Background:CD (cluster of differentiation) 4⁺ T-cell responses to APOB (apolipoprotein B) are well characterized in atherosclerotic mice and detectable in humans. CD4⁺ T cells recognize antigenic peptides displayed on highly polymorphic HLA (human leukocyte antigen)-II. Immunogenicity of individual APOB peptides is largely unknown in humans. Only 1 HLA-II-restricted epitope was validated using the DRB1*07:01-APOB_3036–3050 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.Methods:We selected 20 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 assay, intracellular cytokine staining, IFNγ (interferon gamma) enzyme–linked immunospot and cytometric bead array. High-throughput sequencing revealed TCR (T-cell receptor) clonalities of APOB-reactive CD4⁺ T cells.Results:Using stringent positive, negative, and crossover stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4⁺ T cytokine responses to the APOB₂₀ pool. Ex vivo assessment of AIM⁺CD4⁺ T cells revealed a 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 IFNγ enzyme–linked immunospot led to the discovery of 6 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. Cytometric bead array analysis detected APOB₆-induced secretion of both proinflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease, APOB₆ stimulation induced higher activation and memory phenotypes and augmented secretion of proinflammatory cytokines TNF (tumor necrosis factor) and IFNγ, compared with patients with low coronary artery disease.Conclusions:Using 3 cohorts, each with ≈20 donors, we discovered and validated 6 immunodominant, HLA-II–restricted APOB epitopes. The immune response to these APOB epitopes correlated with coronary artery disease severity.

中文翻译：

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人载脂蛋白 B 中的免疫显性 MHC-II（主要组织相容性复合体 II）限制性表位

背景：CD（分化簇）4 ⁺ T 细胞对 APOB（载脂蛋白 B）的反应在动脉粥样硬化小鼠中得到了很好的表征，并且在人类中也可检测到。CD4 ⁺ T 细胞识别高度多态性 HLA（人类白细胞抗原）-II 上展示的抗原肽。单个 APOB 肽的免疫原性在人类中很大程度上未知。使用 DRB1*07:01-APOB _{3036 –3050}四聚体仅验证了 1 个 HLA-II 限制性表位。我们假设人类 APOB 可能含有离散的免疫显性 CD4 ⁺ T 细胞表位，可在具有不同 HLA 等位基因的供体中触发动脉粥样硬化相关的自身免疫反应。方法：我们从计算机筛选中选择了 20 个 APOB 衍生肽 (APOB 20 ) 并进行了实验_验证与最常见的人类 HLA-II 等位基因结合。我们优化了基于再刺激的工作流程，以评估 HLA 型供体中多种候选肽的抗原性。这包括激活诱导标记物测定、细胞内细胞因子染色、IFNγ（干扰素γ）酶联免疫斑点和细胞计数珠阵列。高通量测序揭示了 APOB 反应性 CD4 ⁺ T 细胞的 TCR（T 细胞受体）克隆。结果：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检测 CD4 ⁺ T 细胞因子反应的特异性到 APOB ₂₀池。^{AIM +} CD4 ⁺ T 细胞的离体评估揭示了对 APOB ₂₀的统计显着的自身免疫反应，但对普遍表达的阴性对照蛋白肌动蛋白没有反应。使用 IFNγ 酶联免疫斑点解析 CD4 ⁺ T 反应对单个肽的水平，发现了 6 个免疫显性表位 (APOB ₆ )，这些表位在大多数供体中触发了强烈的 CD4 ^{+ T 激活。}APOB ₆特异性响应 CD4 ⁺ T 细胞富含独特的扩展 TCR 克隆型并优先表达记忆标记。细胞计数珠阵列分析检测到 APOB ₆诱导的促炎细胞因子和调节细胞因子的分泌。在经血管造影证实的冠状动脉疾病患者的临床样本中，APOB ₆与低冠心病患者相比，刺激诱导更高的激活和记忆表型，并增加促炎细胞因子 TNF（肿瘤坏死因子）和 IFNγ 的分泌。结论：使用 3 个队列，每个队列有约 20 名供体，我们发现并验证了 6 种免疫显性、 HLA-II 限制性 APOB 表位。对这些 APOB 表位的免疫反应与冠状动脉疾病的严重程度相关。