)对病毒配体的耐受性。我们认为，理解转化生长因子的负面调控是如何实现的？限制NK细胞的细胞毒作用将提供一种策略，使NK细胞在面对不断进化的病毒时变得更强大。

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.