CD47 in the tumor microenvironment and CD47 antibody blockade regulate natural killer cell recruitment and activation

Elevated CD47 expression in some cancers is associated with decreased survival and limits phagocytic clearance by engaging its counter-receptor SIRPa, but elevated CD47 mRNA expression in human melanomas is associated with improved survival. Gene expression data suggested that recruitment of natural killer (NK) cells, which highly express CD47, into the tumor microenvironment contribute to this correlation. The CD47 ligand thrombospondin-1 inhibited wildtype but not Cd47-/- murine NK cell proliferation and granzyme B and interferon-? expression in vitro. Cd47-/- NK cells correspondingly showed augmented effector phenotypes. Although, wildtype and Cd47-/- NK cells were equally effective for killing B16 melanoma cells in vitro, Cd47-/- mice exhibited enhanced B16 tumor growth in vivo. Consistent with the human data, tumor-bearing Cd47-/- mice had decreased splenic NK numbers with impaired effector protein expression and elevated exhaustion markers. A pro-apoptotic signature in Cd47-/- NK cells was associated with stress-mediated elevation of mitochondrial proton leak, increased reactive oxygen species and apoptosis. Gene expression profiling identified CD47-dependent transcriptional responses in in NK cells from tumor-bearing mice that regulate systemic NK activation and exhaustion. Treating wildtype mice with a CD47 antibody that blocks thrombospondin-1 binding delayed tumor growth and was associated with increased NK recruitment and increased granzyme B- and interferon-? levels in intratumoral NK but not CD8+ T cells. Therefore, CD47 in the tumor microenvironment regulates NK-mediated tumor immunity, and therapeutic blockade enhances NK immune function. Overall design: Examining natural killer (NK) cell intrinsic role of CD47 in the tumor microenvironment.

在部分癌症中，CD47表达升高与患者生存期缩短相关，并通过结合其配体受体信号调节蛋白α（SIRPa）抑制吞噬清除，但在人类黑色素瘤中，CD47 mRNA表达升高却与患者生存期延长相关。基因表达数据显示，高表达CD47的自然杀伤细胞（natural killer cell，NK）向肿瘤微环境的募集，是该相关性的重要成因。CD47的配体凝血酶敏感蛋白-1（thrombospondin-1）在体外可抑制野生型小鼠NK细胞的增殖以及颗粒酶B与干扰素-γ的表达，但对Cd47基因敲除（Cd47-/-）小鼠的NK细胞无此作用。相应地，Cd47基因敲除小鼠的NK细胞表现出增强的效应细胞表型。尽管体外实验中，野生型与Cd47基因敲除小鼠的NK细胞对B16黑色素瘤细胞的杀伤活性无明显差异，但Cd47基因敲除小鼠体内的B16肿瘤生长速度更快。与人类数据一致，荷瘤Cd47基因敲除小鼠的脾脏NK细胞数量减少，效应蛋白表达受损，且耗竭标志物水平升高。Cd47基因敲除小鼠NK细胞的促凋亡特征，与应激介导的线粒体质子漏升高、活性氧（reactive oxygen species，ROS）水平增加以及细胞凋亡加剧相关。基因表达谱分析发现，荷瘤小鼠的NK细胞中存在CD47依赖的转录应答，该应答可调控系统性NK细胞活化与耗竭过程。使用阻断凝血酶敏感蛋白-1结合的CD47抗体处理野生型小鼠，可延缓肿瘤生长，且该处理与肿瘤内NK细胞的募集增加、颗粒酶B与干扰素-γ水平升高相关，但对CD8+ T细胞（CD8+ T cells）无此影响。因此，肿瘤微环境中的CD47可调控NK细胞介导的肿瘤免疫，而治疗性阻断CD47可增强NK细胞的免疫功能。实验整体设计：探究CD47在肿瘤微环境中NK细胞的内在作用。