Activated Targeted Killer (ATAK) Cells for Invasive Fungal Infections

描述（由申请人提供）：侵袭性真菌感染导致血小板减少症患者不可接受的死亡率。由于侵袭性真菌感染的血小板减少患者的生存率与患者的粒细胞计数呈线性相关，因此外源性吞噬细胞替代对这些感染具有很大的治疗潜力。虽然中性粒细胞输注显示出有希望的结果，但由于一些技术困难，它们在20世纪80年代初失宠。为了解决血小板减少症患者的侵袭性真菌感染，我们正在开发一种基于细胞的免疫疗法，作为中性粒细胞输注的替代方案。我们的策略是基于人吞噬细胞系HL-60，其可以在体外永久生长，但随后可以在输注到受感染的骨髓清除宿主中之前被激活以分化为成熟的中性粒细胞表型。将HL-60细胞活化为活化靶向杀伤（ATAK）细胞降低了它们的复制能力，并显著提高了它们杀死白色念珠菌的效率，这与细胞向成熟粒细胞的成熟一致。ATAK细胞的施用实质上改善了感染C.白色念珠菌或烟曲霉。因此，ATAK细胞能够在体外和体内重演中性粒细胞宿主防御功能。我们假设ATAK细胞在治疗播散性念珠菌病方面是有效的，因为它们循环到受感染的组织，并通过氧化和/或非氧化机制杀死真菌。我们还假设，插入诱导型自杀陷阱将能够在需要时从宿主中快速清除细胞，从而增强该策略的安全性。我们现在试图确定ATAK细胞杀死真菌的机制，确定诱导型自杀陷阱在体内清除细胞的能力，并确定ATAK细胞在感染各种真菌病原体的小鼠中的循环、持久性和功效。本提案的具体目的是：1）确定ATAK细胞抗真菌活性的机制; 2）用含有自杀陷阱和用于跟踪细胞的报告系统的慢病毒构建体转染ATAK细胞; 3）通过两个物种的临床、实验室、代谢组学和组织病理学评价，确定ATAK细胞在血小板减少小鼠中的体内循环、毒性和寿命（小鼠和兔）;和4）确定ATAK细胞对三种高致死性霉菌感染的保护范围：曲霉病、毛霉病或镰刀菌病。ATAK细胞的保护和毒性机制的定义将阐明吞噬宿主对真菌的防御机制，并对未来增强ATAK功效至关重要。此外，插入自杀陷阱和发光标记将使我们能够在需要时消除任何残留的ATAK细胞，并在体内真实的实时监测细胞。我们还将确定ATAK细胞的疗效范围，并使用临床、实验室、代谢组学和组织病理学标志物确定细胞在小鼠模型中的长期毒性。最终，提出的研究将为一种独特的尖端策略提供基础，以重现侵袭性真菌感染的骨髓清除宿主中的中性粒细胞功能。公共卫生相关性：真菌感染是化疗导致白色血细胞计数低的癌症患者死亡的主要原因。几十年来，医生们一直试图通过使用白色血细胞来对抗这种感染。然而，白色细胞输注由于重大的技术障碍而失败。目前的研究将能够开发一种独特的策略，在试管中培养未成熟的白色血细胞，直到感染的癌症患者需要它们的时候。然后用活化因子的组合处理细胞，使它们成熟为活化靶向杀伤细胞（ATAK）。我们建议确定ATAK细胞如何杀死病原体以及细胞如何在受感染宿主体内循环。我们还建议在细胞中插入自杀陷阱，以便在需要时消除它们。完成拟议的研究将使我们能够创建一个安全有效的策略，以克服白色血细胞输注的障碍，彻底改变癌症患者感染的治疗。

DESCRIPTION (provided by applicant): Invasive fungal infections cause unacceptable mortality rates in neutropenic patients. Since survival of neutropenic patients with invasive fungal infections is linearly related to the patients' granulocyte counts, exogenous replacement of phagocytes is of great therapeutic potential for these infections. Although neutrophil transfusions have shown promising results, they fell out of favor in the early 1980s due to several technical difficulties. To address invasive fungal infections in neutropenic patients, we are developing a cell-based immunotherapy as an alternative to neutrophil transfusions. Our strategy is based on a human phagocytic cell-line, HL-60, which can be grown perpetually in vitro, but can then be activated to differentiate towards a mature neutrophil phenotype prior to infusion into an infected, myeloablated host. Activation of HL-60 cells into Activated Targeted Killer (ATAK) cells diminishes their replicative capacity and markedly increases the efficiency with which they kill Candida albicans, consistent with the cells' maturation towards mature granulocytes. Administration of ATAK cells substantively improves the survival of neutropenic mice infected with C. albicans or Aspergillus fumigatus. Therefore, ATAK cells are capable of recapitulating neutrophil host-defense functions in vitro and in vivo. We hypothesize that ATAK cells are effective at treating disseminated candidiasis because they circulate to infected tissues and there kill fungi by oxidative and/or non-oxidative mechanisms. We also hypothesize that insertion of an inducible suicide trap will enable rapid purging of the cells from a host when desired, thereby enhancing the safety of the strategy. We now seek to define the mechanisms by which ATAK cells kill fungi, to define the ability of an inducible suicide trap to purge the cells in vivo, and to define ATAK cell circulation, persistence, and efficacy in mice infected with a variety of fungal pathogens. The specific aims of this proposal are to: 1) define the mechanisms of ATAK cell anti-fungal activity; 2) transfect ATAK cells with a lentiviral construct containing a suicide trap and a reporter system for tracking cells; 3) define the in vivo circulation, toxicities, and life-span of ATAK cells in neutropenic mice using clinical, laboratory, metabolomic, and histopathological evaluations in two species (mice and rabbit); and 4) define the breadth of protection of ATAK cells against three highly lethal mold infections: aspergillosis, mucormycosis, or fusariosis. Definition of the mechanism of protection and toxicities of ATAK cells will elucidate phagocytic host defense mechanisms against fungi in general, and is critical to future enhancement of ATAK efficacy. Furthermore, insertion of a suicide trap and luminescence marker will enable us to eliminate any residual ATAK cells when desired and to monitor the cells in real- time in vivo. We will also determine the breadth of efficacy of ATAK cells, and define long-term toxicity of the cells in the murine model using clinical, laboratory, metabolomic, and histopathological markers. Ultimately, the studies proposed will provide a foundation for a unique, cutting-edge strategy to recapitulate neutrophil functions in myeloablate hosts with invasive fungal infections. PUBLIC HEALTH RELEVANCE: Fungal infections are leading causes of death in cancer patients whose white blood cell counts are low from chemotherapy. For decades, physicians have tried to combat such infections by transfusing white blood cells. However, white cell transfusions have failed due to significant technical barriers. The current studies will enable development of a unique strategy to grow immature white blood cells in the test tube until such time as they are needed for an infected cancer patient. The cells are then treated with a combination of activating factors, which causes them to mature into Activated Targeted Killer (ATAK) cells. We propose to determine how ATAK cells kill pathogens and how the cells circulate through the body of an infected host. We also propose to insert a suicide trap into the cells so we can eliminate them when desired. Completion of the proposed studies will enable us to create a safe and effective strategy to overcome the barriers to white blood cell transfusions, revolutionizing the treatment of infections in cancer patients.