Immunodominant MHC-II (Major Histocompatibility Complex II) Restricted Epitopes in Human Apolipoprotein B.
Immunodominant MHC-II (Major Histocompatibility Complex II) Restricted Epitopes in Human Apolipoprotein B.
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人载脂蛋白B的免疫显性MHC-II限制性表位
DOI：
10.1161/circresaha.122.321116
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发表时间：
2022-07-22
影响因子：
20.1
通讯作者：
Ley, Klaus
中科院分区：
文献类型：
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作者：
Roy, Payel;Sidney, John;Lindestam Arlehamn, Cecilia S.;Phillips, Elizabeth;Mallal, Simon;Armstrong Suthahar, Sujit Silas;Billitti, Monica;Rubiro, Paul;Marrama, Daniel;Drago, Fabrizio;Vallejo, Jenifer;Suryawanshi, Vasantika;Orecchioni, Marco;Makings, Jeffrey;Kim, Paul J.;McNamara, Coleen A.;Peters, Bjoern;Sette, Alessandro;Ley, Klaus
CD4+T细胞对载脂蛋白B的反应在动脉粥样硬化小鼠中有很好的特征，在人类中也能检测到。CD4+T细胞识别高度多态的人类白细胞抗原-II上的抗原肽。在人类中，单个APOB多肽的免疫原性在很大程度上是未知的。仅用DRB1*07：01-APOB3036-3050四聚体验证了一个HLA-II限制性表位。我们假设人类载脂蛋白B可能包含离散的免疫优势的CD4+T细胞表位，在具有不同的HLA等位基因的捐赠者中触发动脉粥样硬化相关的自身免疫反应。我们从电子筛查中选择了20个APOB衍生多肽(APOB20)，并通过实验验证了它们与最常见的人类HLA-II等位基因的结合。我们优化了一种基于再刺激的工作流程，以评估多个候选多肽在HLA型供体中的抗原性。包括活化诱导标记物分析、细胞内细胞因子染色、干扰素У-酶联免疫斑点检测和细胞微珠阵列检测。高通量测序描绘了APOB反应性CD4+T细胞的TCR克隆。使用严格的阳性、阴性和交叉刺激对照，我们确认了基于扩增的方案的特异性，以检测对APOB20池的CD4+T细胞因子反应。对AIM+CD4+T细胞的体外评估显示，对APOB20有统计学上显著的自身免疫反应，但对普遍表达的负控制蛋白-肌动蛋白没有。使用干扰素У-ELISpot对单个多肽水平的CD+T反应进行解析，导致发现了6个免疫优势表位(APOB 6)，它们在大多数捐赠者中引发了强大的CD 4+T活化。APOB6特异性应答的CD4+T细胞富含独特的扩增TCR克隆型，并优先表达记忆标记。CBA分析检测到APOB6诱导的促炎和调节性细胞因子的分泌。在冠状动脉造影证实的冠心病患者的临床样本中，与低冠状动脉疾病患者相比，APOB6刺激诱导了更高的激活和记忆表型，并增加了促炎细胞因子肿瘤坏死因子和干扰素У的分泌。使用三个队列，每个队列有大约20个供体，我们发现并验证了六个免疫显性的、人类白细胞抗原II限制的载脂蛋白B表位。对这些载脂蛋白B表位的免疫应答与冠心病的严重程度相关。
CD4+T cell responses to apolipoprotein B are well characterized in atherosclerotic mice and detectable in humans. CD4+T cells recognize antigenic peptides displayed on highly polymorphic Human Leukocyte Antigen-II. Immunogenicity of individual APOB peptides is largely unknown in humans. Only one HLA-II-restricted epitope was validated using the DRB1*07:01-APOB3036–3050 tetramer. We hypothesized that human APOB may contain discrete immunodominant CD4+T cell epitopes that trigger atherosclerosis-related autoimmune responses in donors with diverse HLA alleles. We selected twenty APOB-derived peptides (APOB20) from an in silico screen and experimentally validated binding to the most commonly occurring human HLA-II alleles. We optimized a restimulation-based workflow to evaluate antigenicity of multiple candidate peptides in HLA-typed donors. This included activation-induced marker (AIM) assay, intracellular cytokine staining (ICS), IFNУ–enzyme-linked immunospot (ELISpot) and Cytometric Bead Array (CBA). High-throughput sequencing delineated TCR clonalities of APOB-reactive CD4+T cells. Using stringent positive, negative and crossover-stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4+T cytokine responses to APOB20 pool. Ex vivo assessment of AIM+CD4+T cells revealed statistically significant autoimmune response to APOB20, but not to a ubiquitously-expressed negative control protein, actin. Resolution of CD4+T responses to the level of individual peptides using IFNУ-ELISpot, led to the discovery of six immunodominant epitopes (APOB6) that triggered robust CD4+T activation in most donors. APOB6-specific responding CD4+T cells were enriched in unique expanded TCR clonotypes and preferentially expressed memory markers. CBA analysis detected APOB6-induced secretion of both pro-inflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease (CAD), APOB6 stimulation induced higher activation and memory phenotypes, and augmented secretion of proinflammatory cytokines TNF and IFNУ, compared to patients with low CAD. Using three cohorts, each with ~20 donors, we discovered and validated six immunodominant, HLA-II-restricted APOB epitopes. Immune response to these APOB epitopes correlated with CAD severity.