Role and Regulation of NKG2D Ligand Expression on Tumor-Infiltrating Myeloid Cells

 描述(申请人提供)：激活对肿瘤细胞的免疫反应的治疗方法使癌症治疗方法发生了革命性的变化。这些治疗方法是数十年来关于免疫系统如何识别和消除癌症的研究的高潮，但需要更多的研究来寻找更多的治疗靶点，并改进现有的治疗靶点。自然杀伤细胞(Natural Killer，NK)是一种具有很强抗肿瘤活性的免疫细胞。NK细胞被描绘成 来自正常组织的癌细胞使用生殖线编码的细胞表面受体识别转化细胞上的分子，但不识别健康细胞。NK细胞通过分泌细胞毒颗粒和强大的抗肿瘤细胞因子来清除肿瘤。由于许多增强NK反应的治疗方法都在临床开发中，因此深入探讨调节NK细胞抗肿瘤活性的机制是至关重要的。我们实验室最近的一项研究提供了证据，表明肿瘤浸润性髓系细胞通过NK免疫受体NKG2D持续刺激NK细胞。这种持续的刺激导致NK细胞不那么活跃，无法杀死癌细胞，从而使肿瘤逃避NK的识别。这种脱敏是由肿瘤浸润性巨噬细胞和单核细胞上表达的NKG2D配体(NKG2D-L)RAE-1δ驱动的。初步数据表明，髓系RAE-1δ的表达是由肿瘤细胞产生的细胞因子mCSF引起的。因此，肿瘤可能利用多细胞轴来规避NK细胞介导的免疫监视，从而促进自身的生长。该应用旨在进一步探索这些发现的分子基础，并测试阻断NK细胞和髓系细胞之间的NKG2D/RAE-1δ相互作用是否可以缓解这种脱敏并促进肿瘤排斥反应。在目的1中，通过阻断mCSF抗体、慢病毒过表达mCSF以及Cas9基因在肿瘤细胞中的敲除来确定肿瘤来源的mCSF是否促进了移植瘤和自体肿瘤模型中肿瘤浸润性髓系细胞RAE-1δ的表达。通过检测巨噬细胞刺激下游的PI3K是否调节巨噬细胞中的RAE-1δ，将探索巨噬细胞诱导RAE-1δ的信号通路。过表达和敲除策略将被用来调节PI3K信号，以检测该途径在RAE-1δ诱导中的作用。最后，我将确定人巨噬细胞集落刺激因子是否能刺激外周血单核细胞NKG2D-L的诱导，并检测人NKG2D-L对人源化小鼠肿瘤浸润性髓系细胞的表达。在目标2中，将通过阻断抗mcsf和RAE-1δ的抗体、基因消融RAE-1δ和巨噬细胞耗竭来确定单独或与其他激活NK细胞的治疗方法联合阻断msf/RAE-1δ/NKG2D脱敏轴是否可以促进表达NKG2D和其他NK受体配体的肿瘤的排斥反应。我们推测，依赖巨噬细胞集落刺激因子和RAE-1δ的NK脱敏是肿瘤的共同特征，也是癌症治疗的潜在靶点。

 DESCRIPTION (provided by applicant): Treatments that activate immune responses against tumor cells have revolutionized cancer therapeutics. These treatments are the culmination of decades of studies into how the immune system recognizes and eliminates cancer, but more research is necessary to find additional therapeutic targets and improve current ones. Natural Killer (NK) cells are one type of immune cell with potent anti-tumor activities. NK cells delineate cancer cells from normal tissue using germline-encoded cell surface receptors that recognize molecules on transformed cells but not healthy cells. NK cells eliminate tumors by secreting cytotoxic granules and powerful anti-tumor cytokines. Because many therapies to boost NK responses are in clinical development, it is crucial to thoroughly explore the mechanisms that regulate NK activity against tumors. A recent study in our laboratory provided evidence that tumor-infiltrating myeloid cells persistently stimulate NK cells through the NK immunoreceptor NKG2D. This persistent stimulation causes NK cells to be less active and unable to kill cancer cells, allowing the tumor to evade NK recognition. This desensitization is driven by the NKG2D ligand (NKG2D-L) RAE-1δ expressed on tumor-infiltrating macrophages and monocytes. Preliminary data suggest that myeloid RAE-1δ expression is caused by the cytokine MCSF, produced by tumor cells. Thus, tumors may facilitate their own growth using a multicellular axis to circumvent NK cell-mediated immunosurveillance. This application seeks to further explore the molecular underpinnings of these findings, and to test whether interrupting NKG2D/RAE-1δ interactions between NK cells and myeloid cells can relieve this desensitization and promote tumor rejection. In Aim 1, blocking antibodies against MCSF, lentiviral overexpression of MCSF, and Cas9-mediated knockout of the MCSF gene in tumor cells will be used to determine whether tumor-derived MCSF promotes RAE-1δ expression on tumor-infiltrating myeloid cells in transplanted and autochthonous tumor models. The signaling pathways responsible for RAE-1δ induction by MCSF will be explored by examining whether PI3K, downstream of MCSF stimulation, regulates RAE-1δ in macrophages. Overexpression and knock-down strategies will be used to modulate PI3K signals to examine the contribution of this pathway to RAE-1δ induction. Finally, I will determine whether human MCSF can stimulate NKG2D-L induction on peripheral blood monocytes, and examine human NKG2D-L expression on myeloid cells infiltrating tumors in humanized mice. In Aim 2, blocking antibodies against MCSF and RAE-1δ, genetic ablation of RAE-1δ, and macrophage depletion will be used to determine whether interrupting the MCSF/RAE-1δ/NKG2D desensitization axis, alone or in combination with other therapies that activate NK cells, can promote rejection of tumors expressing ligands for NKG2D and other NK receptors. We hypothesize that MCSF- and RAE-1δ-dependent NK desensitization is a common feature of tumors and a potentially worthwhile target for cancer therapy.