DataSheet_2_Identification of apolipoprotein B–reactive CDR3 motifs allows tracking of atherosclerosis-related memory CD4+T cells in multiple donors.docx

IntroductionAtherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)–reactive CD4+T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+T cells are unknown.
MethodsIn a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen–typed donors.
ResultsWe identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio ≥1, Fisher’s test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning–based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+ motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire.
DiscussionThe identified APOB-reactive expanded CD4+T cell clones and conserved motifs can be used to annotate and track human atherosclerosis-related autoreactive CD4+T cells and measure their clonal expansion.

引言
动脉粥样硬化（Atherosclerosis）是多种心血管疾病（CVDs）的核心病理基础。其病因学涉及自身耐受的破缺，可导致自身反应性载脂蛋白B（APOB）反应性CD4+T细胞发生克隆扩增，这一过程与临床心血管疾病的发生密切相关。然而，介导APOB特异性CD4+T细胞活化的T细胞受体（TCR）序列至今尚未明确。

方法
在既往研究中，我们已对6种多数供体均可识别的免疫优势APOB表位响应的CD4+T细胞的高可变互补决定区3（CDR3）进行了表征分析。本研究针对来自6例人类白细胞抗原（HLA）分型供体的数据集展开全面分析，该数据集包含149065条APOB反应性CDR3序列与199211条非反应性对照CDR3序列。

结果
相较于对照组，我们在APOB反应性组中鉴定出672个高度扩增的克隆（频率阈值＞1.39×10^-3），这些克隆显著富集（对数10比值比≥1，Fisher精确检验P＜0.01）。对来自相同供体的114755条初始T细胞（naïve）、91001条中央记忆T细胞（TCM）及29839条效应记忆T细胞（TEM）的CDR3序列进行分析后发现，APOB反应性克隆可追溯至未富集的外周血T细胞的复杂克隆库。APOB反应性克隆与记忆CDR3序列的重叠比例介于2.2%至46%之间（平均16.4%），显著高于其与初始T细胞库的重叠比例（0.7%至2%，平均1.36%）。借助基于机器学习的计算机辅助工具GLIPHs（通过抗原结合域热点归类淋巴细胞相互作用，grouping of lymphocyte interactions by paratope hotspots）进行CDR3基序分析，我们共鉴定出532个APOB反应性基序。对初始T细胞与记忆T细胞的CDR3序列开展GLIPH分析显示，约40%（532个中的209个）的APOB反应性基序在记忆细胞库中显著富集。使用Cytoscape进行网络分析发现，隶属于记忆细胞群的APOB反应性基序可在多个供体间广泛共享。我们还鉴定出6种基序，这类基序在超过80%的供体的中央记忆T细胞与效应记忆T细胞CDR3序列中均存在，且在APOB反应性TCR库中高度富集。

讨论
本研究鉴定出的APOB反应性扩增CD4+T细胞克隆与保守基序，可用于注释并追踪人类动脉粥样硬化相关的自身反应性CD4+T细胞，并量化其克隆扩增水平。