Table_1_An Integrated Genomic, Proteomic, and Immunopeptidomic Approach to Discover Treatment-Induced Neoantigens.xlsx

All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires — particularly novel MHC presentation of mutation-bearing peptides (neoantigens) — can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells’ antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.

所有有核哺乳动物细胞均表达主要组织相容性复合体（major histocompatibility complex, MHC）蛋白，此类蛋白可在细胞表面呈递肽段以参与免疫监视。此类由MHC呈递的肽段（即肽-MHC复合物，peptide-MHC complex, pMHC）是介导T细胞应答、靶向携带病原体来源或体细胞突变来源非自身抗原的细胞的关键分子基础。肿瘤特异性抗原谱的改变——尤其是携带突变的肽段的新型MHC呈递（即新抗原，neoantigens）——可成为抗肿瘤免疫应答的强效靶点。本研究采用整合基因组学与蛋白质组学的抗原发现策略，以人淋巴瘤细胞系GRANTA-519为模型，探究γ干扰素（interferon gamma, IFN-γ）对抗原呈递过程的调控作用。经IFN-γ处理后，细胞内共有126个蛋白质呈现差异表达（占所有定量蛋白质的2%），其中涵盖抗原呈递机器组分、干扰素信号通路蛋白以及MHC分子本身。此外，研究发现多个蛋白酶体亚基的表达受到调控，这与此前IFN-γ诱导免疫蛋白酶体形成的研究报道相符。该结果提示，细胞对IFN-γ产生的轻度蛋白质组应答，可对细胞的抗原/表位谱造成更为显著的改变。据此，我们通过MHC免疫沉淀联合洗脱肽段谱的质谱分析，揭示了IFN-γ诱导的特异性分子特征：MHC I类分子可重复呈递951种独特肽段，MHC II类分子则呈递582种独特肽段。此外，另有一组pMHC复合物（包含多个候选新抗原）的相对丰度发生改变，借此可区分对照组与IFN-γ处理组样本。据此，我们构建了一套分类系统，用于区分因表达量改变而呈现差异呈递的肽段与因抗原加工过程改变而产生的新型肽段。综上，本研究数据证实，IFN-γ可通过改变抗原的种类与丰度，重塑细胞的抗原谱。将本研究方法推广至更具临床相关性的模型中，有望开发出可人为重塑免疫肽谱的策略，从而增强内源性或疫苗诱导的抗肿瘤免疫应答，乃至潜在的抗病毒免疫应答。