TCR-seq of tissue-infiltrating lymphocytes in obese patients

White adipose tissue (WAT) plays a role in immune defense, with diverse cell types and fat-associated lymphoid clusters contributing to its functions. WAT contains a significant population of memory T cells reactive to viral antigens but only limited murine research has explored it. Here, we performed TCR-seq from blood, omental WAT, subcutaneous WAT, and liver samples from 11 obese patients to explore the landscape of tissue infiltrating T cells antigen specificity. Briefly, library preparation involves a multiplex PCR with custom designed primers, "fishing" of the molecules of interest by magnetic particles, and addition of the adaptors' sequences for Ion S5 through the extension of the DNA strands covalently bound to the particles. This last step is critical since it avoids ligation bias. TCR beta chain sequencing was performed in the Ion S5 platform using Ion-530 chips (Ion Torrent, Thermo Fisher Scientific).

白色脂肪组织（White Adipose Tissue，WAT）参与免疫防御过程，其功能依赖于多种细胞类群与脂肪相关淋巴簇的协同作用。该组织中存在大量可识别病毒抗原的记忆T细胞，但目前针对该群体的小鼠相关研究仍较为匮乏。本研究从11名肥胖患者的血液、网膜白色脂肪组织、皮下白色脂肪组织及肝脏样本中获取材料开展T细胞受体测序（TCR-seq），以解析组织浸润T细胞的抗原特异性图谱。简言之，文库构建流程如下：采用定制引物进行多重PCR，通过磁珠钓取目标分子，并通过延伸结合于磁珠的DNA链，为Ion S5测序平台添加接头序列。该最后一步至关重要，可有效避免连接偏好性。本研究使用Ion S5测序平台及Ion-530芯片（Ion Torrent，赛默飞世尔科技）完成T细胞受体β链测序。