目的·探究结直肠癌（colorectal cancer，CRC）组织T细胞受体（T cell receptor，TCR）组库多样性与患者临床特征以及具核梭形杆菌（Fusobacterium nucleatum，Fn）丰度之间的关系。方法·收集63例上海交通大学医学院附属仁济医院胃肠外科收治的CRC患者的肿瘤组织及匹配的癌周正常黏膜组织，运用Illumina HiSeq 4000测序平台对符合质量标准的53对组织样本进行深度RNA测序，测序方案为双末端测序（pair-end 150 bp，PE150）。对原始测序数据进行质量控制和基因组注释，并利用TRUST4算法从RNA测序结果中提取肿瘤组织及癌周正常黏膜组织的TCR组库信息。同时对该53对样本16S rDNA的特定序列进行高通量测序分析，通过对测序数据进行质量控制及聚类，获得操作分类单元（operational taxonomic units，OTU）信息，并基于rdp_16s_v16.fa分类学数据库对OTU进行分类学注释以获取细菌的丰度信息，尤其是Fn的丰度信息。利用immunarch R包分析肿瘤组织及癌周正常黏膜组织TCR组库克隆型和多样性的特点。TCR组库多样性采用Chao1指数、逆Simpson指数以及Hill数值曲线等指标评价。根据患者性别、年龄、肿瘤位置、TNM分期等临床特征和Fn丰度因素进行分组，使用Wilcoxon检验比较不同临床特征以及Fn丰度下TCR组库多样性的差异。结果·CRC患者肿瘤组织TCR克隆型的数目低于癌周正常黏膜组织（P=0.000），肿瘤组织Chao1指数、逆Simpson指数以及Hill数值显著小于癌周正常黏膜组织（P=0.000）。不同性别、年龄（≥65岁vs <65岁）、TNM分期（Ⅰ/Ⅱ期 vs Ⅲ/Ⅳ期）分组下CRC肿瘤组织Chao1指数以及逆Simpson指数差异无统计学意义（均P>0.05）。发生于直肠部肿瘤的TCR克隆型数目较非直肠部位肿瘤更高（P=0.040）。Fn高负荷的CRC患者TCR组库Chao1指数显著低于Fn低负荷患者（P=0.030）。结论·TCR组库多样性与CRC患者多项临床特征以及Fn的丰度有关，提示其在CRC的发生和发展中具有重要意义。

Objective ·To analyze the association between T cell receptor repertoire diversity in colorectal cancer (CRC) patients and clinical characteristics and Fusobacterium nucleatum (Fn) abundance. Methods ·Sixty-three surgical CRC specimens and adjacent normal mucosa tissues were collected from the Department of Gastrointestinal Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine. Totally 53-paired qualified samples were subsequently sequenced on an Illumina HiSeq 4000 for paired-end 150 bp (PE150) sequencing. Quality control and genome annotation were carried out for the original sequencing data. Then a TRUST4 algorithm was further used to acquire TCR repertoire information from RNA sequencing results of tumor tissues and adjacent normal tissues. Meanwhile, the specific sequences of all samples' 16S rDNA were selected for high-throughput sequencing analysis. Clustered from the sequencing data after quality control, the operational taxonomic units (OTU) information was annotated based on rdp_16s_v16.fa taxonomic database to acquire bacterial abundance information, especially Fn. Immunarch R package was utilized to evaluate the characteristics of TCR repertoire clonotypes and diversity. Chao1 index, inverse Simpson index and Hill numerical curve were then conducted to assess the TCR repertoire diversity. Clinical features, such as gender, age, tumor location, and TNM stage, and biological factors like Fn load, were included to dichotomize patients into different groups, and repertoire diversity comparison between subgroups within different clinical features and Fn loads was conducted by Wilcoxon test subsequently. Results ·The number of TCR repertoire clonotypes in CRC tissues was lower compared to that in adjacent normal tissues (P=0.000). Chao1 index, inverse Simpson index and Hill number in tumor tissues were significantly lower than those in adjacent normal tissues (P=0.000). The Chao1 indexes, and inverse Simpson indexes between different gender, age (≥65 years vs <65 years) or TNM stage (Ⅰ/Ⅱ stage vs Ⅲ/Ⅳ stage) group showed no statistically significant difference (P>0.05). Tumors located in rectum had higher TCR clonotypes than those located in non-rectum (P=0.040). In addition, CRC patients with high Fn load demonstrated lower Chao1 indexes than those with low Fn load (P=0.030). Conclusion ·The TCR repertoire diversity is correlated with multiple clinical features as well as the abundance of Fn, indicating that the analysis based on TCR repertoire diversity of CRC patients can provide potential value for CRC tumorigenesis and development.