癌细胞的免疫原性受多种因素的影响,包括主要组织相容性复合体I类(MHC-I)的表达,抗原表达,和蛋白酶体产生的表位肽的库。恶性胸膜间皮瘤细胞系ACC-MEOS-4(MESO-4)表达高水平的MHC-I和Wilms肿瘤1(WT1)肿瘤抗原。使用对HLA-A*24:02限制性WT1表位(WT1235,CMTWNQMNL)具有特异性的功能性T细胞报告基因测定,我们寻找增强MESO-4免疫原性的因素,重点是蛋白酶体,在抗原加工机制中起着核心作用。ONX-0914,免疫蛋白酶体亚基β5i的选择性抑制剂,在低浓度下剂量依赖性地增强免疫原性而没有细胞毒性。此外,识别WT1的CD8+T淋巴细胞对用ONX-0914预处理的MESO-4显示更大的细胞毒性。MESO-4表达标准蛋白酶体(SP)和免疫蛋白酶体(IP)。值得注意的是,IP具有与SP不同的催化活性,有利于在抗原呈递细胞和癌细胞中产生对MHC-I具有高亲和力的抗原肽。体外,免疫蛋白酶体消化试验和质谱分析表明,IP在疏水残基后内部切割WT1235。重要的是,ONX-0914减轻了WT1235表位的这种内部切割。这些结果表明,ONX-0914可以防止IP对WT1235的内部破坏性裂解,从而促进MESO-4对WT1表位的特异性呈递。总之,选择性IP抑制剂可能通过指导特定肿瘤表位的呈递提供调节癌细胞免疫原性的手段。
The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.