Harnessing NK cell effector function for immunotherapies

自然杀伤(NK)细胞是人类淋巴细胞的主要谱系，具有先天和适应性的重要功能 血液病免疫和获得性免疫。NK细胞的功能是由不同的 高度多态的人类白细胞抗原-A、-B和-C分子与等位多态的杀伤免疫球蛋白的相互作用 类受体(KIR)。与它们互补的是人类白细胞抗原E与CD94：NKG2的保守相互作用 杂二聚体。编码KIR受体和人类白细胞抗原配体的基因是独立的，因此 在家庭和群体中产生独特和多样化的基因类型。从表观上看，这会产生 功能不同的NK细胞谱系，这是一种对HCT结局有深远影响的自然变异。至 迄今为止，一直没有一个合适的体内异种模型来研究NK细胞的发育和 通过红细胞压积进行教育或确认人类NK细胞的收养转移。从历史上看，曾经有过 研究人类NK细胞植入和功能的两个主要障碍：(1)人类NK细胞重组和 生存依赖于共同的链细胞因子IL-2、IL-7和IL-15，它们之间没有交叉反应 人类NK细胞功能受抑制性受体的教育、调节和维持 与MHC I类分子接触，这些分子也不会在物种之间发生交叉反应。我们现在有了 利用尖端的腺相关病毒(AAV)克服细胞因子和MHC I类屏障 载体介导的基因传递方法转导编码人类白细胞抗原-A、-B、-C和-E多肽的基因 以及某些人类细胞因子，对缺乏鼠源性2的高度免疫缺陷的NsG小鼠- 微球蛋白，命名为NSG-B2M-/-小鼠。在特定目标(SA)1中，我们将建立人类免疫系统(HIS) 表达特定人细胞因子和人类白细胞抗原A*02和/或人类白细胞抗原E的小鼠。细胞因子IL-2、IL-3、IL-6、IL-7、IL-6、IL-2、IL-3、IL-6、IL-2、IL-3、IL-2、IL-3、IL-6、IL-6、IL-2、IL-6、IL-6、IL-2、IL-2、IL-6、IL-2、IL-3、IL-2、IL-3、IL-6、IL-6、IL-2、IL-3、IL-6、IL-3、IL-2、IL-6、IL- 15和GM-CSF将与人T细胞、B细胞、髓系细胞一起促进人NK细胞的发育 巨噬细胞和DC。我们将应用42-plex质量细胞仪抗体板来紧密定位淋巴细胞。 在HCT之后以前所未有的决心进行重建。NK细胞的发展、教育和功能将 在20周的范围内进行纵向研究。在SA2中，我们将用荧光素酶标记的肿瘤感染他的小鼠 以评价NK细胞培养对HCT抗肿瘤活性和结局的影响。我们 将通过质量细胞仪比较来自His小鼠的NK细胞的抗肿瘤功能以及通过 生物发光成像。我们的His小鼠模型将为人类白细胞抗原-E是否可以 直接培养CD94：NKG2A+人NK细胞及其对免疫应答能力的影响 循环中的肿瘤。在SA2中，我们还将测量CMV感染(和重新激活)对 适应性NK细胞的培养及其增强的抗肿瘤反应。以发展为最终目标 他的小鼠表达人类细胞因子以及复杂的人类白细胞抗原I类单倍型，我们将能够有效地 利用NK细胞效应器治疗血液病。

Natural Killer (NK) cells are a major lineage of human lymphocytes with vital functions in innate and adaptive immunity and adaptive immunity to hematologic cancers. NK cell functions are mediated by the diverse interactions of highly polymorphic HLA-A, -B, and -C molecules with equally polymorphic killer immunoglobulin- like receptors (KIR). They are complemented by the conserved interactions of HLA-E with CD94:NKG2 heterodimers. The genes encoding KIR receptors and HLA ligands segregate independently, thereby generating unique and diverse genotypes within families and populations. Phenotypically, this produces functionally distinct NK cell repertoires, a natural variation that has profound effects on the outcome of HCT. To date, there has never been an appropriate in vivo xenogeneic model for studying NK-cell development and education through HCT or for validating the adoptive transfer of human NK cells. Historically, there have been two major barriers to study human NK-cell engraftment and function: (1) human NK-cell reconstitution and survival is dependent upon common -chain cytokines IL-2, IL-7 and IL-15 that are not cross reactive between species; and (2) human NK-cell function is educated, regulated and maintained by inhibitory receptor engagement with MHC class I molecules that are also not cross-reactive between species. We have now overcome the cytokine and MHC class I barrier by utilizing a cutting-edge adeno-associated virus (AAV) vector-mediated gene delivery approach to transduce genes encoding HLA-A, -B, -C and –E polypeptides, as well as certain human cytokines, to highly immunodeficient NSG mice that lack mouse-derived 2- microglobulin, named NSG-B2M-/- mice. In Specific Aim (SA)1 we will establish human immune system (HIS) mice expressing select human cytokines and HLA-A*02 and/or HLA-E. The cytokines IL-2, IL-3, IL-6, IL-7, IL- 15 and GM-CSF will promote development of human NK cells along with human T cells, B cells, myeloid macrophages and DCs. We will apply a 42-plex mass cytometry antibody panel to closely map lymphocyte reconstitution following HCT at an unprecedented resolution. NK cell development, education and function will be studied longitudinally over a range of 20 weeks. In SA2 we will infect HIS mice with luciferase-labeled tumor target cells in order to evaluate the impact of NK cell education on anti-tumor activity and outcome of HCT. We will compare anti-tumor function of NK cells from HIS mice by mass cytometry as well as tumor burden by bioluminescence imaging. Our HIS mouse model will provide a definitive answer on whether HLA-E can directly educate CD94:NKG2A+ human NK cells and how this education impacts their capacity to respond to circulating tumors. In SA2, we will also measure the in vivo effects of CMV infection (and reactivation) on the education of adaptive NK cells and their enhanced anti-tumor response. With the ultimate goal of developing HIS mice expressing human cytokines along with complex HLA class I haplotypes, we will be able to effectively harness NK cell effector for treatment of hematologic cancers.