Activated Targeted Killer (ATAK) Cells for Invasive Fungal Infections

项目摘要 侵袭性真菌感染导致中性粒细胞减少患者不可接受的死亡率。自.以来 中性粒细胞减少的侵袭性真菌感染患者的存活率与患者的存活率呈线性相关 粒细胞计数，外源性替代吞噬细胞具有很大的治疗潜力 这些感染。尽管中性粒细胞输注已经显示出有希望的结果，但它们没有 20世纪80年代初，由于几个技术困难，它受到了青睐。应对侵袭性真菌感染 在中性粒细胞减少的患者中，我们正在开发一种基于细胞的免疫疗法，作为替代 中性粒细胞输注。我们的策略是基于人类吞噬细胞系HL-60，它可以 在体外永久生长，但随后可以被激活分化为成熟的中性粒细胞 在输注到受感染的去髓宿主之前的表型。HL-60细胞活化为 激活的靶向杀伤(ATAK)细胞复制能力减弱，显著增加 它们杀死白色念珠菌的效率，与细胞成熟到 成熟的粒细胞。ATAK细胞的给药显著提高了小鼠的存活率 感染白色念珠菌或烟曲霉菌的中性粒细胞减少的小鼠。因此，ATAK细胞是 能够在体外和体内重现中性粒细胞的宿主防御功能。 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，在那里通过氧化和/或非氧化机制杀死真菌。 我们还假设，插入一个可诱导的自杀陷阱将使细胞能够快速净化 在需要时从主机发送，从而增强了策略的安全性。我们现在试图定义 ATAK细胞杀死真菌的机制，以确定可诱导的自杀陷阱清除真菌的能力 体内细胞，并确定ATAK细胞在感染小鼠后的循环、持久性和有效性 各种真菌病原体。这项提议的具体目的是：1)确定机制 ATAK细胞抗真菌活性的研究；2)慢病毒载体转染ATAK细胞 自杀陷阱和追踪细胞的报告系统；3)定义体内循环、毒性和 用临床、实验室、代谢组学和细胞生物学方法研究中性粒细胞减少症小鼠ATAK细胞的寿命 在两个物种(小鼠和兔)中进行的组织病理学评估；以及4)定义 ATAK细胞对三种高度致命的霉菌感染的保护作用：曲霉病、毛霉病或 羽毛虫病。 ATAK细胞的保护机制和毒性的定义将得到澄清 吞噬宿主对真菌的一般防御机制，对未来至关重要 ATAK疗效的提高。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，并实时监控这些细胞- 在体内的时间。我们还将确定ATAK细胞的疗效广度，并定义长期 应用临床、实验室、代谢组学和生物化学方法研究小鼠模型中细胞的毒性 组织病理学标志物。最终，拟议的研究将为一种独特的、 在伴有侵袭性真菌的骨髓清髓宿主中重现中性粒细胞功能的前沿策略 感染。

Project Summary 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 myeloablated hosts with invasive fungal infections.