Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children’s Hospital, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany.
Haematologica. 2013 Sep;98(9):1388-96. doi: 10.3324/haematol.2012.069708. Epub 2013 May 28.
Current diagnostic approaches that characterize T-cell deficiency by analyzing diversity of T-cell receptor sequences effectuate limited informational gain about the actual restrictiveness. For deeper insight into T-cell receptor repertoires we developed next-generation-sequencing-spectratyping, which employs high coverage Roche/454 sequencing of T-cell receptor (β)-chain amplicons. For automated analysis of high-throughput-sequencing data, we developed a freely available software, the TCR profiler. Gene usage, length, encoded amino acid sequence and sequence diversity of the complementarity determining region 3 were determined and comprehensively integrated into a novel complexity score. Repertoires of CD8(+) T cells from children with idiopathic or hepatitis-induced very severe aplastic anemia (n=7), children two months after bone marrow transplantation (n=7) and healthy controls (children n=5, adults n=5) were analyzed. Complexity scores clearly distinguished between healthy and diseased, and even between different immune deficiency states. The repertoire of aplastic anemia patients was dominated by public (i.e. present in more than one person) T-cell receptor clonotypes, whereas only 0.2% or 1.9% were public in normal children and adults, respectively. The CDR3 sequence ASSGVGFSGANVLT was highly prevalent in 3 cases of hepatitis-induced anemia (15-32% of all sequences), but was only low expressed in idiopathic aplastic anemia (2-5%, n=4) or healthy controls (<1%). Fifteen high frequent sequences were present exclusively in aplastic anemia patients. Next-generation-sequencing-spectratyping allows in-depth analysis of T-cell receptor repertoires and their restriction in clinical samples. A dominating clonotype was identified in hepatitis-induced anemia that may be associated with disease pathogenesis and several aplastic-anemia-associated, putatively autoreactive clonotypes were sequenced.
目前，通过分析 T 细胞受体序列的多样性来描述 T 细胞缺陷的诊断方法，对于实际受限程度的信息增益有限。为了更深入地了解 T 细胞受体库，我们开发了下一代测序-光谱分析，该方法采用 Roche/454 高通量测序对 T 细胞受体（β）链扩增子进行高覆盖测序。为了对高通量测序数据进行自动分析，我们开发了一个免费的软件 TCR profiler。确定了基因使用情况、长度、编码氨基酸序列和互补决定区 3 的序列多样性，并将其综合纳入一个新的复杂性评分中。分析了特发性或肝炎引起的极重度再生障碍性贫血患儿（n=7）、骨髓移植后两个月的患儿（n=7）和健康对照者（儿童 n=5，成人 n=5）的 CD8+T 细胞受体库。复杂性评分清楚地区分了健康和患病者，甚至区分了不同的免疫缺陷状态。再生障碍性贫血患者的受体库由公共（即存在于不止一个人身上）T 细胞受体克隆型主导，而正常儿童和成人的公共克隆型分别仅占 0.2%或 1.9%。CDR3 序列 ASSGVGFSGANVLT 在 3 例肝炎引起的贫血中高度流行（所有序列的 15-32%），但在特发性再生障碍性贫血（2-5%，n=4）或健康对照者中表达水平较低（<1%）。15 个高频序列仅存在于再生障碍性贫血患者中。下一代测序-光谱分析允许对临床样本中的 T 细胞受体库及其受限情况进行深入分析。在肝炎引起的贫血中发现了一种主要的克隆型，可能与疾病发病机制有关，并且对一些与再生障碍性贫血相关的、推测的自身反应性克隆型进行了测序。
