de Beijer Monique T A, Bezstarosti Karel, Luijten Robbie, Doff Wouter A S, Boor Patrick P C, Pieterman Roel F A, Bouzid Rachid, Biesta Paula J, Ijzermans Jan N M, Doukas Michail, de Man Robert A, Woltman Andrea M, Demmers Jeroen A A, Buschow Sonja I
Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center Rotterdam, the Netherlands.
Proteomics Center, Erasmus MC, Rotterdam, the Netherlands.
JHEP Rep. 2022 Sep 5;4(11):100576. doi: 10.1016/j.jhepr.2022.100576. eCollection 2022 Nov.
BACKGROUND & AIMS: Antigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy.
Primary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity.
Using unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 10 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides.
We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general.
Immunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments ( peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses.
抗原特异性免疫疗法是治疗乙型肝炎病毒（HBV）感染和肝细胞癌（HCC）的一种有前景的策略。为了促进对恶性和/或受感染肝细胞的杀伤，了解患者来源的肝细胞上人类白细胞抗原（HLA）-I复合物呈递哪些T细胞靶点至关重要。在此，我们旨在揭示肝细胞特异性HLA-I肽组，重点关注源自HBV蛋白和肿瘤相关抗原（TAA）的肽，以指导抗原特异性免疫疗法的开发。
使用新设计的无灌注程序从（HBV感染的）非肿瘤和HCC组织中高纯度分离原代人肝细胞。通过无偏质谱（MS）鉴定肝细胞衍生的HLA结合肽，之后对来源蛋白进行基因本体论和通路分析。使用靶向MS搜索HBV抗原和TAA衍生的HLA肽，并对所选肽的免疫原性进行测试。
使用无偏数据依赖采集（DDA），我们获得了一个高质量的HLA-I肽组，包含2×10个肽，其中有8个源自HBV的肽和14个来自8种已知HCC相关TAA的肽，这些肽是肿瘤特有的。其中，3个源自HBV的和12个源自TAA的HLA肽在最初通过DDA鉴定它们的样本中通过靶向MS检测到。此外，在未使用无偏MS进行鉴定的样本中检测到2个源自HBV的和2个源自TAA的HLA肽。最后，证明了5个源自HBV的肽和3个源自TAA的肽具有免疫原性。
我们展示了首个分离的无免疫细胞的原代人肝细胞的HLA-I免疫肽组。鉴定出的源自HBV的和源自TAA的肽直接有助于开发针对慢性HBV感染和HCC的抗原特异性免疫疗法。所描述的方法通常也可应用于个性化治疗肝脏疾病的免疫治疗。
旨在诱导针对病毒或肿瘤的免疫反应的免疫疗法是治疗慢性HBV感染和肝癌的一种有前景的新型治疗选择。为了设计成功的疗法，了解患病肝细胞将病毒衍生和肿瘤特异性蛋白的哪些片段（肽）呈递给免疫系统的T细胞，从而成为免疫疗法的良好靶点至关重要。在此，我们从患有慢性HBV感染和/或肝癌的患者中分离出肝细胞，分析这些细胞呈递哪些肽，并评估哪些肽能够驱动免疫反应。
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