Reader-free ELISPOT assay for immuno-monitoring in peptide-based cancer vaccine immunotherapy
Reader-free ELISPOT assay for immuno-monitoring in peptide-based cancer vaccine immunotherapy
复制标题
DOI：
10.3892/br.2020.1289
复制
发表时间：
2020-05-01
影响因子：
2.3
通讯作者：
Oji, Yusuke
中科院分区：
文献类型：
--
作者：
Hayashi, Sae;Imanishi, Rin;Oji, Yusuke
癌症疫苗免疫疗法是诱导针对靶分子的细胞免疫应答以引发临床抗肿瘤作用的疗法。监测针对靶分子的这些细胞免疫应答，以评价在疫苗接种期间是否诱导并维持抗原特异性细胞免疫应答。酶联免疫斑点（ELISPOT）测定被广泛地进行以不仅分析免疫细胞的频率，而且分析它们的效应器功能，如通过它们的细胞因子产生/分泌所确定的。本研究的目的是开发一种无读数ELISPOT检测使用方便的膜打孔设备称为ELI 8。在ImageJ软件的颗粒分析的辅助下，斑点计数的结果是可重复的，具有较高的批间和检查者间一致性。免疫细胞产生和分泌Th 1细胞因子，而无需抗原肽刺激外周血单个核细胞（PBMC）进行检测，其频率在癌症患者中显着高于那些在健康个体。这些频率在个体之间以及每个患者癌症疫苗免疫治疗过程中的时间点之间变化。由于PBMC自发细胞因子产生/分泌的可变性，提出了抗原特异性免疫应答（IR）指数，其是经历抗原刺激的斑点形成细胞（SFC）的数目与具有自发细胞因子分泌而没有抗原刺激的SFC的数目的比率。该指数可用作用癌症免疫疗法治疗的患者中抗原特异性细胞免疫应答的标志物。IR指数成功地检测了用癌症疫苗免疫疗法治疗的癌症患者中Wilms肿瘤1特异性细胞免疫应答的诱导。
Cancer vaccine immunotherapy is a therapy that induces cellular immune responses against a target molecule to elicit clinical anti-tumor effects. These cellular immune responses against the target molecule are monitored to evaluate whether the antigen-specific cellular immune responses are induced and maintained during the vaccination period. Enzyme-linked immunospot (ELISPOT) assay is widely performed to analyze not only the frequency of immune cells, but also their effector functions as determined by their cytokine production/secretion. The present study aimed to develop a reader-free ELISPOT assay using a handy membrane-punching device termed ELI 8. With the assistance of particle analysis by ImageJ software, the results of spot counting were reproducible with high inter-assay and inter-examiner concordance. Immune cells that produce and secrete Th1 cytokines without antigen-peptide stimulation of peripheral blood mononuclear cells (PBMCs) were detected, and their frequencies in patients with cancer were significantly higher compared with those in healthy individuals. These frequencies varied between individuals, as well as between time points during the course of cancer vaccine immunotherapy in each patient. Due to the variability in spontaneous cytokine production/secretion by PBMCs, an antigen-specific immune response (IR) index is proposed, which is a ratio of the number of spot-forming cells (SFCs) subjected to antigen-stimulation to that of SFCs with spontaneous cytokine secretion without antigen-stimulation. This index may be used as a marker for antigen-specific cellular immune responses in patients treated with cancer immunotherapy. The IR index successfully detected the induction of Wilms' tumor 1-specific cellular immune responses in patients with cancer treated with cancer vaccine immunotherapy.