89Zr Immuno-PET Imaging of Tumor PD-1 Reveals That PMA Upregulates Lymphoma PD-1 through NFκB and JNK Signaling

IF 2.8 4区 医学 Q2 Medicine
Kyung-Ho Jung, Jin Hee Lee, Mina Kim, Y. Cho, Kyung-Han Lee
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引用次数: 2

Abstract

Immune therapy of T-cell lymphoma requires assessment of tumor-expressed programmed cell death protein-1 (PD-1). Herein, we developed an immuno-PET technique that quantitatively images and monitors regulation of PD-1 expression on T-cell lymphomas. Methods. Anti-PD-1 IgG underwent sulfhydryl moiety-specific conjugation with maleimide-deferoxamine and 89Zr labeling. Binding assays and Western blotting were performed in EL4 murine T-cell lymphoma cells. In vivo pharmacokinetics, biodistribution, and PET were performed in mice. Results. 89Zr-PD-1 IgG binding to EL4 cells was completely blocked by cold antibodies, confirming excellent target specificity. Following intravenous injection into mice, 89Zr-PD-1 IgG showed biexponential blood clearance and relatively low normal organ uptake after five days. PET/CT and biodistribution demonstrated high EL4 tumor uptake that was suppressed by cold antibodies. In EL4 cells, phorbol 12-myristate 13-acetate (PMA) increased 89Zr-PD-1 IgG binding (305.5 ± 30.6%) and dose-dependent augmentation of PD-1 expression (15.8 ± 3.8 − fold of controls by 200 ng/ml). FACS showed strong PD-1 expression on all EL4 cells and positive but weaker expression on 41.6 ± 2.1% of the mouse spleen lymphocytes. PMA stimulation led to 2.7 ± 0.3-fold increase in the proportion of the strongest PD-1 expressing EL4 cells but failed to influence that of PD-1+ mouse lymphocytes. In mice, PMA treatment increased 89Zr-PD-1 IgG uptake in EL4 lymphomas from 6.6 ± 1.6 to 13.9 ± 3.6%ID/g (P = 0.01), and tumor uptake closely correlated with PD-1 level (r = 0.771, P < 0.001). On immunohistochemistry of tumor sections, infiltrating CD8α+ T lymphocytes constituted a small fraction of tumor cells. The entire tumor section showed strong PD-1 staining that was even stronger for PMA-treated mice. Investigation of involved signaling revealed that PMA increased EL4 cell and tumor HIF-1α accumulation and NFκB and JNK activation. Conclusion. 89Zr-PD-1 IgG offered high-contrast PET imaging of tumor PD-1 in mice. This was found to mostly represent binding to EL4 tumor cells, although infiltrating T lymphocytes may also have contributed. PD-1 expression on T-cell lymphomas was upregulated by PMA stimulation, and this was reliably monitored by 89Zr-PD-1 IgG PET. This technique may thus be useful for understanding the mechanisms of PD-1 regulation in lymphomas of living subjects.
肿瘤PD-1的89Zr免疫PET成像揭示PMA通过NFκB和JNK信号上调淋巴瘤PD-1
T细胞淋巴瘤的免疫治疗需要评估肿瘤表达的程序性细胞死亡蛋白-1(PD-1)。在此,我们开发了一种免疫PET技术,可以定量成像和监测PD-1在T细胞淋巴瘤上的表达调控。方法。抗PD-1 IgG与马来酰亚胺去铁胺和89Zr标记进行巯基部分特异性结合。在EL4小鼠T细胞淋巴瘤细胞中进行结合测定和蛋白质印迹。在小鼠体内进行药代动力学、生物分布和PET。后果89Zr-PD-1 IgG与EL4细胞的结合被冷抗体完全阻断,证实了优异的靶特异性。小鼠静脉注射后,89Zr-PD-1 IgG在五天后显示出双指数血液清除率和相对较低的正常器官摄取。PET/CT和生物分布显示高EL4肿瘤摄取被冷抗体抑制。在EL4细胞中,佛波醇12-肉豆蔻酸13-乙酸酯(PMA)增加了89Zr-PD-1 IgG的结合(305.5±30.6%)和PD-1表达的剂量依赖性增加(对照组的15.8±3.8倍 ng/ml)。FACS在所有EL4细胞上都显示出强的PD-1表达,在41.6±2.1%的小鼠脾淋巴细胞上显示出阳性但较弱的表达。PMA刺激导致最强表达PD-1的EL4细胞的比例增加2.7±0.3倍,但未能影响PD-1+小鼠淋巴细胞的比例。在小鼠中,PMA治疗使EL4淋巴瘤的89Zr-PD-1 IgG摄取量从6.6±1.6增加到13.9±3.6%ID/g(P=0.01),肿瘤摄取量与PD-1水平密切相关(r=0.771,P<0.001)。整个肿瘤切片显示出强的PD-1染色,这对于PMA处理的小鼠来说甚至更强。对相关信号传导的研究表明,PMA增加了EL4细胞和肿瘤HIF-1α的积累以及NFκB和JNK的激活。结论89Zr-PD-1 IgG提供了小鼠肿瘤PD-1的高对比度PET成像。发现这主要代表与EL4肿瘤细胞的结合,尽管浸润的T淋巴细胞也可能起作用。PD-1在T细胞淋巴瘤上的表达通过PMA刺激而上调,并且这通过89Zr-PD-1 IgG PET可靠地监测。因此,这项技术可能有助于了解PD-1在活体受试者淋巴瘤中的调节机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Molecular Imaging
Molecular Imaging 生物-核医学
CiteScore
4.50
自引率
3.60%
发文量
21
审稿时长
>12 weeks
期刊介绍: Molecular Imaging is a peer-reviewed, open access journal highlighting the breadth of molecular imaging research from basic science to preclinical studies to human applications. This serves both the scientific and clinical communities by disseminating novel results and concepts relevant to the biological study of normal and disease processes in both basic and translational studies ranging from mice to humans.
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