与表观遗传药物联合治疗通过上调胶质瘤中MAGE-D4的表达增强MAGE-D4肽特异性T细胞的抗肿瘤活性。

Combined treatment with epigenetic agents enhances anti-tumor activity of MAGE-D4 peptide-specific T cells by upregulating the MAGE-D4 expression in glioma.

作者信息

Bi Shui-Qing, Zhang Qing-Mei, Zeng Xia, Liu Chang, Nong Wei-Xia, Xie Huan, Li Feng, Lin Li-Na, Luo Bin, Ge Ying-Ying, Xie Xiao-Xun

机构信息

Department of Histology and Embryology, School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi, China.

Department of Neurosurgery, The People's Hospital of Guangxi Zhuang Autonomous Region, Guangxi Academy of Medical Sciences, Nanning, China.

出版信息

Front Oncol. 2022 Aug 3;12:873639. doi: 10.3389/fonc.2022.873639. eCollection 2022.

DOI:10.3389/fonc.2022.873639

PMID:35992806

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9382192/

Abstract

OBJECTIVE

The study evaluated the efficacy of combined epigenetic drugs of decitabine (DAC), valproic acid (VPA), and trichostatin A (TSA) on immunotherapy against glioma.

METHODS

The expression and prognosis of MAGE-D4 in glioma were analyzed online, and the expression of MAGE-D4 and HLA-A2 in glioma induced by epigenetic drugs was detected by qRT-PCR, Western blot, and flow cytometry. The methylation status of the MAGE-D4 promoter was determined by pyrosequencing. An HLA-A2 restricted MAGE-D4 peptide was predicted and synthesized. An affinity assay and a peptide/HLA complex stability assay were performed to determine the affinity between peptide and HLA. CCK8 assay, CFSE assay, ELISA and ELISPOT were performed to detect the function of MAGE-D4 peptide-specific T cells. Flow cytometry, ELISA, and cytotoxicity assays were used to detect the cytotoxicity effect of MAGE-D4 peptide-specific T cells combined with epigenetic drugs against glioma . Finally, the glioma-loaded mouse model was applied to test the inhibitory effect of specific T cells on gliomas .

RESULTS

MAGE-D4 was highly expressed in glioma and correlated with poor prognosis. Glioma cells could be induced to express MAGE-D4 and HLA-A2 by epigenetic drugs. MAGE-D4-associated peptides were found that induce DCs to stimulate the highest T-cell activities of proliferation, IL-2 excretion, and IFN-γ secretion. MAGE-D4 peptide-specific T cells treated with TSA only or combining TSA and DAC had the most cytotoxicity effect, and its cytotoxicity effect on glioma cells decreased significantly after HLA blocking. experiments also confirmed that MAGE-D4-specific T cells inhibit TSA-treated glioma.

CONCLUSION

MAGE-D4 is highly expressed in glioma and correlated with the prognosis of glioma. The novel MAGE-D4 peptide identified was capable of inducing MAGE-D4-specific T cells that can effectively inhibit glioma growth, and the epigenetic drug application can enhance this inhibition.

摘要

目的

本研究评估了地西他滨（DAC）、丙戊酸（VPA）和曲古抑菌素A（TSA）联合表观遗传药物对胶质瘤免疫治疗的疗效。

方法

在线分析胶质瘤中MAGE-D4的表达及预后，采用qRT-PCR、蛋白质免疫印迹法和流式细胞术检测表观遗传药物诱导的胶质瘤中MAGE-D4和HLA-A2的表达。通过焦磷酸测序确定MAGE-D4启动子的甲基化状态。预测并合成了一种HLA-A2限制性MAGE-D4肽。进行亲和力测定和肽/HLA复合物稳定性测定以确定肽与HLA之间的亲和力。采用CCK8法、CFSE法、酶联免疫吸附测定（ELISA）和酶联免疫斑点测定（ELISPOT）检测MAGE-D4肽特异性T细胞的功能。采用流式细胞术、ELISA和细胞毒性测定检测MAGE-D4肽特异性T细胞联合表观遗传药物对胶质瘤的细胞毒性作用。最后，应用荷胶质瘤小鼠模型检测特异性T细胞对胶质瘤的抑制作用。

结果

MAGE-D4在胶质瘤中高表达且与预后不良相关。表观遗传药物可诱导胶质瘤细胞表达MAGE-D4和HLA-A2。发现与MAGE-D4相关的肽可诱导树突状细胞（DCs）刺激T细胞产生最高的增殖活性、白细胞介素-2（IL-2）分泌和γ干扰素（IFN-γ）分泌。单独用TSA或联合TSA和DAC处理的MAGE-D4肽特异性T细胞具有最强的细胞毒性作用，HLA阻断后其对胶质瘤细胞的细胞毒性作用显著降低。实验还证实MAGE-D4特异性T细胞可抑制TSA处理的胶质瘤。