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，在先天和适应性免疫中具有重要功能。 免疫和获得性免疫。NK细胞的功能是由多种 高度多态性的HLA-A、-B和-C分子与同样多态性的杀伤免疫球蛋白的相互作用， 类受体（KIR）。它们由HLA-E与CD 94：NKG 2的保守相互作用补充 异二聚体。编码KIR受体和HLA配体的基因独立分离，从而 在家庭和群体中产生独特和多样的基因型。从表型上看， 功能不同的NK细胞库，这是一种对HCT结果具有深远影响的自然变异。到 迄今为止，还没有合适的体内异种模型用于研究NK细胞发育， 通过HCT的教育或用于验证人NK细胞的过继转移。历史上， 研究人NK细胞移植和功能的两个主要障碍：（1）人NK细胞重建和 存活依赖于共同的β-链细胞因子IL-2、IL-7和IL-15，它们在细胞因子之间不交叉反应。 人NK细胞功能受抑制性受体的训练、调节和维持 与MHC I类分子接合，这些分子在物种之间也没有交叉反应性。我们现在已经 利用最先进的腺相关病毒（AAV）克服细胞因子和MHC I类障碍 载体介导的基因递送方法，其将编码HLA-A、-B、-C和-E多肽的HLA-A基因，如 以及某些人细胞因子，对缺乏小鼠源性β 2受体的高度免疫缺陷NSG小鼠， 微球蛋白，命名为NSG-B2 M-/-小鼠。在特定目标（SA）1中，我们将建立人类免疫系统（HIS） 表达选择的人细胞因子和HLA-A*02和/或HLA-E的小鼠。细胞因子IL-2、IL-3、IL-6、IL-7、IL-10、IL-11、IL-12、IL-14、IL-16、IL-18、IL-19、IL- 15和GM-CSF将促进人NK细胞与人T细胞、B细胞、髓样细胞、淋巴细胞和淋巴细胞一起沿着发育。 巨噬细胞和DC。我们将应用42重流式细胞术抗体板来密切定位淋巴细胞 在HCT后以前所未有的分辨率重建。NK细胞的发育、教育和功能将 在20周的时间范围内进行纵向研究。在SA 2中，我们将用酶标记的肿瘤感染HIS小鼠 靶细胞，以评估NK细胞教育对抗肿瘤活性和HCT结果的影响。我们 将通过质谱细胞术比较来自HIS小鼠的NK细胞的抗肿瘤功能，以及通过流式细胞术比较肿瘤负荷。 生物发光成像我们的HIS小鼠模型将提供一个明确的答案，HLA-E是否可以 直接教育CD 94：NKG 2A+人NK细胞，以及这种教育如何影响他们应对 循环肿瘤在SA 2中，我们还将测量CMV感染（和再活化）对细胞的体内影响。 适应性NK细胞及其增强的抗肿瘤反应的教育。最终目标是发展 HIS小鼠表达人类细胞因子沿着复杂的HLA I类单倍型，我们将能够有效地 利用NK细胞效应子治疗血液癌症。

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.