Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vß oligoclonality and CDR3 homology in acquired aplastic anemia

In this work, we investigated the frequency and oligoclonal expansion of effector CD28-CD57+ memory cells in CD8+ T cell compartments and the T-cell Receptor (TCR) Vß repertoire in acquired aplastic anemia (AA) to provide additional evidence to the immune hypothesis in AA pathophysiology. Using flow cytometry, deep sequencing, and computational methods, we examined the blood of 32 AA patients and 29 appropriate controls for Vß usage; eight of these patients showing CD8+CD57+ expansion and three healthy subjects were subjected to TCR deep sequencing to confirm the clonality. We showed that CD8+CD57+ cells were frequently expanded with oligoclonal characteristics in AA, and patients and healthy donors shared CD8+ clonotypes by complementarity-determining region 3 sequences analysis. Overall design: For VDJ combination and CDR3 sequence profiling, DNA was isolated from FACS-sorted CD4+ and CD8+ T cells from nine SAA patients with CD8+CD57+ cell expansion and four healthy subjects (mean, 2.4 µg of DNA; range, 1.2 – 3.4 µg). DNA was also isolated from beads-sorted (Miltenyi Biotec Inc., San Diego, CA) CD8+CD57+ cells from two of the eight SAA patients with enough cells for further analysis (mean, 1.6 µg of DNA; range, 1.0 – 2.3 µg). TCR repertoire sequencing was performed with Illumina HiSeq 2000 sequencer (Illumina, Inc., San Diego, CA).

本研究针对获得性再生障碍性贫血（acquired aplastic anemia, AA）患者CD8+ T细胞亚群内效应性CD28⁻CD57⁺记忆细胞的频率与寡克隆扩增特征，以及T细胞受体（T-cell Receptor, TCR）Vβ谱系分布展开探究，旨在为AA发病机制的免疫假说补充实验证据。
本研究采用流式细胞术（flow cytometry）、深度测序（deep sequencing）及生物信息学分析手段，对32例AA患者与29例匹配对照的血液样本进行Vβ基因取用情况检测；其中8例存在CD8+CD57+细胞扩增的患者及3名健康受试者接受了TCR深度测序，以验证细胞克隆性。
研究结果表明，AA患者体内CD8+CD57+细胞常以寡克隆特性发生扩增；通过互补决定区3（complementarity-determining region 3, CDR3）序列分析发现，患者与健康供者共享部分CD8+ T细胞克隆型。
整体实验设计：为开展VDJ组合与CDR3序列谱型分析，本研究从9例伴CD8+CD57+细胞扩增的重型再生障碍性贫血（Severe Aplastic Anemia, SAA）患者及4名健康受试者的经荧光激活细胞分选（FACS-sorted）的CD4+、CD8+ T细胞中提取基因组DNA（DNA平均质量为2.4 μg，范围1.2~3.4 μg）。此外，本研究还从8例SAA患者中的2例（细胞量满足后续分析需求）的经磁珠分选的CD8+CD57+细胞中提取DNA（DNA平均质量为1.6 μg，范围1.0~2.3 μg），磁珠分选试剂购自美国加利福尼亚州圣地亚哥市美天旎生物科技公司（Miltenyi Biotec Inc., San Diego, CA）。本研究采用Illumina HiSeq 2000测序仪（Illumina, Inc., San Diego, CA）完成TCR谱系测序。