Department of Pathology, Sapporo Medical University, Sapporo, Hokkaido, Japan.
Department of Respiratory Medicine and Allergology, Sapporo Medical University, Sapporo, Hokkaido, Japan.
J Immunol. 2022 Jun 15;208(12):2856-2865. doi: 10.4049/jimmunol.2100717. Epub 2022 May 27.
CD8 T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8 T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.
CD8 T 细胞识别由 HLA Ⅰ类分子呈递的肽，并监测细胞内的肽库。众所周知，蛋白酶体将两个短肽片段拼接在一起。最近使用质谱（MS）和生物信息学分析的研究表明，蛋白酶体生成的拼接肽（PSPs）可能占 HLA Ⅰ类配体的很大一部分。然而，生物信息学方法鉴定的 PSPs 的真实性仍然存在疑问。在这项研究中，我们采用基于 MS 的从头测序技术直接捕获不在基因组中模板化的隐匿 HLA 配体。我们在具有微卫星不稳定性的人结直肠癌细胞系中鉴定了两个源自同一蛋白质的 PSP。来自健康供体的 CD8 T 细胞很容易对这两个 PSP 产生反应，表明它们的天然 HLA 呈递和抗原性。使用 minigene 构建体的实验表明，两种 PSP 分别通过标准和反向拼接的蛋白酶体依赖性加工产生。我们的结果表明，蛋白酶体拼接产生的 HLA Ⅰ类 Ag 库具有更广泛的多样性，支持 MS 方法用于全面鉴定 PSPs 的优势。
