Center for Autoimmune Disease, Laboratory of Inflammation Biology (P.R., S.S.A.S., M.B., J.V., V.S., M.O., J.M., K.L.), La Jolla Institute for Immunology, CA.
Center for Infectious Disease and Vaccine Research (J.S., C.S.L.A., P.R., D.M., B.P., A.S.), La Jolla Institute for Immunology, CA.
Circ Res. 2022 Jul 22;131(3):258-276. doi: 10.1161/CIRCRESAHA.122.321116. Epub 2022 Jun 29.
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 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 20 APOB-derived peptides (APOB) 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.
Using stringent positive, negative, and crossover stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4 T cytokine responses to the APOB pool. Ex vivo assessment of AIMCD4 T cells revealed a statistically significant autoimmune response to APOB 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 (APOB) that triggered robust CD4 T activation in most donors. APOB-specific responding CD4 T cells were enriched in unique expanded TCR clonotypes and preferentially expressed memory markers. Cytometric bead array analysis detected APOB-induced secretion of both proinflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease, APOB 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.
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.
在动脉粥样硬化小鼠中，CD（分化群）4 T 细胞对 APOB（载脂蛋白 B）的反应得到了很好的描述，并且在人类中也可以检测到。CD4 T 细胞识别在高度多态性 HLA（人类白细胞抗原）-II 上呈现的抗原肽。个体 APOB 肽的免疫原性在人类中基本未知。仅使用 DRB1*07:01-APOB 四聚体验证了 1 个 HLA-II 限制性表位。我们假设人类 APOB 可能包含离散的免疫优势 CD4 T 细胞表位，这些表位在具有不同 HLA 等位基因的供体中引发与动脉粥样硬化相关的自身免疫反应。
我们从计算机筛选中选择了 20 个 APOB 衍生肽（APOB），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 HLA 分型供体中多个候选肽的抗原性。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）酶联免疫斑点和流式细胞术珠阵列。高通量测序揭示了 APOB 反应性 CD4 T 细胞的 TCR（T 细胞受体）克隆性。
使用严格的阳性、阴性和交叉刺激对照，我们证实了基于扩增的方案可特异性检测 APOB 池对 CD4 T 细胞因子反应的检测。使用 AIMCD4 T 细胞的体外评估揭示了对 APOB 的统计学上显著的自身免疫反应，但对普遍表达的阴性对照蛋白肌动蛋白没有反应。使用 IFNγ 酶联免疫斑点将 CD4 T 反应分辨率降低到单个肽的水平，导致发现了 6 个免疫显性表位（APOB），这些表位在大多数供体中引发了强烈的 CD4 T 激活。APOB 特异性反应的 CD4 T 细胞在独特的扩增 TCR 克隆型中富集，并优先表达记忆标记物。流式细胞术珠阵列分析检测到 APOB 诱导的促炎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，与低冠状动脉疾病患者相比，APOB 刺激诱导了更高的激活和记忆表型，并增强了促炎细胞因子 TNF（肿瘤坏死因子）和 IFNγ 的分泌。
使用 3 个队列，每个队列约有 20 名供体，我们发现并验证了 6 个免疫显性、HLA-II 限制性 APOB 表位。这些 APOB 表位的免疫反应与冠状动脉疾病的严重程度相关。
