LRRC15来源HLA-A2.1限制性CTL表位肽鉴定及其抗乳腺癌效应评价  

Identification and anti-breast cancer effect validation of a LRRC15-derived HLA-A2.1-restricted CTL epitope

作　　者：汤海潮 刘雯思 于兆进 Tang Haichao;Liu Wensi;Yu Zhaojin(Department of Pharmacology,School of Pharmacy,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学药学院药理学教研室,沈阳110122

出　　处：《国际免疫学杂志》2022年第5期441-448,共8页International Journal of Immunology

基　　金：国家自然科学基金-辽宁联合基金重点支持项目(U1608281);辽宁省教育厅科学研究一般项目(QN2019033);辽宁省自然科学基金项目面上项目(2020-MS-141)。

摘　　要：目的鉴定富含亮氨酸重复蛋白15(leucine-rich repeat-containing protein-15,LRRC15)来源的优势细胞毒性T淋巴细胞(cytosolic T lymphocyte,CTL)表位肽,并验证其诱导CTL特异性抗乳腺癌免疫效应。方法收集中国医科大学附属第一医院乳腺癌外科自2010年1月至2010年7月经手术病理证实的乳腺癌组织及正常组织各30例。利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和免疫组化方法评估LRRC15在乳腺癌中表达情况。利用免疫表位数据库(Immune Epitope Database and Analysis Resource,IEDB)联合SYFPEITHI数据库预测LRRC15蛋白序列中HLA-A2.1限定性CTL表位肽。利用分子对接软件MOE、T2亲和力实验和荧光共聚焦技术确认候选CTL表位肽与HLA-A2.1分子亲和性。利用酶联免疫斑点实验(enzyme-linked immunospot assay,ELISPOT)以及流式细胞术检测CTL表位肽免疫活性。利用钙黄素和Dil标记乳腺癌细胞评价CTL表位肽诱导的杀伤效应。建立乳腺癌移植瘤模型进行免疫回输,在体内评估CTL表位肽诱导的抗肿瘤效应。结果相比于正常组织,LRRC15 mRNA在乳腺癌组织中表达水平明显增高,组间差异有统计学意义[(4.00±1.57)比(1.31±0.72),Z=14.85,P<0.05];LRRC15 mRNA表达与CD8+T细胞浸润呈显著正相关性(r=0.20,P<0.001)。LRRC15蛋白在乳腺癌组织中呈现高表达,可作为乳腺癌潜在靶点。在筛选得到的3条候选CTL表位肽中,L2与HLA-A2.1分子具有最高亲和力(FI=2.490)。与阴性肽组相比,L2能够明显提升CD8+T细胞亚群比例(27.70%比45.50%),L2负载的树突状细胞(dendritic cells,DCs)能够明显提升CD8+T细胞分泌干扰素(interferon,IFN)-γ的水平(50.20%比74.90%)。在最高效靶比时,L2诱导的效应细胞对乳腺癌细胞系MDA-MB-231的杀伤效应明显高于阴性肽对照组,组间差异有统计学意义[(19.30±2.20)%比(8.60±1.40)%,t=1.45,P<0.05],但对乳腺上皮细胞MCF-10A无杀伤效应(P>0.05)。L2负载DCs诱导的CTL细胞对2例乳腺癌�Objective To identify a predominant Leucine-rich repeat-containing protein-15(LRRC15)-derived cytotoxic T lymphocyte(CTL)epitope and validate specific anti-breast cancer effects of the epitope inducing CTL.Methods Total of 30 cases of breast cancer tissues and 30 cases of normal tissues were collected from breast cancer surgery of the First Affiliated Hospital of China Medical University from January 2010 to July 2010.LRRC15 expression was evaluated in breast cancer tissues based The Cancer Genome Atlas(TCGA)database and immunohistochemistry assay.The LRRC15-derived HLA-A2.1-restricted CTL epitopes was predicted by the Immune Epitope Database and Analysis Resource(IEDB)database combined with SYFPEITHI database.Molecular docking software,T2 affinity assay and confocal assay were performed to analyze the affinity of CTL epitopes and HLA-A2.1 molecule.The immunoactivity of CTL epitope peptide was detected by(enzyme-linked immunospot assay,ELISPOT)and flow cytometry.The cytotoxic effect induced by CTL epitope peptide was evaluated by labeling breast cancer cells with calcaxin and Dil.The anti-tumor effect induced by CTL epitope peptide was evaluated in vivo by immunotransfusion in a breast cancer transplanted tumor model.Results The expression level of LRRC15 mRNA in breast cancer tissues was significantly higher than that in normal tissues[(4.00±1.57)vs(1.31±0.72),Z=14.85,P<0.05].LRRC15 mRNA expression was positively correlated with CD8+T cell infiltration(r=0.20,P<0.001).LRRC15 protein is highly expressed in breast cancer tissues and can be used as a potential target of breast cancer.Among the three candidate CTL epitopes screened,L2 had the highest affinity with HLA-A2.1(FI=2.490).Compared with the negative peptide group,L2 significantly increased the proportion of CD8+T cell subsets(27.70%vs 45.50%),and L2-loaded dendritic cells(DCs)significantly increased the interferon(IFN)-γsecretion level of CD8+T cells(50.20%vs 74.90%).At the most efficient target ratio,the killing effect of L2-induced effector cells on b

关 键 词：乳腺癌 毒性T淋巴细胞表位肽 富含亮氨酸重复蛋白15 肿瘤免疫治疗 

分 类 号：R737.9[医药卫生—肿瘤]